U.S. patent application number 14/636059 was filed with the patent office on 2015-06-25 for identifying and modulating molecular pathways that mediate nervous system plasticity.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Gabriel Kreiman, Mriganka Sur, Daniela Tropea.
Application Number | 20150174212 14/636059 |
Document ID | / |
Family ID | 38610217 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150174212 |
Kind Code |
A1 |
Sur; Mriganka ; et
al. |
June 25, 2015 |
IDENTIFYING AND MODULATING MOLECULAR PATHWAYS THAT MEDIATE NERVOUS
SYSTEM PLASTICITY
Abstract
The present invention provides methods for identifying genes and
pathways involved in plasticity. The invention applies some of
these methods to identify genes that are differentially regulated
in at least a portion of the nervous system of an individual
subjected to conditions known to result in altered nervous system
plasticity, i.e., dark rearing (DR) or monocular deprivation (MD).
The genes are targets for pharmacological agents that modify
plasticity. The invention also identifies biological pathways that
are enriched in genes that are differentially regulated under
conditions known to result in altered nervous system plasticity.
The present invention further provides methods and compositions for
modifying plasticity in the nervous system of a subject. The
invention includes a method for modifying plasticity in the nervous
system of a subject comprising administering a plasticity-modifying
agent to the subject, wherein the plasticity-enhancing agent
modulates a gene or pathway that is differentially regulated in
developmental conditions that alter nervous system plasticity
(e.g., DR or MD). The methods and compositions may be administered
to a subject suffering from damage to the nervous system or from a
neuropsychiatric disorder in order to enhance recovery,
reorganization, or function of the nervous system. The methods
optionally include administering a proteolysis-enhancing agent to
the subject.
Inventors: |
Sur; Mriganka; (Cambridge,
MA) ; Tropea; Daniela; (Cambridge, MA) ;
Kreiman; Gabriel; (West Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
38610217 |
Appl. No.: |
14/636059 |
Filed: |
March 2, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12297189 |
Aug 11, 2009 |
8969295 |
|
|
PCT/US2007/009172 |
Apr 12, 2007 |
|
|
|
14636059 |
|
|
|
|
60792275 |
Apr 14, 2006 |
|
|
|
Current U.S.
Class: |
424/85.5 ;
514/423; 514/460; 514/8.6 |
Current CPC
Class: |
A61K 38/49 20130101;
A61K 38/30 20130101; A61P 25/18 20180101; A61P 43/00 20180101; A61K
38/49 20130101; A61K 38/217 20130101; A61K 45/06 20130101; A61P
25/28 20180101; A61P 25/00 20180101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/30 20060101
A61K038/30; A61K 45/06 20060101 A61K045/06; A61K 38/21 20060101
A61K038/21 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government Support under Grant
No. EY014134 awarded by the NIH. The Government has certain rights
in the invention.
Claims
1. A method for modifying plasticity or aiding restoration of
function in the nervous system of a subject comprising the step of:
administering a plasticity-modifying agent to a subject in need
thereof, wherein the agent is administered either alone or in
combination with one or more additional agents in an amount
effective to modify nervous system plasticity, wherein the
plasticity-modifying agent modulates a gene or pathway that is
differentially regulated in at least a portion of the nervous
system of an individual subjected to a plasticity-modifying
condition.
2. The method of claim 1, wherein the condition is a
neurodevelopmental disorder.
3. The method of claim 2, wherein the neurodevelopmental disorder
is selected from the group consisting of autism, Rett Syndrome,
Fragile X syndrome, tuberous sclerosis, or autism spectrum
disorder.
4. The method of claim 1, wherein the condition is a
neuropsychiatric disorder.
5. The method of claim 3, wherein the neuropsychiatric disorder is
selected from the group consisting of schizophrenia and bipolar
disorder.
6. The method of claim 1, wherein the condition is trauma to the
nervous system caused by a condition such as stroke.
7. The method of claim 1, wherein the plasticity-modifying
condition comprises dark rearing (DR) or monocular deprivation
(MD).
8. The method of claim 1, wherein the plasticity-modifying agent is
IGF1 or a modulator of the IGF1 pathway.
9. The method of claim 1, wherein the plasticity-modifying agent is
a modulator of the JAK/STAT pathway.
10. The method of claim 1, wherein the plasticity-modifying agent
is IFN.gamma..
11. The method of claim 1, wherein the plasticity-modifying agent
is a statin.
12. The method of claim 1, which comprises administering a first
agent that activates the IGF1 pathway and a second agent that
activates the JAK/STAT pathway.
13. The method of claim 1, wherein the plasticity-modifying agent
inhibits development, survival, or activity of parvalbumin
expressing interneurons.
14. The method of claim 1, wherein the plasticity-modifying agent
inhibits expression or activity of parvalbumin.
15. The method of claim 1, wherein the plasticity-modifying agent
is delivered in an amount effective to enhance the capacity of the
nervous system, or a portion thereof, to restore lost function in
an activity-dependent manner or in response to a second agent.
16. The method of claim 1, wherein the plasticity-modifying agent
is administered to a subject who has suffered damage to the nervous
system or has been diagnosed with a disorder of the nervous system,
and is administered in an amount and for a time effective to
promote nervous system recovery, reorganization, or function.
17. The method of claim 1, wherein the step of administering
comprises steps of: (1) administering a first plasticity-modifying
agent to a subject following an event that damages the brain or
spinal cord or following diagnosis of a neuropsychiatric or
neurodevelopmental disorder for a first period of time; (2)
administering a second plasticity-modifying agent to the subject
for a second period of time, wherein the second period of time is
separate from the first period of time.
18-66. (canceled)
67. A method for promoting recovery or reorganization in the
nervous system of a subject comprising the step of: administering a
plasticity-modifying agent to a subject in need thereof, wherein
the agent is administered either alone or in combination with one
or more additional agents in an amount effective to promote
recovery or reorganization in the nervous system, wherein the
plasticity-modifying agent modulates a gene or pathway that is
differentially regulated in at least a portion of the nervous
system of an individual subjected to a plasticity-modifying
condition.
68-77. (canceled)
78. A method for promoting recovery or reorganization in the
nervous system of a subject in need thereof comprising the step of:
administering to the subject a proteolysis-enhancing agent and an
agent selected from the group consisting of: agents that activate
the IGF1 pathway, agents that activate the JAKISTAT pathway, agents
that inhibit the development, survival, or activity of parvalbumin
expressing interneurons, and agents that inhibit the expression of
parvalbumin, wherein the proteolysis-enhancing agent is
administered at least 3, 6, 12, 24, or more hours after a specific
damaging event or diagnosis of a disorder and optionally also prior
to 3 hours after the specific damaging event or diagnosis of a
disorder.
79. A drug delivery device for implantation into the nervous system
of a subject, the drug delivery device comprising: a biocompatible
polymer; and a plasticity-modifying agent, wherein the
plasticity-modifying agent is released from the polymer in an
amount effective to modify plasticity in the nervous system of the
subject.
80-101. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. provisional patent application U.S. Ser. No.
60/792,275, filed Apr. 14, 2006, which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Diseases and accidents leading to nervous system damage or
degeneration are among the leading causes of mortality and
morbidity in many countries. For example, approximately 700,000
people suffer a first or recurrent stroke annually in the United
States, resulting in over 150,000 deaths. Although stroke
represents the most common cause of damage to the central nervous
system (CNS), a number of other conditions are also significant
causes of functional deficits due to loss of brain tissue, either
as a direct consequence of injury, or secondary to events such as
swelling. Among these are primary brain tumors, brain metastases,
and surgery for these or other conditions.
[0004] Strokes are a result of a sudden disruption of blood flow to
a part of the brain and occur when a blood vessel that normally
supplies brain tissue either bursts or becomes transiently or
permanently blocked, such as by a blood clot (e.g., a
thromboembolus) or other embolus or obstruction. The resulting
disruption in normal blood flow deprives the affected tissue of
needed oxygen and nutrients and can also impair removal of waste
products, resulting in damage to, or death of, nervous system
cells. Currently the only therapy for ischemic stroke approved by
the U.S. Food and Drug Administration (FDA) is infusion of the
thrombolytic agent tissue type plasminogen activator (tPA) within a
short time window following the causative event. Such thrombolytic
therapy was shown to be both safe and beneficial if delivered
within 3 hours of the onset of symptoms (NINDS, Tissue plasminogen
activator for acute ischemic stroke. The national institute of
neurological disorders and stroke RT-PA stroke study group. N.
Engl. J. Med. 333: 1581-1587, 1995).
[0005] While stroke is the third leading cause of death in
industrialized countries, in most cases stroke is not fatal.
However, stroke is a major cause of morbidity and a leading cause
of serious, long-term disability. About 4.8 million stroke
survivors are alive today in the United States, with a much larger
total number worldwide. Many of these individuals suffer from
functional limitations affecting the senses, motor activity, speech
and/or the ability to understand speech, behavior, thought
patterns, memory, emotions, or other aspects of cognition. Although
functional deficits following stroke may be permanent, in many
cases full or partial recovery is possible. The mainstays of
treatment are supportive care and rehabilitation therapy, which
frequently continues for months or years. Unfortunately, there are
no pharmacological agents that have demonstrated efficacy in
improving the long-term outcome of stroke.
[0006] Approximately 10,000-12,000 individuals suffer spinal cord
injuries (SCI) each year in the United States, bringing the
projected prevalence rate in the United States to nearly 280,000 by
the year 2014 (DeVivo, M. J., 2002) Improvements in supportive care
have greatly increased the survival rate following such injuries,
but therapeutic options remain limited, and efforts focus on
rehabilitation. Tumors affecting the spinal cord or meninges
(either primary tumors or metastases) are also a significant source
of morbidity.
[0007] Disorders of the nervous system also have a massive impact
on society. Disorders of brain development, such as autism, now
afflict about 1 in 166 children. The total number of individuals in
the U.S. afflicted with autism, learning disabilities, and similar
disorders is estimated to exceed 4 million. Neuropsychiatric
disorders such as schizophrenia and bipolar disorders extract a
huge cost in lifetime care for afflicted individuals as well as
emotional toll on caregivers and families. Neurodevelopmental
disorders such as autism are usually treated with behavioral
therapies alone, and these strategies have limited success.
Similarly, neuropsychiatric disorders such as schizophrenia and
bipolar disorder have very limited therapeutic possibilities.
[0008] Thus there is a need in the art for improved treatments,
particularly pharmacological treatments, that would enhance
recovery following damage to the CNS and/or help improve CNS and
cognitive function in neuropsychiatric and neurodevelopmental
disorders. Common to a large range of CNS conditions is the concept
that they centrally involve the function of synapses and their
ability to change (i.e., plasticity). Thus, there is a need for new
approaches to the identification of genes, molecules, cell types,
and biological pathways that play a role in key nervous system
properties such as plasticity and that can be modulated to provide
a therapeutic benefit.
SUMMARY OF THE INVENTION
[0009] The invention provides a method of identifying a gene
involved in plasticity comprising steps of: subjecting an
individual to a condition that modifies nervous system plasticity;
measuring level or activity of each of a plurality of genes in at
least a portion of the individual's nervous system; and identifying
one or more genes whose expression or activity is differentially
regulated in the portion of the individual's nervous system
relative to its expression or activity under alternative
conditions. In some embodiments, the condition comprises depriving
at least a portion of the individual's nervous system of normal
inputs. The method may comprise identifying a biological pathway or
process enriched in genes that are differentially regulated in at
least a portion of the nervous system of an individual subjected to
a plasticity-modifying condition.
[0010] The invention provides genes that are differentially
regulated under conditions that modify plasticity. The invention
provides biological pathways that are enriched in such genes. The
invention identifies a specific cell type, parvalbumin containing
interneurons, as being downregulated under conditions that prolong
plasticity. Based at least in part on the identification of these
genes, pathways, and cell type, the invention provides combinations
of plasticity-modifying agents of particular use. For example, in
one embodiment an activator of the insulin-like growth factor 1
(IGF1) pathway (e.g., IGF1 or an active peptide fragment thereof;
or a modulator of the JAK/STAT pathway, e.g., IFN.gamma. or an
HMG-CoA reductase inhibitor such as a statin) are administered to a
subject either individually or in a single composition.
[0011] The present invention provides a method for modifying
plasticity in the nervous system of a subject comprising the step
of administering a plasticity-modifying agent to a subject in need
thereof, wherein the agent is administered either alone or in
combination with one or more additional agents in an amount
effective to modify nervous system plasticity, wherein the
plasticity-modifying agent modulates a gene or pathway that is
differentially regulated in at least a portion of the nervous
system of an individual subjected to a plasticity-modifying
condition. The agent may be administered once, multiple times,
and/or continuously. The time may be selected in conjunction with
the amount to be effective to modify nervous system plasticity.
Exemplary plasticity-modifying condition comprise dark rearing or
monocular deprivation.
[0012] The invention includes a method for promoting recovery
and/or reorganization in the nervous system of a subject in need of
enhancement of recovery and/or reorganization of the nervous system
comprising administering a plasticity-modifying agent to the
subject, wherein the plasticity-enhancing agent modulates a gene or
pathway that is differentially regulated in the nervous system of
an individual subjected to a plasticity-modifying condition, e.g.,
dark-rearing (DR) or monocular deprivation (MD). The agent is
administered in an amount effective to promote recovery or
reorganization in the nervous system. The agent may be administered
once, multiple times, and/or continuously. The time may be selected
in conjunction with the amount to be effective to promote nervous
system recovery or reorganization. The subject may be in need of
recovery or reorganization of the nervous system as a result of
ischemic, hemorrhagic, neoplastic, degenerative, traumatic, and/or
neurodevelopmental damage to the nervous system. The subject may be
in need of reorganization of the nervous system as a result of a
neurodevelopmental or neuropsychiatric disorder. The method can
include a step of identifying or providing, e.g., diagnosing a
subject as having suffered such damage or having a
neurodevelopmental or neuropsychiatric disorder. The methods can
include a step of identifying or diagnosing the subject as having a
reasonable likelihood (e.g., at least a 5% chance, at least a 10%,
or at least a 50% chance).
[0013] The methods may also include administering a
proteolysis-enhancing agent such as tissue plasminogen activator
(tPA), plasmin, or a PAI inhibitor to the nervous system of the
subject. A plasticity-modifying agent of the present invention is,
in general, distinct from the proteolysis-enhancing agents
described herein. The plasticity-modifying agent and the
proteolysis-enhancing agent may be administered as part of a single
composition or individually. The present invention provides a
composition comprising a plasticity-modifying agent and a
proteolysis-enhancing agent. The composition(s) can be delivered
using a variety of techniques including injection, via infusion
pump, from an implantable microchip, or using a polymeric delivery
vehicle. The composition(s) can be administered, for example, to
one or more subdivisions or areas of the brain, the spinal cord, or
to one or more nerves or nerve tracts innervating diverse regions
of the body.
[0014] In certain embodiments the composition is administered by
implanting into the subject a drug delivery device that releases
the plasticity-modifying agent over a period of time at or in the
vicinity of a desired location. The desired location can be, for
example, an area of ischemic, hemorrhagic, neoplastic,
degenerative, traumatic, and/or neurodevelopmental damage in the
central or peripheral nervous system, or location in a brain
hemisphere opposite to an area of damage. In some embodiments the
drug delivery device comprises a pump. In some embodiments the drug
delivery device comprises a biocompatible polymer, e.g., a
biodegradable polymer. In some embodiments the polymeric matrix of
the drug delivery device comprises a hydrogel. In some embodiments
of the invention the composition comprises a plurality of polymeric
microparticles or nanoparticles having the plasticity-modifying
agent associated therewith (e.g., encapsulated therein, adsorbed
thereon, entangled in a polymer network, etc.).
[0015] The invention also includes a drug delivery device for
implantation into the body of a subject to modify plasticity. In
certain embodiments of the invention the device is implanted to
promote nervous system reorganization and/or recovery following
ischemic, hemorrhagic, neoplastic, traumatic, degenerative, and/or
neurodevelopmental damage.
[0016] An inventive device may include a proteolysis-enhancing
agent, e.g., a proteolytic agent such as a protease. Alternatively
or additionally, a proteolysis-enhancing agent can be administered
separately. In certain embodiments the proteolysis-enhancing agent
is plasmin, a plasminogen activator, and/or an inhibitor of an
endogenous plasminogen activator inhibitor. For example, in certain
embodiments, the proteolysis-enhancing agent is tissue plasminogen
activator (tPA), e.g., human tPA. In certain embodiments of the
invention, the proteolysis-enhancing agent is plasmin. In certain
embodiments, the proteolysis-enhancing agent promotes degradation
of a component of the extracellular matrix (ECM). In certain
embodiments, the proteolytic agent directly or indirectly degrades
fibrin.
[0017] Optionally, the plasticity-modifying agent and/or the
proteolysis-enhancing agent is covalently attached to a polymer by
an optionally cleavable linkage. In some embodiments, one or both
of the plasticity-modifying agent and the proteolysis-enhancing
agent is delivered in a solution that forms a gel following contact
with physiological fluids. The plasticity-modifying agent and,
optionally, a proteolysis-enhancing agent may, for example, be
delivered in an amount effective to promote structural
reorganization of synaptic connections, increase formation of new
synaptic connections, increase dendritic spine motility, promote
growth of axons and synaptic connections, inhibit at least in part
functional and/or structural deterioration or degradation,
stabilize synapses, or any combination of the foregoing.
[0018] In certain embodiments the composition comprises one or more
neural growth enhancing agents, neurotransmitters or analogs
thereof, neurally active growth factors, neural signaling
molecules, neurally active small molecules, and neurally active
metals. Alternatively or additionally, one or more of these agents
can be administered separately, for example, by focal
administration to the nervous system or by an alternate route.
[0019] The invention further provides a method of treating a
subject in need of enhancement of recovery or reorganization in the
nervous system comprising focally administering a composition
comprising a plasticity-modifying agent and a proteolysis-enhancing
agent to the central or peripheral nervous system of the subject.
The subject will typically have suffered nervous system damage as a
result of ischemic, hemorrhagic, neoplastic, degenerative,
traumatic, and/or neurodevelopmental damage. The invention provides
methods of treating a subject in need of enhancement of recovery
and/or reorganization in the nervous system comprising
administering a plasticity-modifying agent, a proteolysis-enhancing
agent, and a neural growth enhancing agent to the subject. One,
more than one, or all of the agents can be administered focally to
the central or peripheral nervous system. Agents can be
administered separately or in a single composition. Any of the
methods for administration contemplated herein can be used.
[0020] In any of the inventive methods, the subject may be engaged
in a program of rehabilitation designed to promote functional
recovery following ischemic, hemorrhagic, neoplastic, traumatic,
and/or neurodevelopmental damage to the nervous system, wherein the
subject is so engaged during at least part of the time interval
during which the agent is administered or during which the agent
remains active in the nervous system of the subject.
[0021] In any of the methods described herein, the subject may be
engaged in a program of behavioral or cognitive therapy to improve
function of the nervous system following from a neurodevelopmental
disorder, wherein the subject is so engaged during at least part of
the time interval during which the agent is administered or during
which the agent remains active in the nervous system of the
subject.
[0022] The present invention provides drug delivery devices
comprising: a biocompatible polymer and a plasticity-modifying
agent, wherein the plasticity-modifying agent is released from the
polymer in an amount effective to promote structural or functional
recovery or reorganization in the nervous system of the subject.
The device may comprise a proteolysis-enhancing agent.
[0023] The present invention provides compositions comprising a
plasticity-modifying agent and a neural growth enhancing agent,
which is optionally selected from among neurotransmitters or
analogs thereof, neurally active growth factors, neural signaling
molecules, and neurally active small molecules, and neurally active
metals. The invention comprises drug delivery devices, e.g.,
polymer-based drug delivery devices, comprising the
composition.
[0024] This application refers to various patents and publications.
The contents of all of these are incorporated by reference. In
addition, the following publications are incorporated herein by
reference: Ausubel, F., (ed.). Current Protocols in Molecular
Biology, Current Protocols in Immunology, Current Protocols in
Protein Science, and Current Protocols in Cell Biology, all John
Wiley & Sons, N. Y., edition as of July 2002; Sambrook,
Russell, and Sambrook, Molecular Cloning: A Laboratory Manual,
3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, 2001; Kandel, E., Schwartz, J. H., Jessell, T. M., (eds.),
Principles of Neural Science, 4.sup.th ed., McGraw Hill, 2000;
Cowan, W. M., Sudhof, T. C., and Stevens, C. F., (eds.), Synapses,
The Johns Hopkins University Press, Baltimore and London, 2001; and
Hardman, J., Limbird. E., Gilman, A. (Eds.), Victor, M. and Ropper,
A. H., Adams and Victor's Principles of Neurology, 7.sup.th ed.,
McGraw Hill, 2000; Grossman, R. I. and Yousem, D. M.,
Neuroradiology: The Requisites, 2.sup.th ed., C. V. Mosby, 2003;
Gillen, G. and Burkhardt, A. (eds.), Stroke Rehabilitation: A
Function-Based Approach, 2.sup.nd ed., C. V. Mosby, 2004; Somers,
M. F., Spinal Cord Injury: Functional Rehabilitation, 2.sup.th ed.,
Prentice Hall, 2001; Goodman and Gilman's The Pharmacological Basis
of Therapeutics, 10.sup.th Ed., McGraw Hill, 2001 (referred to
herein as Goodman and Gilman). In the event of a conflict or
inconsistency between any of the incorporated references and the
instant specification or the understanding of one or ordinary skill
in the art, the specification shall control, it being understood
that the determination of whether a conflict or inconsistency
exists is within the discretion of the inventors and can be made at
any time.
[0025] Where ranges of numerical values are stated herein, the
endpoints are included within the range unless otherwise stated or
otherwise evident from the context. Where a range of values is
provided, it is understood that each intervening value, to the
tenth of the unit of the lower limit unless the context clearly
dictates otherwise, between the upper and lower limits of that
range is also specifically disclosed. Each smaller range between
any stated value or intervening value in a stated range and any
other stated or intervening value in that stated range is
encompassed within the invention. The upper and lower limits of
these smaller ranges may independently be included in or excluded
from the range, and each range where either, neither or both limits
are included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either or both of those included limits are also
included in the invention.
[0026] This application refers to various genes and proteins using
names that are well known in the art. At times one or more
identifiers and/or accession numbers for these genes and proteins
are provided. Such names, identifiers, and/or accession numbers are
utilized in various databases available to one of skill in the art
such as Genbank and Pubmed. For example, one of skill in the art
can search the Entrez Gene database provided by the National Center
for Biotechnology Information (NCBI), available at the web site
having URL
www.ncbi.nlm.nih.gov/entrezlquery.fcgi?CMD=search&DB=gene and
can thereby locate the Gene ID for any particular gene or protein
of interest. The Gene ID entry provides biological information,
alternate names, chromosomal location, etc., as well as links to
database entries for the corresponding nucleotide and protein
sequences and references in the scientific literature. It will be
appreciated that the names and/or sequences of genes mentioned
herein may differ in different species. The invention encompasses
the genes regardless of species. When the methods for modifying
plasticity, nervous system structure or function, nervous system
recovery or reorganization, etc., are applied to a subject it may
be preferable to employ agents that modulate the expression and/or
activity of genes and/or pathways as they exist in the species to
which the subject belongs, although in many cases such agents will
be effective in multiple species. In certain embodiments of the
invention the gene is a human gene. One of skill in the art will be
able to identify the human homologs of mouse genes mentioned herein
in other species such as humans.
BRIEF DESCRIPTION OF THE DRAWING
[0027] FIGS. 1A-D: Analysis and characterization of genes activated
in different paradigms of visual input deprivation. (A) Three
experimental groups were considered: control mice, dark-reared (DR)
mice and monocularly-deprived (MD) mice. From each sample, tissue
from anatomically defined primary visual cortex (V1) was taken at
P27. For control and DR mice, V1 was taken from both hemispheres,
while for MD mice only V1 contralateral to the deprived eye was
used. For each sample, total RNA was extracted and processed for
the microarray procedure. MD and DR samples were compared to the
control independently, each with two different computational
methods (see Example 1): the Significance Analysis of Microarrays
(SAM) for analysis of single genes, and gene set enrichment
analysis (GSEA). Each procedure identified single genes or gene
sets that were up- or down-regulated in deprived groups versus
control. This led to the identification of cellular events involved
in the two models of input deprivation. (B, C) Comparison of gene
expression in (B) dark-reared versus control and (C) monocularly
deprived versus control animals, showing the expression levels of
all probes. Genes showing significantly different expression levels
(p.ltoreq.0.01) are shown in red (overexpression in deprivation
protocol) or in green (overexpression in control). Gene expression
is shown on a logarithmic scale. The dashed white line corresponds
to identity (y=x). (D) Heat map showing the levels of expression of
representative genes that showed differential expression among
those selected for our analysis (p.ltoreq.0.01). Each column
corresponds to a separate sample (n=6 for MD, n=3 for DR and n=3
for control). High levels of expression correspond to brilliant
red, low levels of expression correspond to dark blue (see bottom
of the figure for color scale). For each group, 25 randomly chosen
genes among the significant genes are shown here. Genes within each
group are sorted based on their expression values.
[0028] FIGS. 2A and 2B: Regulation of genes involved in excitatory
and inhibitory transmission in MD and DR animals. (A) Numbers of
inhibitory/excitatory receptor genes that are significantly
upregulated in MD or DR versus control. (B) Representation of the
Microarray Expression Levels (MEL) in control (con), Monocularly
Deprived (MD) and Dark Reared (DR) animals of glutamic acid
decarboxylase genes (GAD65 and GAD67), the synthetic enzymes for
GABA, and different classes of inhibitory neurons. Only the probes
for parvalbumin are significantly downxegulated in DR, while the
other markers are either upregulated or unchanged (star indicates
two-tailed t test, P<0.05).
[0029] FIGS. 3A-C: Confirmation of selected molecules with RT-PCR.
(A) Heat map of the genes confirmed with semi-quantitative PCR. The
level of expression is represented in logarithmic scale; red
corresponds to maximal expression and blue to minimal expression.
The genes are ranked according to their expression level after MD.
(B, C) Representation of the fold increase of selected molecules in
DR (B) and MD (C) versus control, showing the ratio between DR or
MD versus control for Microarray Expression Levels (red) and PCR
values (green). A star indicates that the microarray expression of
the corresponding gene is significantly upregulated (two-tailed t
test P<0.05) in DR vs. control or MD versus control.
[0030] FIGS. 4A-D: Gene Set Enrichment Analysis of gene expression
after DR and MD. (A) Example analysis of enrichment of the ARF
pathway in the MD versus control data set. The hypothesis tested is
that the expression of the ARF gene set (n=19 genes) is enriched in
the MD versus control data set. The genes in the dataset are ranked
according to a correlation statistic (signal-to-noise ratio); genes
up-regulated after MD vs. control appear first while genes
up-regulated in control (that is, downregulated in MD vs. control)
appear late. The straight lines represent genes in the ranked list
that are in the ARF pathway (bottom). The running enrichment score
is plotted in the upper graph (top). The peak enrichment score for
the ARF pathway in the MD versus control data set is 0.48, leading
to a normalized enrichment score (NES) of 6.8. (B) Heat map of the
expression levels of all the probes of the ARF pathway gene set in
the MD and control samples. Highest levels of expression correspond
to brilliant red, while lowest levels of expression correspond to
dark blue. (C) Distribution of normalized enrichment score (NES)
values for the DR versus control data set. The arrows highlight two
pathways that are particularly enriched in DR and are discussed in
the text: the CREB pathway and the Channel Passive Transporter
pathway. The insets show the running enrichment scores for these
two pathways; the red arrows show the positions of Creb and GluR1
probes respectively. (D) Distribution of NES values in the GSEA
analysis for the MD versus control data set. The arrows indicate
two pathways discussed in the text which are particularly enriched
in MD: the EGF pathway and the IGF1 pathway. For each of these
pathways, the insets show the running enrichment score. The red
arrows in the insets point to the positions of Stat1 and
IGF1-IGFBP5 probes respectively.
[0031] FIGS. 5A-E. Immunohistochemistry for molecules that show
increased expression following DR and MD. Immunohistochemistry for
selected molecules was performed on coronal slices containing V1
from P27 control, Dark Reared (DR) and Monocularly Deprived (MD)
mice. In DR mice, the expression of three proteins: (A)
Parvalbumin, (B) GluR1 and (C) Phospho-Creb was examined. The
parvalbumin gene is down-regulated in DR versus control and the
immunohistochemistry shows a decrease in the number of
parvalbuminpositive neurons in DR animals. The histogram on the
right shows a significant decrease (P<0.01) in the number of
parvalbuminergic neurons versus control. GluR1 and P-Creb proteins
were over-expressed in visual cortex of DR animals versus control.
In MD mice, the expression of (D) activated Stat1 and (E) IGFBP5
was examined. Both proteins are selectively up-regulated in V1
after 15 days of MD relative to control. Bars in the right panels
(B-E) show the intensity of the staining in sections of DR or MD
and control animals; for all the molecules examined the intensity
of staining was significantly higher in the deprived conditions
that in controls (P<0.05). For each molecule, low magnification
pictures (scale bar=765 .mu.m) and high magnification pictures
(scale bar=100 .mu.m) are shown. Arrows in the low magnification
pictures demarcate V1.
[0032] FIGS. 6A-C: Application of IGF1 prevents the ocular
dominance shift after MD in mouse V1. (A) Left: Mouse brain showing
the location of V1 (black region). Right: Ocular dominance index
map in mouse V1. The dotted line separates the binocular zone (b)
from the monocular zone (m). Scale bar, 1 mm. (B) Histograms of
ocular dominance index in the binocular zone of three
representative mice. Red line, P27 control mouse; black line, P27
mouse after 7 days of MD; blue line, P30 mouse after 7 days of MD
plus IGF1 application for the same period. The data from each
animal typically includes a region within binocular cortex
containing over 2000 pixels. (C). Mean ocular dominance indices of
the 3 groups of mice. Open circles, mean ocular dominance index of
the binocular zone pixels from each animal; filled circles, average
value of each group.
[0033] FIGS. 7A and 7B: Immunohistochemistry for selected markers
of the IGF1 pathway. (A) Double staining for IGFBP5 (green), and
GAD67 (red) in visual cortex of a P28 mouse. Yellow arrow shows an
overlap between the two colors suggesting that IGFBP5 is present in
GABAergic neurons; however the presence of cells immunopositive for
IGFBP5 but not for GAD67 (green arrow) and vice versa (red arrow)
shows that IGFBP5 is present in other cell classes as well. Scale
bar=17 .mu.m. (B) Immunostaining for selected molecules in three
different conditions: P28 control (animal reared in normal light
conditions), P28 MD (animals monocularly deprived for 4 days), and
P28 MD+IGF1 (animals deprived for 4 days and simultaneously
injected IP daily with IGF1 solution). In all the MD panels the
cortex shown is contralateral to the deprived eye. Bars at right
show the staining intensity of each molecule in the different
conditions. Scale bar=70 .mu.m.
BRIEF DESCRIPTION OF THE TABLE APPENDIX
[0034] The Appendix, which is a part of the instant specification,
consists of the following Tables:
[0035] Table 4 lists genes whose expression is downregulated in
visual cortex under conditions of DR.
[0036] Table 5 lists genes whose expression is upregulated in
visual cortex under conditions of DR.
[0037] Table 6 lists genes whose expression is downregulated in
visual cortex under conditions of long term MD.
[0038] Table 7 lists genes whose expression is upregulated in
visual cortex under conditions of long term MD.
[0039] Table 8 lists genes whose expression is downregulated in
visual cortex under conditions of short term MD
[0040] Table 9 lists genes whose expression is upregulated in
visual cortex under conditions of short term MD.
[0041] Table 10 lists genes that are downregulated in visual cortex
under conditions of short term MD in subjects treated with an
activator of the IGF1 pathway.
[0042] Table 11 lists genes that are upregulated in visual cortex
under conditions of short term MD in subjects treated with an
activator of the IGF1 pathway.
DEFINITIONS
[0043] Approximately: As used herein, the term "approximately" in
reference to a number is generally taken to include numbers that
fall within a range of 10% in either direction of the number
(greater than or less than) unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0044] Agonist: As used herein, the term "agonist" generally refers
to a substance that can directly interact with (e.g., bind to) a
receptor and initiate a physiological or a pharmacological response
characteristic of the activity of that receptor, e.g., the activity
that is normally induced by interaction of an endogenous
positively-acting ligand with the receptor. Substances generally
recognized in the literature as agonists of a particular receptor
are of use in the methods described herein. The term "agonist" also
refers to partial agonists, i.e., compounds that are capable of
partially activating a receptor, e.g., activating it to a lesser
extent than its endogenous ligand. The term also encompasses
substances that indirectly stimulate a receptor, e.g., by
inhibiting reuptake or breakdown/metabolism of an endogenous direct
agonist and/or by stimulating the production or release of an
endogenous direct agonist.
[0045] Antagonist: As used herein, the term "antagonist" generally
refers to a substance that opposes the receptor-associated
responses normally induced by another bioactive agent such as an
endogenous positively-acting ligand. Typically, an antagonist binds
to a receptor and prevents binding of an endogenous ligand that
would normally activate the receptor, or prevents binding of an
exogenous agonist to the receptor. The antagonist may or may not
induce an effect itself. The activity of a receptor is generally
taken to be the activity associated with binding of an endogenous
positively-acting ligand. Substances generally recognized in the
literature as antagonists of a particular receptor are of use in
the methods described herein. The term also encompasses substances
that indirectly inhibit a receptor, e.g., by inhibiting reuptake or
by stimulating breakdown/metabolism of an endogenous direct agonist
and/or by stimulating the production or release of an endogenous
direct antagonist.
[0046] Biocompatible: A material is considered "biocompatible" if
it is substantially non-toxic to the recipient, in the quantities
and at the location used, and also does not elicit or cause a
significant deleterious or untoward effect on the recipient's body,
e.g., a significant immunological or inflammatory reaction,
unacceptable scar tissue formation, etc.
[0047] Biodegradable: As used herein, the term "biodegradable,"
refers to a material that is capable of being broken down
physically and/or chemically within the body of a subject, e.g., by
hydrolysis under physiological conditions, by natural biological
processes such as the action of enzymes present within the body,
etc., to form smaller chemical species which can be metabolized
and/or excreted.
[0048] Biological information resource: As used herein, the term
"biological information resource" refers to a compilation of
reliable information about biochemical species (e.g., genes or
their expression products, substrates, cofactors, physiologically
important ions or small molecules), biological processes, and
optionally, biological pathways, from which it is possible to
conveniently determine information such as (i) whether a
biochemical species is a component of a particular biological
process; (ii) which biochemical species are components of a
particular biological process; (iii) which biological processes
include a particular biochemical species as a component; (iv)
whether a particular biological process includes a particular
biochemical species as a component, etc. A biological information
resource can also include any type of additional biological
information. For example, information such as identifiers of
compounds known to interact with a biochemical species or known to
influence a biological pathway can be included. Names of diseases
or clinical conditions that are related to a biological process or
biochemical species, e.g., in which the biological process or
biochemical species plays a causative role, or in which a defect in
the biological process or biochemical species plays a causative
role, can be included. By "reliable information" is meant
information that is generally recognized in the art as being
substantially accurate. Typically such information will have been
published in the scientific literature and described therein in
sufficient detail to be capable of being independently verified and
will have been replicated and/or acknowledged as being accurate in
one or more additional scientific publications. A biological
information resource will typically comprise a database and will
provide one or more software tools that allow a user to readily
obtain access to the information and to search the information
using one or more query terms, e.g., an identifier for a
biochemical species, biological process, etc. An "identifier"
refers to any term or combination of terms that is used to refer to
a biochemical species, biological process, etc. The identifier can
be, for example, the name of a gene or the name of a biological
process.
[0049] Biological pathway: As used herein, the term "biological
pathway" refers to a sequence of reactions (e.g., physical
interactions between molecules, enzyme reactions) that takes place
in a living organism, typically resulting in a biological effect. A
pathway typically involves a cascade of events in which molecules
involved in the pathway (referred to as "components" of the
pathway) signal to or act upon each other, often in a
characteristic and ordered manner. Many of the components of the
pathway are RNA or polypeptide expression products of genes (also
referred to as "gene products"). Such genes may also be referred to
as components of the pathway. Biological pathways of interest
herein include the IGF1 pathway, the JAK/STAT pathway, the PI3
kinase pathway, and subpathways thereof.
[0050] Biological process: As used herein, the term "biological
process" refers to a series of events accomplished by one or more
biochemical species or ordered assemblies of biochemical species.
The biochemical species or assemblies thereof are referred to as
"components" of the biological process. The components are said to
be "involved in" the biological process. For example, a gene
product that is a component of a biological process, i.e., plays a
role in carrying out that biological process, is said to be
involved in that biological process. Genes whose expression
product(s) are components of a biological process may also be
referred to as components of the pathway. The series of events
making up a biological process is typically directed towards
achieving a biological goal of significance to the biological
system. Examples of biological processes include, without
limitation, cell communication, metabolism, morphogenesis,
secretion, etc. It will be appreciated that a biological process
may comprise a plurality of biological processes (subprocesses). A
biological process may comprise or be performed by one or more
biological pathways. The "central nervous system" (CNS) includes
the brain, spinal cord, optic, olfactory, and auditory systems. The
CNS comprises both neurons and glial cells (neuroglia), which are
support cells that aid the function of neurons. Oligodendrocytes,
astrocytes, and microglia are glial cells within the CNS.
Oligodendrocytes myelinate axons in the CNS, while astrocytes
contribute to the blood-brain barrier, which separates the CNS from
blood proteins and cells, and perform a number of supportive
functions for neurons. Microglial cells serve immune system
functions.
[0051] Concurrent administration: The term "concurrent
administration," as used herein with respect to two or more agents,
e.g., therapeutic agents, is administration performed using doses
and time intervals such that the administered agents are present
together within the body, or at a site of action in the body such
as in the CNS in amounts sufficient to have a biological effect
over a time interval of minutes, hours, days, weeks, etc. The
agents may, but need not be, administered together as part of a
single composition. In addition, the agents may, but need not be,
administered simultaneously (e.g., within less than 5 minutes, or
within less than 1 minute) or within a short time of one another
(e.g., less than 1 hour, less than 30 minutes, less than 10
minutes, approximately 5 minutes apart).
[0052] Critical period: As used herein, the term "critical period"
refers to a time period during the development of an organism in
which the organism's nervous system is particularly able to acquire
a specific functional ability and/or structural configuration,
typically at least in part in response to external environmental
stimuli. Absence of the appropriate stimuli during the critical
period typically results in failure to develop the functional
ability and/or structural configuration that would develop had
these stimuli been present. The timing and duration of the critical
period may depend upon the environmental stimuli received. For
example, lack of certain environmental stimuli prolongs the
critical period.
[0053] Deprived condition: As used herein, the term "deprived
condition" refers to an environment that fail to provide adequate
environmental stimuli needed to allow normal development of one or
more functional or structural features of the nervous system. An
individual subjected to a deprivation condition typically receives
fewer and/or less intense or varied stimuli of one or more types
than an individual subjected to "normal conditions." In the case of
an animal raised in a laboratory, "normal conditions" are standard
laboratory conditions typically used for the maintenance of such
animals.
[0054] Effective amount: As used herein, an "effective amount" of
an active agent refers to the amount of the active agent sufficient
to elicit a desired biological response. As will be appreciated by
those of ordinary skill in this art, the absolute amount of a
particular agent that is effective may vary depending on such
factors as the desired biological endpoint, the agent to be
delivered, the target tissue, etc. Those of ordinary skill in the
art will further understand that an "effective amount" may be
administered in a single dose, or may be achieved by administration
of multiple doses. A desired biological response may be, for
example, (i) functional or structural reorganization of synaptic
connections, dendrites, or axon projections; (ii) maintenance of
synaptic connections, dendrites, or axon projections under
conditions in which they would otherwise deteriorate; (iii)
regeneration of a nerve or an axonal projection system or its
maintenance under conditions in which it would otherwise
deteriorate; (iv) an improvement in performance of a task requiring
motor or sensory function; (v) an improvement in performance of a
task requiring cognitive function, e.g., improved performance on a
test that measures learning and/or memory; (vi) a slowing in the
rate of decline in motor, sensory, and/or cognitive function.
[0055] Enriched condition: As used herein, the term "enriched
condition" refers to an environment that provides receives more
stimuli and/or more intense or varied stimuli of one or more types
than an individual subjected to "normal conditions."
[0056] Expression product: As used herein, the term "expression
product" or "gene product" refers to an RNA transcribed from a gene
or a polypeptide translated from an RNA transcribed from a gene.
RNAs or polypeptides that are modified following their
transcription or translation are considered expression products of
the gene that encodes them. Modifications include, e.g., splicing,
cleavage, addition of phosphate or fatty acid groups, etc.
[0057] Focal delivery: As used herein, the term "focal delivery"
(or "focal administration" in reference to delivery of a
pharmacological agent), refers to delivery that does not rely upon
transport of the agent to its intended target tissue via the
vascular system, e.g., the agent is not administered directly into
a blood vessel. The agent is delivered directly to its intended
target tissue or in the vicinity thereof, e.g. by injection through
a needle, catheter, or cannula, or by implantation of a delivery
vehicle or device containing the agent. If the agent is delivered
to the vicinity of its target tissue rather than into the target
tissue itself, the agent may reach its target tissue by diffusion.
For purposes of the present invention, any method that achieves
delivery of an agent to the CNS or portion thereof without
requiring transport via the vascular system from a site outside the
skull or meninges (the membranes that cover the brain and the
spinal cord), is considered to achieve focal delivery of the agent.
Specifically included are delivery by use of an implanted or
external pump, and/or delivery directly into one or more ventricles
of the CNS. It will be understood that once having been focally
delivered a portion of the agent (typically only a minor fraction
thereof) may in part enter the vascular system and be transported
to another location.
[0058] Function: As used herein, the term "function," with
reference to the nervous system or a component thereof, is used
broadly herein to refer to any function, role, task, or activity
performed by the nervous system or a component thereof. The term
includes, without limitation, the ability to process and recall
information, regulate behavior, stimulate release of endogenous
chemicals, control motor functions, receive and process sensory
input, maintain consciousness, etc.
[0059] Functional recovery: As used herein, the term "functional
recovery" refers to the process in which a nervous system or
component thereof that has at least in part lost the ability to
perform a function that it previously performed, regains at least
in part the ability to perform the function. Functional recovery
may take place in at least two different ways: (i) the recovery in
function may involve partial or complete recovery of the portion of
the nervous system that previously performed the function; (ii) the
recovery in function may involve a portion of the nervous system
performing a function that it did not previously perform. Of course
in some instances both processes may take place. Functional
recovery can also refer to preservation of the ability of the
nervous system or a portion thereof to perform a function that it
previously performed, after the nervous system or component thereof
has been physically altered, disrupted, or otherwise subjected to a
physical or chemical insult or neurodegenerative disease, when such
physical alteration, disruption, physical or chemical insult or
neurodegenerative disease would otherwise be expected to lead to
deterioration or loss of the ability of the nervous system or
portion thereof to perform the function.
[0060] Functional reorganization: The term "functional
reorganization," as used in reference to the nervous system or a
portion thereof, refers to the process in which a portion of the
nervous system wholly or partially assumes, i.e., takes on, a
function (e.g., a sensory, motor, or cognitive function) that was
not previously performed by that portion of the nervous system. The
function or task may, but need not have been, previously performed
by a different portion of the nervous system. Functional
reorganization may, but need not, entail one or more aspects of
structural reorganization. Functional reorganization may also be
referred to as functional rearrangement.
[0061] An example of functional reorganization is the capacity of
an area of sensory or motor cortex adjacent to an area of injury or
necrosis of CNS tissue to control CNS output to a portion of the
body that was previously controlled by the injured or necrotic
tissue, or to receive and process input from a region of the body
from which input was previously received and processed by the
injured or necrotic tissue. Another example is the capacity of an
area of sensory or motor cortex corresponding in location to an
area of injury or necrosis of CNS tissue, but located in the
opposite hemisphere of the brain, to control CNS output to a
portion of the body that was previously controlled by the injured
or necrotic tissue, or to receive and process input from a region
of the body from which input was previously received and processed
by the injured or necrotic tissue. Yet another example is provided
by the nervous system's response to monocular deprivation, which is
further discussed below.
[0062] Infarct: As used herein, the term "infarct" refers to an
area of localized tissue necrosis resulting from inadequate blood
supply, e.g., due to obstruction of a blood vessel. Also referred
to as an infarction. When the necrotic tissue is brain tissue, the
infarct may be referred to as a cerebral infarct or cerebral
infarction.
[0063] Modulate: As used herein, the term "modulate" means to
alter, e.g., to increase or enhance, to decrease or inhibit, or to
cause a variation in a temporal pattern. To "modulate a gene" means
to modulate the level and/or activity of an RNA or polypeptide
expression product of the gene, e.g., by administering an agonist
or antagonist. "Level" of an expression product refers to amount,
e.g., concentration by weight or volume, number of molecules per
cell or by weight or volume, etc. To "modulate a pathway" means to
modulate at least one reaction and/or gene involved in the pathway,
typically resulting in an alteration in the biological effect or
outcome of the pathway. To "modulate a cell" means to increase or
enhance, or to decrease or inhibit, the development, survival,
and/or activity of the cell.
[0064] Neural tissue: As used herein, the term "neural tissue"
refers to one or more components of the central nervous system
and/or peripheral nervous system. Such components include brain
tissue and nerves, which may be present in bundles or tracts. In
general, brain tissue and nerves contain neurons (which typically
comprise cell body, axon, and dendrite(s)), glial cells (e.g.,
astrocytes, oligodendrocytes, and microglia in the CNS; Schwann
cells in the PNS). It will be appreciated that brain tissue and
nerves typically also contain various noncellular supporting
materials such as basal lamina (in the PNS), endoneurium,
perineurium, and epineuriun in nerves, etc. Additional nonneural
cells such as fibroblasts, endothelial cells, macrophages, etc.,
are typically also present. See Schmidt and Leach, 2003, for
further description of the structure of various neural tissues.
[0065] Peripheral nervous system: As used herein, the term
"peripheral nervous system" (PNS) includes the cranial nerves
arising from the brain (other than the optic and olfactory nerves),
the spinal nerves arising from the spinal cord, sensory nerve cell
bodies, and their processes, i.e., all nervous tissue outside of
the CNS. The PNS comprises both neurons and glial cells
(neuroglia), which are support cells that aid the function of
neurons. Glial cells within the PNS are known as Schwann cells, and
serve to myelinate axons by providing a sheath that surrounds the
axons. In various embodiments of the invention the methods and
compositions described herein are applied to different portions of
the PNS.
[0066] Plasticity: As used herein, the term "plasticity" refers to
the capacity of the nervous system, or a portion thereof, to change
(e.g., to reorganize) its structure and/or function, generally in
response to an environmental condition, injury, experience, or
ongoing nervous system activity. Plasticity may involve the
proliferation of neurons or glia, the growth or movement of
neuronal processes and/or alterations in their shape. Plasticity
may involve formation of new synaptic connections between neurons
and/or strengthening or weakening of existing synaptic connections.
Formation of new synaptic connections may involve growth or
movement of neuronal processes. Plasticity may also involve
alterations in non-neuronal components of the nervous system, e.g.,
astrocytes or other glial cells.
[0067] Plasticity-modifying agent: As used herein, the term
"plasticity-modifying agent" refers to a substance whose
administration to a subject, either alone or in combination with
one or more other substances or non-pharmacological therapy,
results in a detectable alteration in the plasticity of at least a
portion of the nervous system. The alteration may be evidenced by
an alteration in nervous system function and/or structure as
compared with the function and/or structure that would be observed
in the absence of the agent. The agent has a clinically significant
effect on the nervous system to modify plasticity and is not
administered simply for nutritional or dietary purposes. The agent
may increase, decrease, and/or prolong plasticity.
[0068] Plurality: As used herein, the term "plurality" means more
than one.
[0069] Polypeptide: As used herein, the term "polypeptide" refers
to a polymer of amino acids. As used herein, the term "protein"
refers to a molecule composed of one or more polypeptides. The
terms "protein," "polypeptide," and "peptide" may be used
interchangeably herein. Polypeptides as described herein typically
contain only natural amino acids, although non-natural amino acids
(i.e., compounds that do not occur in nature in polypeptides but
that can be incorporated into a polypeptide chain) and/or amino
acid analogs as are known in the art may also be employed.
[0070] Proteolysis: As used herein, the term "proteolysis" refers
to the breakdown, or degradation, of proteins into smaller
polypeptides, typically by cleavage of peptide bonds. Ultimately
proteolysis may result in breakdown of the protein into individual
amino acids.
[0071] Proteolysis-enhancing agent: As used herein, the term
"proteolysis-enhancing agent" refers to a substance, e.g., a
protease, that increases, contributes to, or causes proteolysis of
one or more proteins or inhibits an inhibitor of proteolysis.
[0072] Purified: As used herein, the term "purified" means
separated from many other compounds or entities. A compound or
entity may be partially purified, substantially purified, or pure,
where it is pure when it is removed from substantially all other
compounds or entities (other than solvents, ions, etc.), i.e., it
is preferably at least about 90%, more preferably at least about
91%, 92%; 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99%
pure. A partially or substantially purified compound or entity may
be removed from at least 50%, at least 60%, at least 70%, or at
least 80% of the material with which it is naturally found, e.g.,
cellular material such as cellular proteins and/or nucleic acids.
In a preferred embodiment a purified protein is removed from at
least 90%, preferably at least 95%, more preferably at least 99%,
or more, of the other proteins in a preparation, so that the
purified protein constitutes at least 90%, preferably at least 95%,
more preferably at least 99%, of the material in the preparation on
a dry w/w basis.
[0073] Recovery: As used herein, the term "recovery" refers to
structural and/or functional recovery.
[0074] Reorganization: As used herein, the term "reorganization"
refers to structural and/or functional reorganization.
[0075] RNAi agent: As used herein, the term "RNAi agent" refers to
a nucleic acid that inhibits gene expression by an RNAi
interference mechanism. Examples include short interfering RNAs
(siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs) and
nucleic acids that are processed intracellularly, e.g., by a member
of the RNase III family of nucleases such as DICER that cleaves
double-stranded RNAs, to produce an siRNA, shRNA, or miRNA. It will
be appreciated that an RNAi agent, if produced using chemical
synthesis, can include one or more deoxyribonucleotides or
nucleotide analogs, modified backbone structures, etc., in addition
to or instead of ribonucleotides linked by phosphodiester
bonds.
[0076] Sequential administration: As used herein, "sequential
administration" of two or more agents refers to administration of
two or more agents to a subject such that the agents are not
present together in the subject's body at greater than de minimis
concentrations. Thus the agents are not present together in the
subject's body in concentrations sufficient for the agents to each
have a separate biological effect. In certain embodiments a first
agent is administered to a subject. A second agent is administered
at a later time at which the concentration of the first agent has
declined to less than 1%, less than 5%, or less than 10% of its
peak concentration in the CNS or PNS. Administration of the agents
may, but need not, alternate. Each agent may be administered
multiple times.
[0077] Small molecule: As used herein, the term "small molecule"
refers to organic compounds, whether naturally-occurring or
artificially created (e.g., via chemical synthesis) that have
relatively low molecular weight and that are not proteins,
polypeptides, or nucleic acids. Typically, small molecules have a
molecular weight of less than about 1500 g/mol. Also, small
molecules typically have multiple carbon-carbon bonds.
[0078] Spine dynamics: As used herein, the term "spine dynamics"
refers to a change in any of various structural properties of
spines over time. The properties include spine shape, size, number,
density, and motility. Spine dynamics may be examined with respect
to the individual spine or with respect to a'plurality (i.e., more
than one) of spines.
[0079] Spine motility: As used herein, the term "spine motility"
refers to a change in spine length over time. When examined with
respect to a plurality of spines, spine motility refers to the
average change in spine length over time.
[0080] Structural recovery: The term "structural recovery," as used
in reference to the nervous system or a portion thereof, refers to
the partial or complete restoration of a structure that has
physically altered, disrupted, or otherwise subjected to a physical
or chemical insult, which is intended to include deprivation of
oxygen and/or nutrients. "Structural recovery" can also refer to
preservation of a structure that has been physically altered,
disrupted, or otherwise subjected to a physical or chemical insult,
when such physical alteration, disruption, physical or chemical
insult would otherwise be expected to lead to deterioration and/or
loss or alteration in normal structural features. The structure can
be, for example, a synaptic connection, a nerve, nerve bundle,
nerve tract, nucleus, brain region, connection between brain
regions, etc.
[0081] Structural reorganization: The term "structural
reorganization," as used in reference to the nervous system or a
portion thereof, refers to an alteration in the pattern of
connections between two or more neurons or between one or more
neurons and one or more glial cells (e.g., astrocytes,
oligodendrocytes, microglia, Schwann cells) that takes place over a
period of time or an alteration in the position of two or more
neuronal or glial cell bodies or cell processes (axons, dendrites,
dendritic spines) with respect to one another. The alteration may
include the formation of synapses between neurons that did not
synapse with each other at the beginning of the time period. The
alteration may include the formation of additional synapses between
neurons that had at least one synaptic connection at the beginning
of the time period. The alteration may also or alternatively
include loss of synapses that existed at the beginning of the time
period. Reorganization may entail growth or retraction of neural
processes such as axons (e.g., axonal sprouting or regeneration),
dendrites, or dendritic spines, migration of neurons or glia,
and/or neuronal or glial cell division. Structural reorganization
may also be referred to as structural rearrangement.
[0082] Subject: As used herein, the term "subject" or "individual"
refers to an individual to whom an agent is to be delivered, e.g.,
for experimental, diagnostic, and/or therapeutic purposes and/or an
individual who is subjected to a condition that modifies
plasticity. Preferred subjects are mammals, particularly
domesticated mammals (e.g., dogs, cats, etc.), primates, or
humans.
[0083] Synapses: As used herein, the term "synapses" refer to
"specialized intercellular junctions between neurons or between
neurons and other excitable cells where signals are propagated from
one cell to another with high spatial precision and speed" (De
Camilli, in Cowan, supra). They are the primary sites of
intercellular communication in the mammalian nervous system. In
general, the basic structure of a synapse consists of a close
juxtaposition of specialized regions of the plasma membrane of two
neurons, referred to as the presynaptic and postsynaptic neurons,
to form a synaptic junction. The presynaptic neuron is the nerve
cell transmitting a signal while the postsynaptic neuron is the
recipient of the signal. Most neurons in the vertebrate nervous
system possess a cell body and two types of cell processes, axons
and dendrites. Signals, i.e., action potentials, are initiated and
transmitted by the axon while dendrites (and also the cell body)
receive inputs via synaptic contacts from other neurons.
[0084] Treating: As used herein, the term "treating" generally
refers to medical and/or surgical management of a patient for
purposes of bringing about an improvement in the state of a subject
with respect to a disease, disorder, or condition from which the
subject suffers and/or reducing or slowing further deterioration of
the subject's condition. Treating can include reversing,
alleviating, and/or inhibiting the progress of, the disease,
disorder, or condition to which such term applies, and/or
reversing, alleviating, inhibiting the progress one or more
symptoms or manifestations of such disease, disorder or
condition.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
Methods for Identifying Genes, Biological Pathways, and Cells
Involved in Plasticity
[0085] The invention provides methods to identify molecular targets
(e.g., genes and their expression product(s)) that may be modulated
in order to modify plasticity in the nervous system of an
individual. The genes are differentially regulated in at least a
portion of the nervous system of individuals subjected to a
condition that modifies plasticity. For example, in certain
embodiments, inventive methods identify a gene wherein the level
and/or activity of an expression product of the gene differs in at
least a portion of the nervous system of a subject if the subject
has been subjected to a condition known to modify plasticity
relative to its expression or activity in that portion of the
nervous system in a subject who has not been subjected to the
condition or who has been subjected to an alternate condition. In
some embodiments, inventive methods identify a gene wherein the
level and/or activity of an expression product of the gene differs
in a portion of the nervous system that has been subjected to a
condition that modifies plasticity relative to its expression or
activity in a portion of the nervous system that has not been
subjected to a condition that modifies plasticity (e.g., a portion
located at a corresponding position of the opposite brain
hemisphere of a subject). The portion of the nervous system may be
any functionally or structurally defined part, area, region, unit,
or component of the nervous system (which terms are used
interchangeably herein). Portions of the nervous system include
cortex, cerebellum, thalamus, hypothalamus, hippocampus, amygdala,
basal ganglia (caudate nucleus, putamen and globus pallidus),
midbrain, pons, medulla, nerve tracts, etc., and any sub-portion of
the foregoing. For example, subregions of the cortex include visual
cortex, auditory cortex, somatosensory cortex, entorhinal cortex,
olfactory cortex, Broca's area, Wernicke's area, etc. It will be
appreciated that these regions themselves may be composed of
smaller subregions. For example, the primate cortex has been
divided into Brodmann areas 1-49 and 52, some of which include
subareas, based on cytoarchitectural distinctions. Important areas
of the primate visual cortex are referred to as V1, V2, V3, V4, and
MT (also referred to as V5). Portions of the nervous system also
include the six major cortical layers (I-VI) and their sublayers.
Portions of the nervous system also include cortical columns, a
term that refers to collections of cells arranged vertically from
the surface of the cortex to the white matter that comprise
functional and/or anatomical units. Thus, a cortical column can be
defined on the basis of anatomical features (e.g. stereotyped
patterns of pyramidal cell apical dendrite bundles), functional
features (e.g. columns of cortical cells all responding to the same
stimulus orientation) or both. Cortical columns include ocular
dominance, orientation, spatial frequency, and color columns. In
certain embodiments, the portion of the nervous system comprises
cells of one or more types, e.g., one or more neuronal cell types.
Cells may be excitatory or inhibitory. Exemplary cell types found
in the nervous system include pyramidal cells, stellate cells,
interneurons (e.g., chandelier cells, neurogliaform cells, basket
cells, double basket cells, Purkinje cells, granule cells,
Cajal-Retzius cells, Meynert cells, etc.).
[0086] Inventive methods are applied herein to identify genes that
are differentially regulated in the visual cortex under monocular
deprivation or dark rearing, both of which are conditions known to
modify plasticity. The invention identifies biological pathways
enriched in such genes.
[0087] The invention provides a method of identifying a gene
involved in plasticity (referred to herein as a "plasticity-related
gene") comprising steps of: (i) subjecting an individual to a
condition that modifies plasticity; (ii) measuring level or
activity of each of a plurality of genes in at least a portion of
the individual's nervous system; and (iii) identifying one or more
genes whose expression or activity is differentially regulated in
the portion of the individual's nervous system relative to its
expression or activity under alternative conditions. Conditions may
be environmental conditions that are deficient in one or more
environmental stimuli that the individual would normally
experience. Conditions may include one or more environmental
stimuli that the individual would not normally experience.
Alternative conditions may be normal environmental conditions,
e.g., standard laboratory conditions. Conditions suitable for
maintaining animals are discussed in Guide for the Care and Use of
Laboratory Animals, Institute for Laboratory Animal Research (ILAR)
Commission on Life Sciences, National Research Council, National
Academies Press, Washington, D.C. (1996). It will be appreciated
that a range of conditions may be considered "normal" but will
generally not include specific efforts to deprive or supplement the
nervous system inputs that typically would be received be animal
maintained as described in the foregoing reference.
[0088] Inventive methods may include identifying one or more
biological processes or pathways involving one or more of the
plasticity-related genes. The biological process pathway may be
enriched for genes identified by the method. For example, the
biological process or pathway may include a higher proportion of
genes identified by the method than would be expected based on the
number of genes in the process or pathway and the number of known
genes in an individual of that particular species. In other words,
genes identified as being differentially regulated are
over-represented among the genes in the biological process or
pathway. See Examples for further details.
[0089] In certain embodiments of the invention, the individual is
subjected to the condition during at least a portion of a critical
period for development of one or more nervous system structure(s),
functions, or properties. Nervous system structures, functions, or
properties for which a critical period has been well documented in
one or more species include ocular dominance, orientation bias,
development of the neuromuscular junction, climbing fiber
refinement, whisker barrel map formation, whisker RF tuning,
cortical tonotopic map, sound localization, birdsong, and human
language. The conditions may include depriving the individual of
normal inputs needed for the establishment of any of these
structures, functions, or properties. The timing of critical
periods and the effects of specific environmental conditions are
known in the art (see, e.g., Hensch, 2004, Annu. Rev. Neurosci.,
27:549).
[0090] In certain embodiments of the invention, conditions include
subjecting a subject to an alteration in visual input, optionally
during a critical period for development of the visual cortex.
Alteration of visual input during postnatal development causes
adaptive changes in the maturation of visual cortex circuitry. One
method of use for identifying genes, biological pathways, and cells
involved in activity-dependent plasticity is to alter visual
experience during a critical period of development. The timing of
such critical periods for development of the visual system is known
in the art.sup.4. One example of altering visual experience is to
raise animals in complete darkness from birth (dark rearing).
Dark-rearing (DR) has diverse effects on the visual cortex, causing
an upregulation of miniature synaptic potentials in subsets of
neurons.sup.5, a reduction in spine number together with an
increase in area of the spines that remain.sup.6, a change in the
threshold for eliciting synaptic potentiation and
depression.sup.7,8, and a prolongation of the critical period for
eliciting experience dependent changes in visual
function.sup.9.
[0091] One example of manipulation of use to study the influence of
activity on visual cortex neurons and networks and to identify
genes, biological pathways, and cells involved in plasticity is
monocular deprivation (MD). In animals with binocular vision,
inputs to a portion of the visual cortex become anatomically and
functionally segregated into alternating stripes of input from the
two eyes, referred to as ocular dominance columns. As a
consequence, individual cortical neurons that were originally
responsive to both eyes become responsive to only one eye. However,
if one eye is deprived of visual input during a critical period
(monocular deprivation), that eye loses most of its ability to
activate the cortex, and the responses of cells shift towards the
nondeprived eye, i.e., ocular dominance (OD) shifts in favor of the
nondeprived eye. The rapid appearance of the functional deficit is
followed by structural changes including a reduction in cortical
area driven by the deprived eye and expansion of the area driven by
the nondeprived eye, which take place on a timescale of weeks to
months. The extent and complexity of thalamocortical axonal arbors
from the deprived eye are reduced, while the extent and complexity
of arbors from the nondeprived eye increase. MD, which can be
achieved by suturing the lids of one eye during the critical
period, causes an increase in the proportion of neurons in the
binocular part of the V1 region of the cortex that respond to the
open eye.sup.13. Short-term MD causes a reorganization of
intracortical connections both functionally and
structurally.sup.14-17, whereas long-term MD leads in addition to a
reduction of thalamocortical arbors from the deprived eye and an
expansion of arbors from the non-deprived eye.sup.18,19.
[0092] The individual can be subjected to the condition during all
or part of a critical period, e.g., for a total of between 10% and
100% of the critical period. The individual can be subjected to the
condition intermittently or continuously. In certain embodiments of
the invention the critical period is, e.g., between 24 hours and 1
year in length, e.g., between 24 hours and 60 days in length. The
critical period can commence at any time after birth or even prior
to birth and may terminate at any later time, depending upon the
particular nervous system structure(s), functions, or properties
under consideration.
[0093] Any suitable method can be used to identify the
differentially regulated genes. In general, the methods involve
obtaining samples of nervous system tissue (e.g., samples of tissue
from a portion of the brain such as cortex, hippocampus, etc.) from
a subject who has been subjected to a condition (e.g., a reduction
in or increase in inputs) that modifies plasticity in at least a
portion of the nervous system. The level and/or activity of each of
a plurality of gene products is measured in the sample and is
compared with the level and/or activity that would exist under
alternate conditions. The method can involve obtaining a sample of
nervous system tissue from a different subject who has not been
subjected to the condition or obtaining a sample of nervous system
tissue from the same subject but from a portion of the nervous
system that has not been subjected to the condition. The level
and/or activity in the two samples can be compared in an experiment
performed on the two samples. Alternatively or additionally, a
comparison with previously gathered data on expression levels
and/or activity can be used.
[0094] Methods for determining the level of a gene product are well
known in the art, and any suitable method can be used. For example,
if the gene product is an RNA, its level can be measured using cDNA
or oligonucleotide microarrays, subtractive hybridization, Northern
blots, quantitative reverse transcription polymerase chain reaction
(RT-PCR), etc. If the gene product is a polypeptide, its level can
be measured using a variety of immunologically based methods such
as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA),
Western blot, protein array technology (e.g., antibody arrays or
arrays using other specific binding agents, etc.).
[0095] Activity of a gene product can also be measured in a variety
of ways that will typically depend upon the specific gene product
whose activity is being measured. For example, if the gene product
is a kinase or phosphatase, the extent to which an endogenous
substrate is phosphorylated provides an indication of the activity
of the gene product. The substrate is isolated from cell in which
it is expressed, and its phosphorylation state is evaluated.
Alternatively or additionally, in vitro kinase or phosphatase
assays can be performed. If the gene product is a transcription
factor, an assay that involves measuring expression of a reporter
construct that contains a DNA element responsive to the
transcription factor can be used. The activity of certain
polypeptides is regulated by post-translational modification,
localization, and/or physical association (typically noncovalent
binding) with one or more cellular structures or molecules. For
example, certain polypeptides are activated or inactivated by
phosphorylation. Activity may be assessed using binding assays,
assays that determine subcellular localization or association with
particular intracellular structures or molecules, assays that
determine modification state, electrophoresis, mass spectrometry,
etc. One of skill in the art will be able to select appropriate
methods for determining and comparing the activity of gene
products.
[0096] In certain embodiments of the invention a highly parallel
method is used. By "highly parallel" is meant that the method
determines the level or activity of at least 10 gene products
essentially simultaneously and/or in a single experiment. Examples
include microarray analysis and protein array analysis, wherein the
array comprises at least 10 features (e.g., at least 10 specific
binding agents such as oligonucleotides or antibodies are affixed
to the array). In certain embodiments of the invention the highly
parallel method determines the level or activity of at least 100,
at least 1000, at least 10,000, or at least 100,000 gene products
essentially simultaneously and/or in a single experiment.
[0097] Many of the genes that have been or will be identified using
the above methods are components of one or more biological
processes or pathways. Such biological processes or pathways may be
identified using a variety of methods. One of skill in the art will
be familiar with processes and pathways in which some of the genes
play a role or will be able to identify such processes and pathways
by searching the literature or by using readily available
biological information resources.
[0098] One biological information resource of particular use is the
Gene Ontology project (www.geneontology.org). The Gene Ontology
(GO) provides a list of three structured, controlled vocabularies
(ontologies) that describe gene products and their associated
biological processes and cellular constituents using a uniform
terminology. In particular, the Gene Ontology database annotates
(and thereby associates) identifiers of gene products (e.g., gene
names) with identifiers of biological processes of which those gene
products are components. The Gene Ontology database can thus be
used to identify the gene products that carry out a particular
biological process and/or to identify the biological processes in
which any gene product of interest plays a role. While the Gene
Ontology database is used herein to exemplify the identification of
biological processes and pathways that involve genes that are
differentially regulated in the nervous system of an individual
subjected to a plasticity-modifying condition, any similar
compilation of information that associates identifiers of
biochemical species with identifiers of biological processes and/or
pathways could be used instead of, or in addition to, the Gene
Ontology database. For example, the Kyoto Encyclopedia of Genes and
Genomes (KEGG) offers somewhat similar facilities. Numerous
additional computer-based resources that provide convenient,
unified access to biological information are available on the World
Wide Web.
[0099] In certain embodiments, biological processes or pathways
whose components (e.g., genes) are over-represented among the
plasticity-related genes are identified as likely to be involved in
modifying plasticity, i.e., they are identified as
plasticity-related processes or pathways. A gene (or other
biochemical species) that is a component of a biological process is
over-represented among the plasticity-related genes if the
likelihood that the number of plasticity-related genes that are
associated with that biological process is greater than the number
of plasticity-related genes that would be expected to be associated
with that biological process based on the number of
plasticity-related genes identified and the number of genes that
are components of the biological process or pathway. Genes that are
components of a biological process or pathway identified as being a
plasticity-related process or pathway are candidate
plasticity-related genes even if they are not themselves
differentially regulated under plasticity-modifying conditions. For
example, a first polypeptide that acts as a ligand, receptor,
substrate, or binding partner for a second polypeptide whose
expression is differentially regulated under plasticity-modifying
conditions may be a component of a biological pathway of which the
first polypeptide is a component and may be modulated instead of,
or in addition to, modulating the first polypeptide.
[0100] In certain embodiments of the invention, once a gene,
pathway, or process is identified using the methods described
above, its role in nervous system structure(s), functions, or
properties is more precisely evaluated using any of a variety of
approaches. Certain of these approaches are also useful to modulate
plasticity for therapeutic purposes, e.g., to improve recovery or
reorganization of the nervous system in a subject in need of
recovery or reorganization. For example, an agent that modulates
the gene, pathway, or process can be administered to an individual
and the effect of the agent on the nervous system is determined.
The individual may or may not be subjected to a
plasticity-modifying condition such as a deprived or enriched
condition. The agent can be administered during all or part of the
period of time over which the individual is subjected to the
condition. In certain embodiments, a transgenic non-human animal
(e.g., a mouse or rat) that has temporally and/or spatially altered
expression of the gene (e.g., that lacks or has reduced expression
of the gene or has elevated or ectopic expression of the gene) is
analyzed to determine whether the animal has altered nervous system
structure or function and/or altered plasticity relative to an
animal in which expression of the gene is not altered (e.g., a
"wild type" animal). The transgenic animal can be generated using
standard methods known in the art and is an aspect of this
invention. In certain embodiments, an agent that modulates a gene,
pathway, or process that is differentially regulated in individuals
subjected to a plasticity-modifying condition is administered to a
non-human animal. The animal may or may not be subjected to a
plasticity-modifying condition or an event that damages the nervous
system. The animal exhibits altered plasticity relative to an
animal to which the agent is not administered. The animal is used
as a model to screen for additional agents that are useful to alter
plasticity and/or promote reorganization or recovery of the nervous
system.
[0101] In certain embodiments of the invention, an agent that
modulates a gene that is a component of a plasticity-related
biological process or pathway is administered. The gene itself may
or may not be differentially regulated under a plasticity-modifying
condition. In some instances, agents that modulate particular
genes, pathways, or pathways will be known to those of skill in the
art. Any such agent can be used. In certain embodiments of the
invention an RNAi agent such as an siRNA or shRNA is used to
inhibit expression of a gene, e.g., by triggering degradation of
mRNA transcribed from the gene. RNA-mediated interference (RNAi)
has recently emerged as a powerful method to reduce the expression
of any target transcript in mammalian cells (see, e.g., Elbashir,
2001; Brummelkamp, 2002; McManus & Sharp, 2002; and U.S. Patent
Publications 2005/0026278, 2004/0259248, and 2003/0108923).
Briefly, it has been found that the presence within a cell of a
short double-stranded RNA molecule referred to as a short
interfering RNA (siRNA), one strand of which is substantially
complementary to transcript present in the cell (the target
transcript) over a length of about 17-29 nucleotides, results in
inhibition of expression of the target transcript. The mechanism
typically involves degradation of the transcript by intracellular
machinery that cleaves RNA (although translational inhibition can
also occur). Short hairpin RNAs are single-stranded RNA molecules
that include a stem (formed by self-hybridization of two
complementary portions of the RNA) and a loop. The stem-loop
structure can be processed intracellularly into an siRNA. In some
embodiments, an antibody, aptamer, or other molecule with specific
binding properties is used to modulate activity of a polypeptide.
In some embodiments, a ligand (e.g., an agonist or antagonist) is
used to modulate activity of a receptor. In certain embodiments of
the invention, the agent is one that can cross the blood brain
barrier so as to achieve an effective concentration in the CNS when
administered to the subject at a location outside the nervous
system (e.g., orally, intravenously, intraperitoneally) at
concentrations that do not cause unacceptable side effects.
[0102] In certain embodiments, antisense oligonucleotides
complementary to an mRNA transcript that encodes a polypeptide, or
ribozymes that cleave the mRNA transcript, are used to decrease
expression. Antisense oligonucleotides, or a vector that provides a
template for intracellular synthesis of an antisense
oligonucleotide, or cells that synthesize the oligonucleotide, can
be administered. Antisense technology and its applications are well
known in the art and are described in Phillips, M. I. (ed.)
"Antisense Technology," Methods Enzymol., Vol. 313 and 314,
Academic Press, San Diego, 2000, and references mentioned therein.
See also Crooke, S. (ed.) "Antisense Drug Technology: Principles,
Strategies, and Applications" (1.sup.st ed), Marcel Dekker, ISBN:
0824705661, 1st edition (2001), and references therein.
[0103] In some embodiments, an aptamer that binds to a polypeptide
and inhibits its activity is used. An aptamer is an oligonucleotide
(e.g., DNA, RNA, which can include various modified nucleotides,
e.g., 2'-O-methyl modified nucleotides) that binds to a particular
protein. Aptamers are typically derived from an in vitro evolution
process (SELEX), and methods for obtaining aptamers specific for a
protein of interest are known in the art (see, e.g., Brody,
2000).
[0104] Ribozymes and deoxyribozymes are RNA and DNA molecules that
can act as enzymes by folding into a catalytically active structure
that is specified by the nucleotide sequence of the molecule. Such
molecules have been shown to catalyze the sequence-specific
cleavage of RNA molecules. The cleavage site is determined by
complementary pairing of nucleotides in the RNA or DNA enzyme with
nucleotides in the target RNA. Thus, RNA and DNA enzymes can be
designed to cleave to any RNA molecule, thereby increasing its rate
of degradation (Cotten and Birnstiel, 1989; Usman, 1996; and Sun,
2000).
[0105] It will be appreciated that synthetic nucleic acids such as
siRNA, antisense oligonucleotides, aptamers, ribozymes, etc., can
include RNA, DNA, nucleoside analog(s), and/or may included
modified sugars, or modified backbone structures.
[0106] Expression or activity of a gene, pathway, or process
identified using the methods of the invention can be modulated as
described above for purposes of modifying nervous system
structure(s), functions, or properties. These approaches are of use
to modulate plasticity for therapeutic purposes, e.g., to improve
recovery or reorganization of the nervous system in a subject in
need of nervous system recovery or reorganization.
[0107] The invention provides methods for modifying plasticity by
modulating particular cell types present in the nervous system.
Cells present in the nervous system have been classified into a
number of different cell types based on their level of expression
of a molecule or portion thereof, or a set of two or more molecules
or portions thereof (referred to herein as "markers"). The molecule
or portion thereof may be, e.g., a particular gene product, a
lipid, a carbohydrate modification of a polypeptide or lipid, etc.,
(referred to herein as "markers"). The marker(s) are said to be
characteristic of the cell type. Cells may be classified into types
with varying degrees of specificity. For example, the cell type may
be an interneuron or may be more specifically classified as being a
particular type of interneuron. Certain cell types may be
identified based on their expression of a single marker. Other cell
types may be identified based on their expression of two or more
markers (referred to as a "set" of markers), in which case each
marker may be expressed in more than one cell type with specific
sets of markers serving to identify specific cell types. In some
instances the cell is identified based on whether or not the marker
is detectably present in the cell or at its surface at significant
levels (above background). In some instances the cell is identified
as being of a particular type based on the level at which the
marker is present in the cell relative to the level at which it is
present in cells of other types. Markers include molecules and
portions thereof, wherein absence of the molecule or portion
thereof may in part be used to classify cells into different types.
Expression of a marker or a specific set of markers may correlate
with various parameters such as morphology (e.g., branching pattern
of neuronal processes), location, and/or electrophysiologic
properties.
[0108] The invention provides methods for selecting a cell type as
a target for modulation to regulate plasticity based on identifying
genes that are differentially regulated under plasticity-modifying
conditions. Cells of the cell type are involved in regulating one
or more aspects of plasticity. Cells of the cell type may play a
role in maintaining or terminating a critical period. They may play
a role in modifying the ability of other cells to respond to
inputs, e.g., nerve impulses arising as a result of environmental
stimuli. They may regulate formation of new synaptic connections
between neurons and/or regulate the strengthening or weakening of
existing synaptic connections. The invention provides methods of
selecting a cell type as a target for modulation comprising steps
of: (i) subjecting an individual to a condition that modifies
plasticity; (ii) measuring level or activity of each of a plurality
of genes in at least a portion of the individual's nervous system;
(iii) identifying one or more genes whose expression or activity is
differentially regulated in the portion of the individual's nervous
system relative to its expression or activity under alternative
conditions; and (iv) selecting a cell type as a target for
modulation, wherein a product of at least one of the genes is a
marker of the cell type. "Product" here refers to an expression
product of the gene or to a molecule or molecular modification that
is present in a cell or at its surface as a result of the
expression of the gene. For example, if the gene encodes a kinase,
the "product" may be the phosphorylated form of a substrate of the
kinase. In certain embodiments of the invention, the cell type
expresses at least two of the differentially regulated genes or
expresses at least one of the differentially regulated genes and
does not significantly express at least one of the differentially
regulated genes. The method may include determining that the number
of cells of the cell type is altered in at least a portion of the
nervous system of an individual subjected to a plasticity-modifying
condition. For example, immunohistochemistry or in vivo imaging can
be used to evaluate cell number.
[0109] The marker may be any marker recognized in the art as useful
to classify cells present in the nervous system into different cell
types. In certain embodiments of the invention, the marker is a
calcium binding protein. A variety of calcium binding proteins
(CBPs) such as calbindin, parvalbumin, and calretenin are
recognized in the art as being markers of different types of
interneurons (Markram et al., 2004, Nat. Rev. Neurosci., 5:793; and
Flames et al., 2005, Neuron, 46:377). The marker may be a
neuropeptide such as somatostatin, vasoactive intestinal peptide,
neuropeptide Y, or cholecystokinin. These neuropeptides are
recognized in the art as being markers of different types of
interneurons (Markram, 2004; and Flames and Marin, 2005). Certain
cell types are identified based on their expression of one or more
CBPs and one or more neuropeptides.
[0110] In illustrative embodiments, as described in the Examples,
inventive methods are applied to identify the gene that encodes
parvalbumin (PV) as being downregulated (underexpressed) in the
visual cortex under conditions of DR, which conditions prolong the
state of plasticity associated with the critical period. The
invention further identifies PV expressing interneurons as being
reduced in number in visual cortex under conditions of DR. Thus in
certain embodiments of the invention, the cell type selected as a
target for modulation is a PV-expressing interneuron, i.e.,
parvalbumin is a marker of the cell type selected as a target for
modulation. In the cortex, interneurons that express PV are
inhibitory interneurons that utilize .gamma.-aminobutyric acid
(GABA) as their neurotransmitter and are morphologically classified
as basket cells and chandelier cells (Markram, 2004).
[0111] The invention includes computer-readable media (e.g., a hard
disk, floppy disk, compact disk, zip disk, flash memory, magnetic
memory, etc.) that store information related to any of the methods
described above. Information may be organized in the form of a
database, i.e., a collection of data that is organized so that its
contents can easily be accessed, managed and updated. Information
may identify one or more genes that are differentially regulated in
at least a portion of the nervous system of an individual subjected
to plasticity-modifying conditions, optionally under conditions in
which an agent is administered to an individual during or after the
time period in which the individual is subjected to
plasticity-modifying conditions. Genes can be identified by name,
by sequence, by accession number(s), etc. It will be appreciated
that the information about expression and/or activity may relate to
the genes themselves and/or to any of their expression products
(RNA or protein). The information may indicate the nature of the
conditions under which differential regulation was observed, may
identify genes whose expression is altered by a
plasticity-modifying agent, etc. Genes may be listed in order or
ranked, e.g., according to the significance of their differential
regulation. Exemplary collections of such information are provided
in Tables 4-11. Computer-readable media may store information
identifying genes that are not differentially regulated, provided
that they also include information pertaining to genes that are
differentially regulated and identifies those genes as being
relevant to plasticity, to nervous system structure, function,
recovery or reorganization, etc. Additional information related to
the gene(s) and/or to their role in plasticity or nervous system
recovery or reorganization can be included, e.g., (i) quantitative
information related to the extent to which the gene(s) is/are
differentially regulated and/or its significance; (ii) information
identifying a biological pathway or process enriched in one or more
of the genes; (iii) results obtained by administering an agent that
modulates expression or activity of one or more of the genes to a
subject, etc. The invention also includes methods comprising the
step of electronically sending or receiving any of the
afore-mentioned information and, optionally, storing at least part
of the information and/or creating a new computer-readable medium
or copy containing at least part of the information.
Compositions and Methods for Modulating Plasticity and Promoting
Nervous System Reorganization and Recovery
[0112] The present invention is based in part on the identification
of genes that are differentially regulated in response to
particular environmental conditions that modify plasticity, namely
dark rearing and monocular deprivation. The invention is based in
part on the identification of biological processes and pathways
that are enriched for one or more of these differentially regulated
genes and are therefore considered herein to be differentially
regulated pathways. In some embodiments, the present invention
encompasses the recognition that expression products' of certain
genes that are differentially regulated in response to DR and/or MD
are involved in plasticity. In some embodiments, the present
invention encompasses the recognition that certain of these genes
are implicated as being involved in structural and/or functional
nervous system reorganization following nervous system damage and
can be manipulated to achieve therapeutic benefit. In some
embodiments, the present invention encompasses the recognition that
certain of these expression products, and agents that modulate
their expression and/or activity, are of use to promote nervous
system recovery and/or reorganization following nervous system
damage, e.g., following ischemic, hemorrhagic, neoplastic,
degenerative, traumatic, and/or neurodevelopmental damage and/or to
inhibit nervous system deterioration that would otherwise occur,
e.g., as a result of deprivation of input.
[0113] The invention identifies (i) genes whose expression is
downregulated in visual cortex under conditions of DR (Table 4),
(ii) genes whose expression is upregulated in visual cortex under
conditions of DR (Table 5), (iii) genes whose expression is
downregulated in visual cortex under conditions of long term MD
(Table 6), (iv) genes whose expression is upregulated in visual
cortex under conditions of long term MD (Table 7), (v) genes whose
expression is downregulated in visual cortex under conditions of
short term MD (Table 8), and (vi) genes whose expression is
upregulated in visual cortex under conditions of short term MD
(Table 9). The invention identifies genes that are differentially
regulated in visual cortex under conditions of short term MD in
subjects who are treated with a plasticity-modifying agent, namely
an activator of the IGF1 pathway (Tables 10 and 11). These genes
are identified as candidates for modulation to modify plasticity
and/or to promote functional and/or structural nervous system
reorganization or recovery of the nervous system. The genes were
identified at least in part by hybridizing mRNA to a microarray
from Affymetrix (www.affymetrix.com) that contained probes for a
large number of mouse genes (see Example 1). The numbered rows in
Tables 4-11 list (from left to right, separated by spaces or tabs)
the Affymetrix identifier of the probe, the p value, the data for
experimental condition (e.g., MD or DR) and control, the gene
symbol corresponding to the probe (where available), accession
number(s) for the genes and/or proteins, and Reference Sequence
(RefSeq) identifier. Items that are not available or not included
are indicated by ---. It will be appreciated that the entries in
the tables can be arranged in a number of different ways and the
specific ordering presented in the tables is not intended to be
limiting. For example, the entries can be listed and/or ranked on
the basis of ascending p value, on the basis of the absolute or
relative magnitude of the difference in expression between the
experimental and control conditions, etc.
[0114] One of ordinary skill in the art will be able to obtain
additional information about the genes and their expression
product(s) listed in Tables 4-11 and/or discussed herein, e.g.,
their sequences, by searching public databases such as those
available through Entrez, the search and retrieval system used at
the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov) for databases, including PubMed, Nucleotide
and Protein Sequences (e.g., Genbank), Protein Structures, Complete
Genomes, Taxonomy, etc., (www.ncbi.nlm.nih.gov/gquery/gquery.fcgi).
These databases can be searched using the symbols or names of the
genes. One of skill in the art will also recognize that additional
information can be found at the publicly available Affymetrix
website, Netaffx Analysis Center
(www.affymetrix.com/analysis/index.affx), visited Apr. 12, 2006,
which allows one to correlate GeneChip.RTM. array results with
array design and annotation information and can be queried by ID.
The website includes libraries for each microarray that provide the
IDs of the probes and accession numbers for the corresponding genes
and proteins.
[0115] The invention provides methods for modifying plasticity in
the nervous system of a subject comprising steps of administering a
plasticity-modifying agent to a subject in need thereof, wherein
the agent is administered either alone or in combination with one
or more additional agents in an amount effective to modify nervous
system plasticity, wherein the plasticity-modifying agent modulates
a gene or pathway that is differentially regulated in at least a
portion of the nervous system of an individual subjected to a
plasticity-modifying condition. In other words, when administered
to the subject, the agent modulates a gene or pathway, wherein the
gene or pathway is a gene or pathway that is differentially
regulated in the nervous system of an individual subjected to a
plasticity-modifying condition, e.g., a gene or pathway identified
using the methods of the present invention. The subject to whom the
agent is administered may or may not be subjected to a
plasticity-modifying condition. In certain embodiments of the
invention, the plasticity-modifying condition is DR or MD. In
certain specific embodiments, the plasticity-modifying condition is
MD. In certain embodiments of the invention the agent modifies
plasticity in a manner that depends on nervous system activity,
e.g., the extent to which the nervous system undergoes structural
or functional alteration in the presence of the agent will depend
on the type of inputs received by the nervous system and/or the
type of stimuli to which the nervous system is subjected. In
certain embodiments of the invention, the agent enhances the
ability of the nervous system to modify its structure or function
in response to the presence of a second agent such as a neural
growth enhancing agent. Thus the plasticity-enhancing agent may at
least in part play a permissive role, contributing to structural or
functional recovery or reorganization in the nervous system when
administered to a subject who is receiving rehabilitative therapy
that modifies nervous system inputs or who is receiving a neural
growth enhancing agent.
[0116] The invention further provides methods of promoting
reorganization or recovery in the nervous system of a subject
comprising steps of: administering a plasticity-modifying agent to
a subject in need thereof, wherein the agent is administered either
alone or in combination with one or more additional agents in an
amount effective to promote nervous system reorganization or
recovery, wherein the plasticity-modifying agent modulates a gene
or pathway that is differentially regulated in at least a portion
of the nervous system of an individual subjected to a
plasticity-modifying condition, e.g., conditions of DR or MD. The
subject may have suffered ischemic, hemorrhagic, neoplastic,
traumatic, neurodegenerative, toxic, and/or neurodevelopmental
damage to the nervous system. The agent may contribute to (e.g.,
enhance) recovery or reorganization in the subject's nervous system
and/or promote normalization of function. In other words, the
degree of reorganization or recovery of the nervous system, or
improvement of function, is greater than would have been the case
if the agent had not been administered to the subject. In certain
embodiments of the invention, the agent does not act solely or
primarily by exerting a neuroprotective effect, e.g., does not act
solely or primarily by inhibiting cell death or dysfunction (e.g.,
necrosis or apoptosis). In certain embodiments of the invention,
the agent exerts both a neuroprotective effect and a
plasticity-enhancing effect. According to certain embodiments of
the invention, the agent is capable of exerting a neuroprotective
effect but is administered within a particular time window
subsequent to a specific damaging event such as a stroke, at a time
that falls outside the time window during which the agent would
exert a neuroprotective effect.
[0117] The above methods may modify plasticity and/or promote
recovery or reorganization in any one or more portions of the
nervous system. For example, in certain embodiments of the
invention, a method modifies plasticity, e.g., promotes plasticity,
and/or promote recovery or reorganization in at least a portion of
the visual cortex. In certain embodiments of the invention, the
portion of the nervous system is one located in proximity to an
implanted drug delivery device. For example, the portion of the
nervous system may be located up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 centimeters (cm) away from the surface or border of the
device.
[0118] Typically, agents and compositions in accordance with the
invention promote structural reorganization and/or functional
reorganization of the nervous system or a portion thereof or
maintain the nervous system in a state in which such reorganization
can occur. In certain specific embodiments, agents of the invention
promote structural and/or functional recovery of the nervous system
or a portion thereof. It will be appreciated that often there will
be a correlation between (i) structural reorganization and/or
recovery and (ii) functional reorganization and/or recovery, e.g.,
both structural reorganization and/or recovery as well as
functional reorganization and/or recovery take place. However, in
some embodiments of the invention, functional reorganization and/or
recovery take place without detectable evidence of structural
reorganization and/or recovery. In some embodiments of the
invention, structural reorganization and/or recovery take place
without detectable evidence of functional reorganization and/or
recovery during a particular time period of evaluation. In such
embodiments, functional reorganization and/or recovery may occur at
a later time, and/or the recovery may not be detected using the
particular measurement tools and methods used for the evaluation.
It will also be appreciated that reorganization is typically
associated with recovery, but reorganization can precede noticeable
evidence of recovery, sometimes by a significant period of
time.
[0119] Functional recovery from damaging events may involve
regrowth of physical connections (e.g., synapses) between surviving
nervous system cells (e.g., neurons, glial cells) and/or
establishment of new connections. Certain of the
plasticity-modifying agents may interact directly with cells (e.g.,
neurons, glial cells, etc.) to enhance their plasticity and/or
stimulate their capacity for structural and/or functional
reorganization. Agents may be administered in conjunction with an
agent that causes degradation of molecule(s) present in the ECM
that would otherwise impede beneficial structural changes or would
exert inhibitory effects on nervous system cells. In certain
embodiments of the invention, two or more agents are administered
concurrently or sequentially to a subject. Either or both of the
agents may be focally administered to the nervous system of the
subject.
[0120] Plasticity-Modifying Agents
[0121] The invention identifies a number of genes and biological
pathways that may be modulated to modify plasticity. Before
discussing certain of these genes and pathways it should be noted
that certain of the genes and their encoded polypeptides discussed
herein are members of families, and in some cases multiple isoforms
of a particular polypeptide exist, as well as post-translationally
modified forms (e.g., forms that have been modified by
phosphorylation, glycosylation, acylation, etc.). In such cases a
single name may be used to collectively refer to multiple genes or
polypeptides. For example, "PI3K" refers to any member or set of
members of the PI3K family. "AKT" refers to at least Akt1, Akt2,
and/or Akt3, etc. "STAT" refers to at least STAT12, 3, 4, 5a, 5b,
6, and/or 7, etc. "JAK" refers to at least JAK1, JAK2, JAK3, and/or
Tyk2, etc. Similarly, the "JAK/STAT pathway" refers to any pathway
involving at least one JAK and at least one STAT. It will be
appreciated that in certain embodiments of the invention it will be
desirable to selectively modulate one or more members of a family,
e.g., one or more members that is/are present in the nervous
system. It will be also be appreciated that multiple variant
polypeptides encoded by a single gene may arise from RNA and/or
protein splicing and that gene editing can also give rise to
variants, all of which may be referred to by the same name or
symbol herein. The invention thus includes embodiments in which any
one or more members of a family, isoforms, splice variants that
arise from RNA or protein splicing or gene editing,
post-translationally modified forms, etc., are modulated.
[0122] One of ordinary skill in the art will readily understand
which particular genes and gene products (e.g. mRNA and
polypeptides) are referred to using the names listed herein and
will be able to retrieve the sequences of these genes and gene
products and relevant information such as sources from which the
molecule can be purified or obtained using, e.g., publicly
available databases such as Genbank and PubMed. For example, one of
skill in the art can search the Entrez Gene database provided by
the National Center for Biotechnology Information (NCBI), available
at the web site having URL
www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=gene and
can thereby locate the Gene ID for any particular gene or
polypeptide of interest. It will be appreciated that allelic
variants, homologs, and biologically active fragments or variants
of the molecules described herein also be used.
[0123] In some embodiments (described in more detail in the
Examples), IGFBP5 is identified as being differentially regulated
under a particular deprived condition (MD). IGFBP5 is a component
of the IGF1 pathway. The invention contemplates modulating one or
more components of the IGF1 pathway in order to modify plasticity.
The invention contemplates modulating one or more components of the
IGF1 pathway to promote recovery or reorganization of the nervous
system in a subject in need thereof.
[0124] As described in the Examples, IGFPB5 is significantly
upregulated under conditions of MD in the visual cortex of subjects
that are subjected to MD. IGFBP5 is one of the most upregulated
genes after MD both at the mRNA and protein level. Furthermore, the
IGF1 pathway is one of the biological pathways that is most
enriched for genes that are differentially regulated after MD, and
both IGFBP5 and IGF1 are constituents of several highly enriched
pathways after MD. Therefore, the IGF1 pathway is identified as
being a plasticity-related pathway of particular interest. As
described in Example 4, administration of an activator of the IFG1
pathway prevented many of the effects of monocular deprivation on
the V1 region of the cortex. To the best of the inventors'
knowledge, these results represent the first evidence showing the
possible functional involvement of the IGF1/IGFBP5 system in
experience-dependent plasticity in the cortex. The results
demonstrate that IGF1 and/or pathways and mechanisms involving IGF1
stabilize synapses and alter plasticity.
[0125] IGF1 is a member of a superfamily of growth-promoting
peptides related to insulin in sequence and biological activity.
The actions of IGF1 are mediated by the type I IGF receptor
(IGF1R), which transmits binding of IGF1 to an intracellular
signaling cascade. Binding of IGFs to the IGF1R enhances the
receptors's tyrosine kinase activity, resulting in phosphorylation
of insulin receptor substrates IRS1-IRS4, which leads to activation
of two major downstream signaling pathways, the mitogen activated
protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K)
pathways. The PI3K pathway is discussed further below. Six IGF
binding proteins (IGFBP1-IGFBP6) regulate the biological activity
of IGF1 by a variety of mechanisms, and some of the IGFBPs have
effects independent of IGF1. IGF1, IGF1R, and certain of the IGFBPs
are expressed in the CNS and have been postulated to have a variety
of different functions therein (Russo, 2005). IGF1 interacts with a
variety of different proteins, and activation of the IGF1 pathway
results in phosphorylation of a large number of downstream
substrates.
[0126] The IGF1 pathway can be modulated using a variety of
different methods. In certain embodiments of the invention, the
pathway is modulated so as to increase the activity of the pathway.
IGF1 or a biologically active fragment thereof can be administered
to the subject to activate the pathway. In some embodiments, the
tripeptide GPE is used. Alternatively or additionally, a different
ligand of an IGF receptor can be administered. The ligand can be an
agonist or antagonist, depending on whether it is desired to
inhibit or activate the receptor. In some embodiments, methods
include (i) administering agent that disrupts the physical
association between IGF1 and an IGFBP; (ii) administering an agent
that activates or inhibits a kinase that phosphorylates one or more
IGF1 substrates; (iii) administering an agent that activates or
inhibits a phosphatase that dephosphorylates one or more IGF1
substrates; (iv) administering an agent that upregulates expression
of IGF1 or IGF1R; (v) administering an agent that upregulates or
downregulates expression of an IGFBP; (vi) administering an agent
that increases the expression or activity of a component of the
PI3K, and/or Akt signaling cascade. etc. In one embodiment, an RNAi
agent is used to inhibit expression of one or more genes in the
pathway, e.g., a gene encoding an IGF binding protein such as
IGFBP5.
[0127] In certain embodiments of the invention, the
phosphoinositide 3-kinase (PI3K) signal transduction pathway is
modulated. Phosphoinositide 3-kinase, also referred to as
phosphatidylinositol 3-kinase, is a lipid kinase and a
serine/threonine kinase that is a component of a signal
transduction pathway involving Src-like or receptor tyrosine
kinases such as the IGF1 receptor. Thus, the PI3K pathway is
responsible at least in part for the actions of IGF1. The PI3K
kinase superfamily includes a large number of structurally related
enzymes with differing regulation and substrates (see Foster, 2003
and Paez et al., 2003, for reviews). "Classical" PI3K comprises a
regulatory subunit (p85) and a 110-kDa catalytic subunit (p110).
PI3K acts through a downstream effector protein kinase B (PKB, also
named Akt) to regulate many cellular processes including cell
survival, cell proliferation, vesicular trafficking, inflammation
and apoptosis inhibition. Three isoforms of Akt (Akt1, Akt2, and
Akt3) are known. When activated, PI3K phosphorylates
phosphoinositides at the 3' position of the inositol ring.
Following their phosphorylation the phosphoinositides promote Akt
activation by phosphorylation. Activated Akt (phosphoAkt) then
phosphorylates a variety of substrates.
[0128] As described in the Examples, PI3K, which is activated by
IGF1, was significantly diminished in expression after MD, but
expression was fully restored after MD when IGF1 treatment was
administered, suggesting that the plasticity-related effects of
IGF1 may at least in part be mediated through PI3K. The present
invention encompasses modulating the PI3K pathway, optionally by
modulating the expression or activity of Akt, to modify plasticity
in a subject in need thereof. For example, the invention
encompasses administering an agent that inhibits or enhances
phosphorylation of Akt. The invention contemplates modulating one
or more components of the PI3K pathway, e.g., Akt, to promote
recovery or reorganization of the nervous system in a subject in
need thereof. Agents that modulate activity of PI3K and/or Akt are
known in the art (see, e.g., U.S. Patent Publication 2003/0236271,
which describes bicyclic or tricyclic fused heteroaryl derivatives
useful to inhibit PI3K; and U.S. Patent Publication 2004/0176385,
describing small molecule inhibitors of PI3K). In some embodiments,
the agent is an RNAi agent, such as an siRNA that is targeted to a
component of the PI3K signal transduction pathway (see, e.g., U.S.
Patent Publication 2005/0272682).
[0129] In certain embodiments (described in more detail in the
Examples), STAT1 is identified as being differentially regulated
under a particular deprived condition (monocular deprivation), and
the JAK/STAT pathway is identified as being a plasticity-related
pathway. In particular, STAT1 is upregulated in the visual cortex
of subjects that are subjected to MD. Furthermore, phosphorylated
STAT1 was upregulated, indicating activation of the JAK-STAT
cascade. The invention contemplates modulating one or more
components of the JAK/STAT pathway in order to modify plasticity in
a subject in need thereof. The invention also contemplates
modulating one or more components of the JAK/STAT pathway to
promote recovery or reorganization of the nervous system in a
subject in need thereof. The JAK/STAT pathway is the major
signaling mechanism for a diverse group of cytokines and growth
factors (reviewed in Rawlings et al., 2004, J. Cell Sci.,
117:1281). Binding of these ligands to their receptors induces
multimerization of receptor subunits that are associated with Janus
tyrosine kinases (JAKs), allowing transphosphorylation of the JAKs.
Activated JAKs phosphorylate signal transducers and activators of
transcription proteins (STATs), transcription factors that are
present in the cytoplasm in latent form until activated.
Phosphorylated STATs dimerize and are translocated into the
nucleus, where they activate or repress transcription of target
genes. In addition to these main components of the JAK/STAT
pathway, other proteins that contribute to JAK/STAT signaling
include signal-trans adapter molecules (STAMs), STAT-interacting
protein (StIP), and the SH2B/Lnk/APS family. There are three main
classes of negative regulators of JAK/STAT signaling: suppressor of
cytokine signaling (SOCS) proteins, protein inhibitors of activated
STATs (PIAS) proteins, and protein tyrosine phosphatases
(PTPs).
[0130] The JAK/STAT pathway can be modulated using a variety of
different methods. A component of the JAK/STAT pathway (e.g., a
STAT or JAK polypeptide), or a ligand of a JAK-binding cytokine
receptor can be administered. For example, a receptor agonist can
be administered to activate the pathway, or an antagonist can be
administered to inhibit the pathway. Other methods to modulate the
JAK/STAT pathway include administering an agent that (i) disrupts
or inhibits the physical association between a JAK and a STAT; (ii)
activates or inhibits a kinase that phosphorylates one or more JAK
substrates; (iii) activates or inhibits a phosphatase that
dephosphorylates one or more JAK substrates; (iv) upregulates
expression of a component of the JAK/STAT pathway; (v)
downregulates expression of a component of the JAK/STAT pathway;
(vi) disrupts the physical association between a JAK-binding
cytokine receptor and a JAK; (vii) activates or inhibits a
JAK-binding cytokine receptor; (viii) inhibits or enhances
translocation of a STAT to the nucleus; (ix) inhibits association
of a STAT with DNA; (x) disrupts the physical association between a
JAK-binding cytokine receptor and an endogenous JAK regulating
protein such as a SOCS or PIAS protein; (xi) induces or inhibits
expression of an endogenous JAK regulating protein, etc. As noted
above, RNAi agents are of use to inhibit expression of genes in the
pathway, e.g., one or more JAK, STAT, SOCS, or PIAS proteins. In
general, inhibiting expression of a JAK or STAT will inhibit the
JAK/STAT pathway, while inhibiting expression of a negative
regulator such as a SOCS or PIAS protein will activate the
pathway.
[0131] The present invention encompasses the discovery that
phosphorylated STAT1 is upregulated after MD. Without wishing to be
bound by any theory, this upregulation may be a response of the
brain to remove or reduce deprived eye connections as well as
possibly expand non-deprived eye connections. Thus, upregulating
STAT1 or otherwise activating the pathway in which it acts would
enhance plasticity and/or increase the ocular dominance shift in a
MD model.
[0132] In some embodiments, the agent that modulates the JAK/STAT
pathway is a cytokine. Cytokines are polypeptides secreted by
immune system cells (e.g., lymphocytes, macrophages, etc.) that
exert a biological effect on other immune system cells and/or on
other cells in the body. Examples include interferons,
interleukins, chemokines, etc. The cytokine may upregulate a
component of the JAK/STAT pathway such as STAT1. IFN.gamma. is an
exemplary cytokine of use in the invention to activate the JAK/STAT
pathway. In some embodiments, the agent reduces STAT1 expression or
activity. Exemplary agents that reduce STAT1 expression or activity
include ionomycin and fludarabine. Without wishing to be bound by
any theory, administration of these agents may alter the ocular
dominance shift in an MD model. In some embodiments, the agent is a
peroxisome proliferator receptor (PPAR)-gamma agonist. Examples
include various prostoaglandins such as 15-deoxy-delta
12,14-prostaglandin J(2), thiazolidinediones such as rosiglitazone,
etc. In certain embodiments of the invention, one or more of these
agents is administered to inhibit phosphorylation of one or more
STAT or JAK proteins. In some embodiments, the agent is an
reductase inhibitor. HMG-CoA reductase inhibitors include statins
such as simvastatin, atorvastatin, lovastatin, etc. These agents
may be administered to inhibit the JAK/STAT pathway. Agents that
inhibit STAT1 phosphorylation by inhibiting JAKs include
tryphostins such as AG490 which blocks the action of JAK2 (Meydan
et al., 1996, Nature, 379:645) and WHI-P131, which blocks the
action of JAK3 (Sudbeck et al., 1999, Clin. Cancer Res., 5:1569).
Tyrphostins are low molecular weight compounds that specifically
inhibit protein tyrosine kinases. See also U.S. Pat. No. 6,080,748,
which describes a variety of dimethoxyquinazoline compounds useful
as inhibitors of JAK3. See also U.S. Patent Publications
2003/0236244, 2004/0209799, 2004/0097504, 2005/0159385, and
2005/0148574.
[0133] The invention provides methods of modifying plasticity
comprising steps of modulating a cell type characterized in that
one or more markers of the cell type is a product of a gene that is
differentially regulated in at least a portion of the nervous
system of an individual subjected to a condition that modifies
plasticity. The invention provides methods of modifying plasticity
comprising steps of: modulating a marker of a cell type
characterized in that one or more markers of the cell type is a
product of a gene that is differentially regulated in at least a
portion of the nervous system of an individual subjected to a
condition that modifies plasticity.
[0134] As noted above, the invention identifies the gene that
encodes PV as being downregulated (i.e. underexpressed) in the
visual cortex under conditions of DR, which prolong the state of
plasticity associated with the critical period. The invention
identifies PV expressing interneurons as being reduced in number in
visual cortex under conditions of DR. Based at least in part on
these discoveries, the invention provides methods of modifying
plasticity in the nervous system of a subject comprising
administering a plasticity-modifying agent to the subject, wherein
the plasticity-modifying agent modulates development, survival,
and/or activity of parvalbumin expressing interneurons in at least
a portion of the brain. In some embodiments, the agent inhibits
development, survival, and/or activity of parvalbumin expressing
interneurons in at least a portion of the brain. In certain
embodiments of the invention, the plasticity-modifying agent
inhibits expression or activity of parvalbumin.
[0135] Exemplary methods of inhibiting development, survival,
and/or activity of parvalbumin expressing interneurons include
administering L-type calcium channel antagonists such as nimodipine
or nifedipine (Jiang et al., 2005, Neuroscience, 135:839). In some
embodiments, PV expressing interneurons are targeted for
elimination by administering a complex comprising a cytotoxic agent
and a targeting moiety, wherein the targeting moiety specifically
binds to a marker of PV expressing interneurons, e.g., a molecule
or portion thereof present at the cell surface of PV expressing
interneurons. The complex or a portion thereof may be internalized.
The cytotoxic agent selectively kills interneurons that have the
marker present at their cell surface. "At the cell surface" is used
herein to mean that a molecule or portion thereof is exposed to the
extracellular environment and accessible to binding by a suitable
binding agent.
[0136] The cytotoxic agent may be covalently or noncovalently
associated with the targeting moiety. Alternatively or
additionally, both the cytotoxic agent and the targeting moiety may
be covalently or noncovalently associated with a third entity. For
example, in some embodiments, the cytotoxic agent and the targeting
moiety are covalently attached to one another either directly or
via a linker moiety to form a conjugate. In some embodiments, the
cytotoxic agent and the targeting moiety are associated with a
delivery vehicle such as a polymeric scaffold, polymeric particle,
or liposome. A variety of cytotoxic moieties can be used. Exemplary
classes include alkalizing or alkylating agents, alkyl sulfonates,
aziridines, ethylenimines and methylamelamines, nitrogen mustards,
certain antibiotics, anti-metabolites, folic acid analogues, purine
analogs, pyrimidine analogs, arabinosides, platinum analogs,
microtubule inhibitors (e.g., microtubule depolymerizing agents or
stabilizers), topoisomerase inhibitors, proteasome inhibitors,
proapoptotic agents, kinase inhibitors, radioisotopes, toxins such
as diphtheria toxin, Pseudomonas exotoxin A (PE), cholera toxin
(CT), pertussis toxin (PT), ricin A chain, botulinum toxin A,
conotoxins, etc. The marker may be, e.g., an ion channel or
receptor subunit that is expressed by PV expressing interneurons.
Typically, the marker is present at the cell surface of PV
expressing interneurons at a higher average level than the level at
which it is present at the cell surface of most or all other cell
types in the nervous system. Examples include a subunits of L-type
calcium channels (e.g., subunit 1.2 or 1.3; Jiang and Swann, 2005),
NR2A subunits of NMDA receptors (Kinney, 2006), and the following
ion channel subunits: HCN2, Kv3.1, Kv1.2, Kv1.6, Kv1.1, Kv3.2,
HCN1, KV.beta.1, and Ca.alpha.1A (Markram, 2004). The targeting
moiety can be ligand of a receptor or channel that includes any of
the foregoing subunits, an antibody or other specific binding agent
(e.g., an aptamer or a binding peptide selected through phage
display) that binds to a marker such as any of the foregoing
subunits, etc.
[0137] Alternatively or additionally, in certain embodiments of the
invention, it is desirable to reduce plasticity by accelerating or
enhancing the development, survival, and/or activity of PV
expressing interneurons. For example, agonists of L-type calcium
channels such as BayK 8644 can be used.
[0138] In some embodiments, the present invention relates to
administering combinations of multiple plasticity-modifying agents
to a subject. The agents may be administered together in a single
composition or separately. In some embodiments, an agent that
modulates the IGF1 pathway and an agent that modulates the JAK/STAT
pathway are administered. In some embodiments, an agent that
modulates the IGF1 or JAK/STAT pathway and that inhibits
development, survival, and/or activity PV expressing interneurons
is administered. In some embodiments, an agent that modulates the
IGF1 pathway, an agent that modulates the JAK/STAT pathway, and an
agent that inhibits development, survival, and/or activity PV
expressing interneurons are administered.
[0139] In some embodiments, the invention relates to compositions
comprising multiple plasticity-modifying agents. One such
composition comprises an agent that activates the IGF1 pathway and
an agent that activates or inhibits the JAK/STAT pathway. The
composition can comprise any agent that activates the IGF1 pathway
and any agent that activates or inhibits the JAK/STAT pathway. In
some embodiments, the composition comprises IGF1 or a biologically
active variant or fragment thereof such as GPE, and an HMG-CoA
reductase inhibitor such as a statin. In some embodiments, the
composition comprises IFN.gamma. or a biologically active fragment
or variant thereof and an HMG-CoA reductase inhibitor.
[0140] Combined Administration of Plasticity-Modifying Agent and
Proteolysis-Enhancing Agent
[0141] In certain embodiments of the invention, one or more
plasticity-modifying agents and one or more proteolysis-enhancing
agents are administered to a subject. As described in co-pending
patent application U.S. Ser. No. 11/205,501, entitled COMPOSITIONS
AND METHODS FOR ENHANCING STRUCTURAL AND FUNCTIONAL NERVOUS SYSTEM
REORGANIZATION, now published as U.S. Patent Publication
2006/0104969, the inventors have shown that focal administration of
proteolysis-enhancing agents such as tPA, plasmin, or agents with
plasmin-like activity to the nervous system of a subject promotes
reorganization and recovery in the subject's nervous system. The
invention provides methods for modifying plasticity in the nervous
system of a subject comprising the step of: administering a
plasticity-modifying agent and a proteolysis-enhancing agent to a
subject in need thereof, wherein the agents are administered in an
amount and for a time effective to modify nervous system
plasticity, wherein the plasticity-modifying agent modulates a gene
or pathway that is differentially regulated in at least a portion
of the nervous system of an individual subjected to a
plasticity-modifying condition. For example, in certain embodiments
of the invention, the agent modulates a gene or pathway that is
differentially regulated in at least a portion of the nervous
system of an individual subjected to conditions of dark rearing
(DR) or monocular deprivation (MD). The plasticity-modifying agent
can be, e.g., any of the agents described herein.
[0142] Without wishing to be bound by any theory, proteolysis of
one or more ECM component(s), mediated by a proteolysis-enhancing
agent such as tPA and/or plasmin, creates an environment that is
permissive for structural reorganization and may enhance activity
of a plasticity-modifying agent. Thus, the present invention
encompasses the recognition that enhancing proteolytic activity in
the nervous system following nervous system damage in combination
with administering a plasticity-modifying agent may permit
increased structural remodeling relative to either therapy alone,
thereby contributing to improved functional recovery. The following
sections describe proteolysis-enhancing agents of use in the
invention, drug delivery devices, methods and locations for the
focal administration of plasticity-promoting agents and
proteolysis-enhancing agents, and various other features of the
invention.
[0143] A variety of different proteolysis-enhancing agents, or
combinations thereof, are of use in the invention. In certain
embodiments of the invention, the proteolysis-enhancing agent is a
polypeptide. In certain embodiments of the invention, the
polypeptide is a protease. In certain embodiments of the invention,
the proteolysis-enhancing agent enhances proteolysis of fibrin. The
agent may directly cleave fibrin or may activate an endogenous
protease that cleaves fibrin. In certain embodiments of the
invention, the agent enhances proteolysis of a component of the ECM
other than fibrin in addition to, or instead of, enhancing
proteolysis of fibrin. For example, the proteolysis-enhancing agent
may cleave one or more extracellular matrix components including,
but not limited to, collagen, laminin, fibronectin, and
proteoglycans. It is noted that the classification of a particular
agent as a plasticity-promoting agent or a proteolysis-enhancing
agent should not be understood to be limiting in any way. Thus the
effect(s) of the proteolysis-enhancing agent on the nervous system
may result wholly or in part from one or more activities that does
not involve proteolysis. While the plasticity-promoting agents of
the present invention are not recognized as having proteolytic
activity, such activity is not excluded, and the effect(s) of the
plasticity-promoting agent on the nervous system may result wholly
or in part from proteolysis that occurs as an indirect effect of
their administration. For example, administration of the
plasticity-promoting agent may increase expression of an endogenous
proteolysis-enhancing agent such as plasmin or inhibit the
expression of an endogenous inhibitor of a proteolysis-enhancing
agent.
[0144] Suitable agents for use in the present invention include
components of the tPA/plasmin cascade. Components of the
tPA/plasmin cascade include plasminogen activators such as tissue
plasminogen activator (tPA) and variants thereof, plasminogen, and
plasmin. Plasminogen activators (PAs) are serine proteases that
catalyze the conversion of plasminogen to plasmin (Vassalli, 1991)
by cleavage of a single peptide bond (R561-V562) yielding two
chains that remain connected by two disulfide bridges (Higgins and
Bennett, 1990). Plasmin is a potent serine protease whose major
substrate in vivo is fibrin, the proteinaceous component of blood
clots. Plasminogen activation by tPA is stimulated in the presence
of fibrin. Plasmin has a broad substrate range and is capable of
either directly or indirectly cleaving many other proteins,
including most proteins found in the ECM. "Direct," as used herein,
means that the protease physically interacts with the polypeptide
that is cleaved, while "indirect" means that the protease does not
usually physically interact with the polypeptide that is cleaved,
but tends to interact with another molecule, e.g., another
protease, which in turn directly or indirectly cleaves the
polypeptide. Plasmin is also capable of activating metalloprotease
precursors. Metalloproteases in turn degrade ECM molecules.
Metalloproteases are of use in certain embodiments of the present
invention. In addition to the aforementioned substrates, plasmin
cleaves and activates various growth factors and growth factor
precursors. Although the liver is the major site of plasmin
synthesis, plasminogen mRNA and protein have been detected in
numerous brain regions. Thus, plasminogen is available to be
cleaved by tPA administered to the nervous system.
[0145] Two PAs, tissue-type PA (tPA) and urokinase-type PA (uPA)
have been identified in mammals. A major physiological function of
PAs to trigger the lysis of clots by activating plasminogen to
plasmin, which degrades fibrin. In the body, PA activity is
regulated in part by various endogenous serine protease inhibitors
that inhibit PAs, a number of which have been identified.
Neuroserpin (Gene ID 5274) belongs to the serpin family of the
serine protease inhibitors and is expressed by neurons of both the
developing and the adult nervous system. Neuroserpin is present in
regions of the brain where either tPA message or tPA protein are
found, suggesting that neuroserpin may be the selective inhibitor
of tPA in the CNS. Plasminogen activator inhibitor 1 (PAI-1; Gene
ID 5054) is the main plasminogen activator inhibitor (PAI) in
plasma but is also found in the nervous system. Protease-nexin I
(Gene ID 5270), PAI-2 (Gene ID 5055), and PAI-3 (Gene ID 268591,
Mus musculus) are other endogenous PAIs. Protease-nexin I and
neuroserpin inhibit plasmin in addition to PAs.
[0146] While not wishing to be bound by any theory, there are a
number of potential substrates for tPA and/or plasmin whose
proteolysis may contribute to structural reorganization in the
nervous system. Among these are various ECM proteins such as
fibrin, fibronectin, tenascin, and laminin. In addition to plasmin,
tPA may activate other proteases such as the plasmin-like protein
hepatocyte growth factor (HGF), which may in turn cleave additional
substrates.
[0147] tPA for use in the present invention may be from any
species, although for administration to humans, it is generally
desirable to use human tPA or a variant thereof. tPA and useful
variants thereof, including variants with improved properties are
described in U.S. Pat. Nos. 6,284,247; 6,261,837; 5,869,314;
5,770,426; 5,753,486 5,728,566; 5,728,565; 5,714,372; 5,616,486;
5,612,029; 5,587,159; 5,520,913; 5,520,911; 5,411,871; 5,385,732;
5,262,170; 5,185,259; 5,108,901; 4,766,075; 4,853,330, and other
patents assigned to Genentech, Inc. (see also Higgins 1990). For
example, and without limitation, the tPA variant may have an
alteration in the protease domain, relative to naturally occurring
tPA, and/or may have a deletion of one or more amino acids at the
N-terminus, relative to naturally occurring tPA. The tPA variant
may have one or more additional glycosylation sites relative to
naturally occurring tPA and/or may have an alteration that disrupts
glycosylation that would normally occur in naturally occurring tPA
when expressed in eukaryotic cells, e.g., mammalian cells.
Properties that may be of use include, but are not limited to,
increased half-life, increased activity, increased affinity or
specificity for fibrin, etc.
[0148] Human tPA has been assigned Gene ID 5327 in the Entrez Gene
database (National Center for Biotechnology Information; NCBI) and
the GenBank entry for the full length amino acid, mRNA, and gene'
sequences are AAA98809, K03021, and NM.sub.--000930, respectively.
However, it is noted that it may be preferable to use the mature
form of tPA, lacking the signal sequence peptide (as described,
e.g., in U.S. Pat. No. 4,853,330 and Yelverton 1983) or a variant
thereof.
[0149] The chymotrypsin family serine proteases, of which tPA is a
member, are normally secreted as single chain proteins and are
activated by a proteolytic cleavage at a specific site in the
polypeptide chain to produce a two chain form (Renatus, 1997, and
references therein). Both the single chain and two chain forms are
active towards plasminogen, although the activity of the two-chain
form is greater. Plasmin activates single-chain tPA to the
two-chain form, thus resulting in a positive feedback loop. The
single chain, the two chain form of tPA, and/or combinations
thereof, may be used in the present invention.
[0150] tPA and variants thereof are commercially available and have
been approved for administration to humans for a variety of
conditions. For example alteplase (Activase.RTM., Genentech, South
San Franciso, Calif.) is recombinant human tPA. Reteplase
(Retavase.RTM., Rapilysin.RTM.; Boehringer Mannheim, Roche
Centoror) is a recombinant non-glycosylated form of human tPA in
which the molecule has been genetically engineered to contain 355
of the 527 amino acids of the original protein. Tenecteplase
(TNKase.RTM., Genentech) is a 527 amino acid glycoprotein
derivative of human tPA that differs from naturally occurring human
tPA by having three amino acid substitutions. These substitutions
decrease plasma clearance, increase fibrin binding (and thereby
increase fibrin specificity), and increase resistance to
plasminogen activator inhibitor-1 (PAI-1). Anistreplase
(Eminase.RTM., SmithKline Beecham) is a commercially available
human tPA.
[0151] Additional plasminogen activators include streptokinase
(Streptase.RTM., Kabikinase.RTM.) and urokinase (Abbokinase.RTM.),
both of which are commercially available.
[0152] Alternatively or additionally, proteolysis-enhancing agents
of use in the invention include tPA activators such as Desmodus
rotundus salivary plasminogen activator (DSPA) Desmoteplase (Paion,
Germany) which is derived from vampire bat saliva (Liberatore,
2003, and references therein). Four distinct proteases have been
characterized and are referred to as D rotundus salivary
plasminogen activators (DSPAs). Full-length vampire bat plasminogen
activator (DSPA1) is the variant most intensively studied and
exhibits >72% amino acid sequence identity with human tPA.
However, 2 important functional differences are apparent. First,
DSPAs exist as single-chain molecules that are not cleaved into 2
chain forms. Second, the catalytic activity of the DSPAs appears to
be dependent on a fibrin cofactor. Urokinase plasminogen activators
such as rescupase (Saruplase.RTM., Grunenthal), and microplasmin (a
cleavage product of plasminogen) are also of use in various
embodiments of the invention. Alfimeprase (Nuvelo) is yet another
proteolysis-enhancing agent of use in the present invention.
Alfimeprase is a recombinantly produced, truncated form of
fibrolase, a known directly fibrinolytic zinc metalloproteinase
that was first isolated from the venom of the southern copperhead
snake (Agkistrodon contortrix contortrix) (Toombs, 2001). These
enzymes breaks down fibrin directly. Fibrolase itself is of use in
the present invention. Also of use is staphylokinase (Schlott,
1997).
[0153] In some embodiments of the invention plasmin or mini-plasmin
is administered instead of, or in addition to, tPA. A variety of
other agents that have plasmin-like activity may be used. In
general, such substances are able to cleave typical plasmin
substrates, such as the synthetic substrate S-2251.TM.
(Chromogenix-Instrumentation Laboratory, Milan, Italy), which is a
conveniently assayed chromogenic substrate for plasmin and
activated plasminogen. Other agents that have tPA-like activity
(e.g., they are able to cleave plasminogen and activate it in a
similar manner to tPA) can be used.
[0154] Lumbrokinase is an enzyme or group of enzymes derived from
earthworms Lumbricus rubellus which has been known for some time
(see, e.g., reporting cloning of a gene encoding lumbrokinase,
PI239, GenBank Accession No. AF433650; Ge, 2005). Other
fibrinolytic proteases isolated from earthworms are of use (Cho,
2004). Also of use is nattokinase.
[0155] In some embodiments, a variety of fibrinolytic enzymes that
have been isolated from various worms, insects, and parasites can
be used in accordance with the present invention. For example,
destabilase, an enzyme present in the leech, hydrolyzes fibrin
cross-links (Zavalova, 1996; Zavalova, 2002).
[0156] In some embodiments of the invention, plasminogen is
administered instead of, or in addition to, tPA.
[0157] Instead of, or in addition to, administering a molecule that
itself has plasminogen activator activity, plasmin activity, or
plasmin-like activity, substances that increase endogenous
expression of plasminogen activators or plasmin may be
administered. Such substances may act by increasing transcription
or translation of the mRNA that encodes the molecule, stabilizes
the molecule, etc. They include, but are not limited to, brain
derived neurotrophic factor (BDNF), transforming growth
factor-.beta. (TGF-.beta.), phorbol esters, and retinoic acid.
[0158] A variety of other agents can be administered to enhance
protolysis in the central or peripheral nervous system in order to
treat nervous system damage due to ischemic, hemorrhagic,
neoplastic, traumatic, degenerative, and/or neurodevelopmental
conditions. Certain of these agents are administered focally while
others are administered using an alternate route of administration,
e.g., oral, intravenous, intraperitoneal, intramuscular,
intradermal, transdermal, subcutaneous, pulmonary (e.g., by
inhalation into the lungs), nasal, etc. For example, sulodexide is
a fibrinolytic agent that releases cellular tPA and thus is of use
to increase tPA activity. In certain embodiments of the invention
it is administered orally (Harenberg, 1998). Other agents of use in
the invention to inhibit PAI include enalapril (Sakata, 1999) and
ampotherin (Parkinnen, 1993).
[0159] Aspirin, which has been reported to stimulate plasmin
activity, is of use in the invention (Milwidksy, 1991). In certain
embodiments aspirin is not used, or if the subject is receiving
aspirin, a different agent is used in addition to aspirin.
[0160] Another strategy that may be used to increase the level of
plasminogen activator activity, plasmin activity, or plasmin-like
activity is to administer a substance that inhibits one or more of
the endogenous inhibitors of tPA or plasmin. Such endogenous
inhibitors include PAI-1, PAI-2, PAI-3, and neuroserpin. A
plasminogen activator inhibitor will be referred to as a PAI
herein. In some embodiments, an inactive form of a PAI, which is
unable to inhibit plasminogen activators, is used (see, e.g., PCT
Publication WO 97/39028; and Lawrence et al., 1997, J. Biol. Chem.,
272:7676; both of which describe various inactive forms of PAI).
Without wishing to be bound by any theory, an inactive form of PAI
may compete with an active form and thereby prevent inhibition of
tPA. Small molecules and peptides that inhibit one or more PAIs are
known in the art and are of use in the present invention. Examples
include PAI-039 (Hennan, 2005), ZK4044 (Liang, 2005), tiplaxtinin
(Elokdah, 2004), piperazine-based derivatives (Ye, 2004), T-686
(Ohtani, 1996), fendosal (HP129), AR-H029953XX, XR1853, XR5118 and
the peptide TVASS (Gils, 2002).
[0161] RNAi may be used to reduce expression of a transcript that
encodes an inhibitory protein, e.g., an endogenous PAL siRNAs or
shRNAs targeted to a transcript that encodes an endogenous PAI can
be delivered together with a proteolysis-enhancing agent or
administered separately. Alternatively or additionally, a vector
that provides a template for intracellular synthesis of one or more
RNAs that hybridize to each other or self-hybridize to form an
siRNA or shRNA that inhibits expression of an inhibitory protein,
or cells that synthesize such RNAs, can be administered.
[0162] Antisense oligonucleotides complementary to an mRNA
transcript that encodes an inhibitory protein, or ribozymes that
cleave the transcript, or vector that provide template for
intracellular synthesis of an antisense RNA or ribozyme can also be
used to downregulate expression of the inhibitor. In some
embodiments of the invention, an aptamer that binds to a PAI and
inhibits its inhibitory activity is used. In some embodiments, an
RNA or DNA enzyme that cleaves a transcript that encodes a PAI and
thus inhibits its inhibitory activity is used.
[0163] In certain embodiments, an antibody or antibody fragment
that binds to a PAI is used to inhibit its activity, or any
polypeptide having a similar binding specificity, e.g., an
affibody. The antibody or antibody fragment can be any
immunoglobulin or immunoglobulin-like molecule that binds to an
antigen and can be monoclonal or polyclonal.
[0164] Any substance that acts to counteract the effect of a
molecule that is inhibitory for activity of a proteolysis-enhancing
agent, whether by causing degradation, by sequestering, by reducing
expression, or by blocking interaction of the molecule with another
molecule or with a cell will be said to counteract the inhibitory
molecule and is within the scope and spirit of the invention.
[0165] The present invention encompasses the recognition that
enhancing proteolytic activity in the nervous system following
nervous system damage may permit increased structural remodeling,
thereby contributing to improved functional recovery and will
increase the efficacy of a plasticity-enhancing agent. However, the
invention described herein does not require any particular
mechanism of action. The invention encompasses use of variants or
modified forms of the proteolysis-enhancing agents, wherein the
variants or modified forms do not enhance proteolysis. For example,
the invention encompasses variants of proteases (e.g., variants
having a mutation in an active site region) in which the sequence
has been altered, such that the variant is no longer an active
proteolytic agent. The invention also encompasses embodiments in
which the proteolysis-enhancing agent has been chemically
inactivated, such that it no longer enhances proteolysis. Thus in
some embodiments of the invention an inactive form of a
proteolysis-enhancing agent is focally administered. However, in
general, a proteolysis-enhancing agent is active or capable of
being activated when used according to the present invention.
[0166] It will be appreciated that various agents have been focally
administered to the nervous system of a subject suffering from
ischemic, hemorrhagic, neoplastic, traumatic, toxic,
neurodegenerative, and/or neurodevelopmental damage to the nervous
system, for purposes other than enhancing proteolysis. For example,
analgesic agents are commonly administered. Should it be the case
that any of such previously administered agents enhance
proteolysis, such agent may be explicitly excluded from the present
invention or, if used in the present invention, its use in the
context of the present invention differs from such previous use.
For example, its use in the context of the present invention
involves administration to a different location, uses a different
administration means, involves administration in combination with a
plasticity-modifying agent, and/or employs a different dose and/or
time course, etc.
[0167] The ability of PAs to trigger the lysis of clots has led to
the use of PAs and other plasminogen-activating proteases such as
streptokinase as thrombolytic agents for the treatment of
myocardial infarction and stroke, as mentioned above. However,
studies have suggested that tPA, which is released by neurons
following excitotoxicity such as occurs in ischemia, could increase
neuronal damage. Furthermore, release or leakage of tPA out of the
vascular system and the attendant potential for damage to nervous
system tissue, is a recognized hazard of thrombolytic therapy. Thus
the invention described herein, which demonstrates that appropriate
administration of plasmin and/or plasminogen-activating proteases
such as tPA can actually contribute to structural and/or functional
nervous system reorganization and recovery, is particularly
noteworthy.
[0168] It will be appreciated that various embodiments of the
present invention differ from previously reported uses of tPA
(e.g., for purposes of thrombolysis) in at least one of the
following ways, which are described in further detail below: (i)
administration as described herein is focally directed to the
nervous system and does not typically take place via the vascular
system; (ii) administration as described herein is typically
performed at least 3 hours following the onset of a stroke or other
damaging event and typically at least 12 hours or more following
the onset of the damaging event; (iii) administration as described
herein may occur multiple times (e.g., 2, 3, or more times)
following the onset of a damaging event and/or may occur either
intermittently or continuously over a prolonged time period
following the onset of a damaging event (e.g., over at least 1
week, 4 weeks, 1 month (30 days), 3 months, 6 months, 1 year, 2
years, 3 years, or even longer); (iv) administration as described
herein typically does not use doses that would be sufficient to
cause effective blood clot lysis at the site of administration when
administered using methods that are intended to achieve blood clot
lysis.
[0169] Variants and Fragments
[0170] It will be appreciated that most proteins can tolerate a
certain amount of sequence variation without substantial loss of
functional activity, provided that such sequence variation does not
affect key residues that are required for such functional activity.
The present invention therefore encompasses variants of the
plasticity-enhancing or proteolysis-enhancing polypeptides (and
other polypeptides disclosed herein), wherein such variants retain
a significant amount of biological activity. For example, the
fragment can have substantially similar activity (e.g., at least
about 10-20% of the relevant activity) to the original polypeptide,
at least about 50% of the relevant activity, etc. The term
"variants" includes fragments, i.e., polypeptides whose sequence is
a continuous subset of a polypeptide disclosed herein. Biologically
active variants or fragments of certain polypeptides of interest
herein are known in the art. The invention contemplates the use of
any such variant or fragment. For example, GPE is a biologically
active fragment of IGF1 of use in the invention. Specifically
encompassed are variants or fragments in which one or more kringle
domains of a polypeptide disclosed herein, e.g., plasmin or tPA, is
removed. Certain fragments of use in this invention contain a
protease domain and, optionally, at least one kringle domain
[0171] As is well known in the art, certain amino acids are
generally similar with respect to particular properties and can
frequently be substituted for one another in a polypeptide without
significantly altering the functional and structural properties of
the polypeptide. For example, the variants may contain one or more
conservative amino acid substitutions, which may be defined in
accordance with Stryer, Biochemistry, 3rd ed., 1988. Amino acids in
the following groups possess similar features with respect to side
chain properties such as charge, hydrophobicity, aromaticity, etc.,
and can be substituted for one another in accordance with certain
embodiments of the invention: (1) Aliphatic side chains: G, A, V,
L, I; (2) Aromatic side chains: F, Y, W; (3) Sulfur-containing side
chains: C, M; (4) Aliphatic hydroxyl side chains: S, T; (5) Basic
side chains: K, R, H; (6) Acidic amino acids: D, E, N, Q; (7)
Cyclic aliphatic side chain: P (P may be considered to fall within
group (1)). One of ordinary skill in the art will recognize that
other definitions of conservative substitutions can also be used.
Amino acid abbreviations used herein are in accordance with common
usage in the art.
[0172] The present invention encompasses administration of variants
that are at least 80% identical, at least 85% identical, at least
90% identical, at least 95% identical, or at least 98% identical to
one or more of the polypeptides disclosed herein over a number of
amino acids equal to at least 50% of the number of amino acids the
polypeptide. Percent identity may be calculated by standard
methods. For example, the percent identity between first and second
polypeptides over a window of evaluation may be computed by
aligning the polypeptides, determining the number of polypeptides
within the window of evaluation that are opposite an identical
polypeptides allowing the introduction of gaps to maximize
identity, dividing by the total number of amino acid positions in
the window, and multiplying by 100. Various computer programs such
as BLAST2, BLASTP, Gapped BLAST, etc., generate alignments and
provide % identity between sequences of interest. Algorithms
employed in those programs (utilizing default values) can be
used.
[0173] The present invention encompasses variants in which up to
20%, up to 15%, up to 10%, up to 5%, or up to 2% of the amino acid
residues are either substituted (e.g., conservatively substituted),
deleted, or added, relative to a polypeptide disclosed herein.
Specifically encompassed are allelic variants that exist within a
population. The invention encompasses variants that are
specifically recognized by immunological reagents (e.g., monoclonal
or polyclonal antibodies) that recognize a polypeptide disclosed
herein, i.e., the immunological reagent binds to the variant with a
substantially similar affinity (e.g., having a K.sub.a at least 50%
as great) as that with which it binds to the polypeptide.
[0174] The invention encompasses variants that have a substantially
similar overall structure to the polypeptides disclosed herein. For
example, certain variants possess sufficient structural similarity
to a protein disclosed herein so that when its 3-dimensional
structure (either actual or predicted structure) is superimposed on
the structure of the protein the volume of overlap is at least 70%,
at least 80%, or at least 90% of the total volume of the structure.
Furthermore a partial or complete 3-dimensional structure of a
variant may be determined by crystallizing the protein using
methods known in the art. Alternatively or additionally, an NMR
solution structure can be generated (see, e.g., Heinemann, 2001;
Wishart D. 2005; and references therein). A modeling program such
as MODELLER (Sali and Blundell, 1993), or any other modelling
program, can be used to generate a predicted structure. The
PROSPECT-PSPP suite of programs can be used (Guo, 2004).
[0175] In certain embodiments of the invention, the variant has
substantially similar plasticity-modifying or proteolysis-enhancing
activity as the polypeptide of which it is a variant. In certain
embodiments of the invention, the variant does not have a
substitution at an active site residue. Active site residues of
serine proteases such as the proteases disclosed herein are well
known in the art.
Methods of Preparing the Agents of the Invention
[0176] The agents disclosed herein are all known in the art, and it
is believed that appropriate methods for their manufacture are well
within the skill of those in the art and therefore need not be
described here in detail. For example, and without limitation, many
of the small molecules described herein can be chemically
synthesized using known methods, as can siRNAs and antisense
oligonucleotides, and peptides. Certain agents can be purified from
natural sources.
[0177] Plasticity-modifying agent such as IGF1, IFN.gamma., and
proteolysis-enhancing agents, e.g., tPA, or other polypeptides such
as plasmin, growth factors, etc., for use in the present invention,
may be purified from natural sources, manufactured using
recombinant DNA technology (e.g., recombinant tPA), synthesized
using purely chemical synthesis (i.e., synthesis not requiring the
use of cells to produce the polypeptide), etc.
[0178] Methods for producing a polypeptide of interest using
recombinant DNA technology are well known in the art. Briefly, such
methods generally involve inserting a coding sequence for the
polypeptide into an expression vector, operatively associated with
expression signals such as a promoter, such that mRNA encoding the
protein is transcribed when the expression vector is introduced
into a suitable host cell. The host cell translates the mRNA to
produce the polypeptide. The polypeptide can include a secretion
signal sequence so that the polypeptide is secreted into the
medium. The polypeptide may be harvested from the cells or from the
medium. Transgenic animals and plants are commonly used to produce
polypeptides. Plants into which viral vectors have been introduced
are also used to produce polypeptides.
[0179] Small molecules such as non-peptide neurotransmitters and
analogs thereof, small peptides, neurally active metals, and other
compounds disclosed herein are typically either purified from
natural sources or chemically synthesized, as appropriate,
according to standard methods.
[0180] Any of the agents disclosed herein can be provided as
pharmaceutically acceptable salts, prodrugs, etc. Furthermore, any
of the polypeptides disclosed herein can be modified using a
variety of methods known in the art. For example, they can be
modified by addition of polyethylene glycol (PEG) or variants
thereof. Such modifications may increase the active half-life of
the polypeptide (see, e.g., Nektar Advanced Pegylation 2005-2006
Product Catalog, Nektar Therapeutics, San Carlos, Calif., which
describes a number of such modifying agents and provides details of
appropriate conjugation procedures). For administration by
injection or infusion, compositions of the invention will typically
be mixed with pharmaceutically acceptable carriers or diluent such
as sodium chloride (e.g., 0.9%) or dextrose (e.g., 5% dextrose)
aqueous solutions. Agents can be provided for administration either
in solution or in lyophilized or otherwise dried form. They can be
reconstituted in water, saline, etc., followed by dilution in an
appropriate pharmaceutically acceptable carrier or diluent.
Polymer-Based Drug Delivery Devices
[0181] The invention provides a drug delivery device for
implantation into the nervous system of a subject to promote
recovery or reorganization, e.g., following ischemic, hemorrhagic,
neoplastic, traumatic, and/or neurodevelopmental damage to the
nervous system. The drug delivery device comprises a release
material, a plasticity-modifying agent, and, optionally, one or
more additional active agents such as a proteolysis-enhancing
agent. The term "release material" is used to refer to any matrix
or material that releases incorporated molecules by diffusion or
disintegration of the matrix. In certain embodiments of the
invention the release material is a biocompatible polymer. The
proteolysis-enhancing agent is released from the release material
in an amount effective to promote reorganization and/or recovery of
the nervous system. A drug delivery device in which an active agent
is physically associated with a polymeric material such as those
disclosed herein is referred to as a "polymer-based drug delivery
device" in order to distinguish such devices from mechanical drug
delivery devices such as infusion pumps, which are used in various
embodiments of this invention, though it should be recognized that
materials other than polymers could also be used.
[0182] In certain embodiments of the invention, the
plasticity-modifying agent and, optionally, the
proteolysis-enhancing agent, is/are incorporated into or otherwise
physically associated with a biocompatible polymeric matrix, which
may be biodegradable or nonbiodegradable. Any form of physical
association is acceptable provided that the association remains
stable under conditions of storage and implantation and for
sufficient time to release the active agent over a desired time
period. For example, the active agent may be encapsulated within a
polymeric matrix, entrapped or entangled within a polymeric matrix,
adsorbed to the surface of a polymeric matrix, covalently attached
to a polymeric matrix, etc. The matrix is delivered to or implanted
into the body at the location of the target tissue or in the
vicinity thereof. The agent is released from the polymeric matrix
over a period of time, e.g. by diffusion out of the matrix or
release into the extracellular environment as the matrix degrades
or erodes. In some embodiments, the active agent is incorporated
into liposomes.
[0183] The polymeric matrix may have a number of different shapes.
For example, microparticles of various sizes (which may also be
referred to as beads, microbeads, microspheres, nanoparticles,
nanobeads, nanospheres, etc.) can be used. Polymeric microparticles
and their use for drug delivery are well known in the art. Such
particles are typically approximately spherical in shape but may
have irregular shapes. Generally, a microparticle will have a
diameter of less than 500 microns, more typically less than 100
microns, and a nanoparticle will have a diameter of 1 micron or
less. If the shape of the particle is irregular, then the volume
will typically correspond to that of microspheres or nanospheres.
Methods for making microspheres are described in the literature,
for example, in U.S. Pat. No. 4,272,398; Mathiowitz and Langer,
1987; Mathiowitz et a/., 1987; Mathiowitz et al., 1988; Mathiowitz
et al., 1990; Mathiowitz et al., 1992; and Benita et al., 1984.
Solid nanoparticles or microparticles can be made using any method
known in the art including, but not limited to, spray drying,
nanoprecipitation, phase separation, single and double emulsion
solvent evaporation, solvent extraction, and simple and complex
coacervation. Preferred methods include spray drying and the double
emulsion process. Solid agent-containing polymeric compositions can
also be made using granulation, extrusion, and/or
spheronization.
[0184] The conditions used in preparing the particles may be
altered to yield particles of a desired size or property (e.g.,
hydrophobicity, hydrophilicity, external morphology, "stickiness,"
shape, etc.). The method of preparing the particle and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may also depend on the agent being encapsulated
and/or the composition of the polymer matrix. If the particles
prepared by any of the above methods have a size range outside of
the desired range, the particles can be sized, for example, using a
sieve.
[0185] Solid nanoparticles or microparticles can be suspended or
dispersed in a pharmaceutically acceptable fluid such as
physiological saline and focally administered by injection or
infusion (e.g., using a pump) to the nervous system.
[0186] Solid polymer-agent compositions (e.g., discs, wafers,
tubes, sheets, rods, etc.) can be prepared using any of a variety
of methods that are well known in the art. For example, in the case
of polymers that have a melting point below the temperature at
which the agent is to be delivered and/or at which the polymer
degrades or becomes undesirably reactive, a polymer can be melted,
mixed with the agent to be delivered, and then solidified by
cooling. A solid article can be prepared by solvent casting, in
which the polymer is dissolved in a solvent, and the agent is
dissolved or dispersed in the polymer solution. Following
evaporation of the solvent, the substance is left in the polymeric
matrix. This approach generally requires that the polymer is
soluble in organic solvent(s) and that the agent is soluble or
dispersible in the solvent. In still other methods, a powder of the
polymer is mixed with the agent and then compressed to form an
implant. Microparticles or nanoparticles comprising a polymeric
matrix and a proteolysis-enhancing agent and optionally one or more
other active agents can be compressed, optionally with the use of
binders, to form an implant.
[0187] A polymeric matrix can be formed into various shapes such as
wafers, tubes, discs, rods, sheets, etc., which may have a range of
different sizes and volumes. For example, prior to polymerization,
a polymer solution may be poured into a mold having the appropriate
shape and dimension. Following polymerization the material assumes
the shape of the mold and is usable as an implant. The agent(s) may
be present in the solution prior to polymerization, or the implant
may be impregnated with the agent following its fabrication.
[0188] Suitable biocompatible polymers, a number of which are
biodegradable include, for example, poly(lactides),
poly(glycolides), poly(lactide-co-glycolides), poly(lactic acids),
poly(glycolic acids), poly(lactic acid-co-glycolic acids),
polycaprolactone, polycarbonates, polyesteramides, polyanhydrides,
poly(amides), poly(amino acids), polyethylene glycol and
derivatives thereof, polyorthoesters, polyacetals,
polycyanoacrylates, polyetheresters, poly(dioxanones),
poly(alkylene alkylates), copolymers of polyethylene glycol and
polyorthoesters, biodegradable polyurethanes. Other polymers
include poly(ethers) such as poly(ethylene oxide), poly(ethylene
glycol), and poly(tetramethylene oxide); vinyl
polymers-poly(acrylates) and poly(methacrylates) such as methyl,
ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and
methacrylic acids, and others such as poly(vinyl alcohol),
poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes);
cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers,
esters, nitrocellulose, and various cellulose acetates;
poly(siloxanes), etc. Other polymeric materials include those based
on naturally occurring materials such as polysaccharides (e.g.,
alginate), chitosan, agarose, hyaluronic acid, gelatin, collagen,
and/or other proteins, and mixtures and/or modified forms thereof.
Chemical derivatives of any of the polymers disclosed herein (e.g.,
substitutions, additions of chemical groups, for example, alkyl,
alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art) are encompassed.
Furthermore, blends, graft polymers, and copolymers, including
block copolymers of any of these polymers can be used. It will be
appreciated that a vast number of different polymer variations are
available. It will be understood that certain of these polymers
require use of appropriate initiators or cross-linking agents in
order to polymerize.
[0189] One of skill in the art will understand that in choosing an
appropriate polymer and method of manufacture, it is important to
select materials and methods that are compatible with stability of
the agent. For example, it may be desirable to avoid processing
temperatures that are likely to result in substantial degradation
or denaturation of the agent, which may result in loss of
bioactivity. It will also be desirable to test the composition so
as to ensure that the agent is released in significant amounts over
the desired period of time.
[0190] In general, the following criteria are important for
selection of a material to be used for delivery of the active
agent(s): (1) minimal or no cytotoxicity, (2) minimal or no
elicitation of immune responses and inflammation, (3) compatibility
with aqueous solutions and physiological conditions, and (4)
compatibility of the material and its processing methods with the
stability of the agent to be incorporated. It may be desirable to
utilize a material with a controlled rate of biodegradation.
Features such as cross-linking and monomer concentration may be
selected to provide a desired rate of degradation and release of
the agent. It will be appreciated that a polymeric drug delivery
device of the invention may include one or more pharmaceutically
acceptable materials such as buffers, spheronizing agents, fillers,
surfactants, disintegrants, binders, or coatings. Exemplary
materials are described in U.S. Pat. No. 5,846,565.
[0191] Methods for purifying or synthesizing the various polymers
for use in drug delivery systems of the invention are known in the
art. Methods for incorporating therapeutically active agents into
polymeric matrices are likewise known in the art. For example, the
active agent can be combined in solution with the polymer prior to
polymerization or can be provided in solid form and encapsulated as
the polymer polymerizes. A number of different agents have been
delivered to the CNS using such polymer matrices. For example,
chemotherapeutic agents have been delivered to tumors in the
nervous system by encapsulating the agent in a polymeric matrix,
which is made into a shaped form, and surgically implanting the
matrix into the brain (see, e.g., U.S. Pat. Nos. 5,626,862;
5,651,986; and 5,846,565). Additional drug delivery devices in
which an active agents is provided in a polymeric matrix are
described (see, e.g., U.S. Pat. Nos. 4,346,709 and 5,330,768; Wu,
1994; Dang, 1996; Fleming, 2002; and Westphal, 2002).
[0192] Similar methods to those used in the afore-mentioned
references are of use to focally deliver the agents of the
invention. In certain embodiments of the invention, the drug
delivery device provides controlled or sustained release, i.e., the
proteolysis-enhancing agent and any other agents contained in the
device are released over a prolonged period of time, e.g., hours to
days, weeks, or months.
[0193] Preparation of polymer-agent drug delivery devices can be
performed using standard methods known in the art. Briefly, drug
delivery devices are typically prepared in one of several ways. For
example, the polymer can be melted, mixed with the substance to be
delivered, and then solidified by cooling. Such melt fabrication
processes generally utilize polymers having a melting point that is
below the temperature at which the substance to be delivered and
the polymer itself degrade or become reactive. Alternatively or
additionally, the device can be prepared by solvent casting, where
the polymer is dissolved in a solvent, and the substance to be
delivered dissolved or dispersed in the polymer solution. The
solvent is then evaporated, leaving the substance in the polymeric
matrix. Solvent casting typically utilizes a polymer that is
soluble in organic solvents, and the drug to be encapsulated should
be soluble or dispersible in the solvent. Similar devices can be
made by phase separation or emulsification or even spray drying
techniques. In still other methods, a powder of the polymer is
mixed with the agent and then compressed to form an implant.
[0194] Methods of producing implants also include granulation,
extrusion, and spheronization. A dry powder blend is produced
including the desired excipients and microspheres. The dry powder
is granulated with water or other non-solvents for microspheres
such as oils and passed through an extruder forming "strings" or
"fibers" of wet massed material as it passes through the extruder
screen. The extrudate strings are placed in a spheronizer which
forms spherical particles by breakage of the strings and repeated
contact between the particles, the spheronizer walls and the
rotating spheroniter base plate. The implants are dried and
screened to remove aggregates and fines.
[0195] These methods can be used to make microimplants
(microparticles, microspheres, and microcapsules encapsulating drug
to be released), slabs or sheets, films, tubes, and other
structures. A preferred form for infusion or injection is
microimplants, as described elsewhere herein.
[0196] Proteins and peptides have been successfully incorporated
into polymeric matrices. For example, insulin has been incorporated
into biodegradable polymeric microcapsules and retains essentially
the same bioactivity as the free form (Takenaga 2004). Natural and
synthetic collagenous matrices have been used as carriers of a
variety of different growth factors (Kanematsu, 2004).
[0197] Of particular interest in the present invention are polymers
that form hydrogels, i.e., gels that contain a substantial
proportion of water. Hydrogels may, for example contain 30%, 40%,
50%, 60%, 70%, 80%, 90%, or an even greater amount of water on a
w/w basis. Polymeric materials can be formed into hydrogels either
prior to or following administration to a subject. An exemplary
material comprises hPLA-b-PEG-PLA macromers. The agent is mixed
with the polymer solution prior to initiating polymerization. Other
suitable hydrogel-forming polymers are known in the art. For
example, a variety of polysaccharides, polypeptides, and
derivatives thereof can be used. Exemplary polysaccharides include
alginate, collagen, cellulose, hyaluronic acid, dextran, chitosan,
derivatives of any of the foregoing, etc. Other materials that form
hydrogels include synthetic polymers such as polyethylene
oxide-polypropylene glycol block copolymers such as Pluronics.TM.
or Tetronics.TM., poly(vinyl alcohol), silicones, polypeptides such
as gelatin, polyethylene glycol and related molecules, polyethylene
oxide and related molecules or derivatives, etc. The hydrogel
precursor materials may contain or be modified to contain
functional groups that become crosslinked to one another.
Optionally, photopolymerization is employed. In some embodiments, a
drug delivery device comprising biodegradable macromers such as
those described in U.S. Pat. No. 6,153,211 is used.
[0198] In some embodiments of the invention, a plasticity-modifying
agent, a proteolysis-enhancing agent, or both, is covalently
attached to the polymer, optionally via a moiety that is cleavable
in vivo, such as an ester linkage or disulfide bond.
[0199] The polymer-based drug delivery devices of the invention may
be implanted at any desired location within the CNS. For example,
and without limitation, the polymer-based drug delivery device can
be implanted either in the brain (e.g., close to a site of damage
such as an ischemic region following stroke, or in the opposite
brain hemisphere), or in the base of the brain, in or near a
CSF-filled space such as ventricle, etc. In the case of a device
implanted into a CSF-filled space, the device releases the agent
into the CSF, allowing it to diffuse to a region of the brain
surround the space. Depending on the size of the device, it can
also be implanted at or adjacent to a nerve, nerve tract, ganglion,
etc., of the PNS. For example, microimplants can be implanted
within or internal to the epineurium or perineurium of a nerve.
Implantable Microchip-Based Delivery
[0200] In certain embodiments of the invention, one or more
agent(s) is delivered to the nervous system using an external or
implantable silicon or polymeric microchip, which contains from
dozens to up to hundreds or thousands of microreservoirs, each of
which can be filled with any combination of drugs, reagents, or
other chemicals. Micro-reservoirs can be opened at predetermined
times and/or on demand using preprogrammed microprocessors, remote
control, or biosensors. If desired, complex chemical release
patterns can be achieved using these approaches. In some
embodiments, micro-reservoirs have "caps" that degrade over time.
Release can be controlled by varying the thickness and/or
composition of the cap, thereby allowing release to occur at
predictable and substantially predetermined times. The cap material
can be, e.g., a degradable polymer. In some embodiments, the cap
material is non-degradable and is permeable to the molecules to be
delivered. The physical properties of the material used, its degree
of crosslinking, and its thickness will determine the time
necessary for the molecules to diffuse through the cap material. If
diffusion out of the release system is limiting, the cap material
delays release. If diffusion through the cap material is limiting,
the cap material determines the release rate of the molecules in
addition to delaying release time.
[0201] In some embodiments, the agent(s) to be delivered are
inserted into the reservoirs in their pure form, as a liquid
solution or gel, or they may be encapsulated within or by a release
material. The release material may be, for example, a biodegradable
or non-biodegradable polymer. Representative polymers include those
mentioned above (see, e.g., Santini et al., 2000; U.S. Pat. Nos.
5,797,898 and 6,808,522; and U.S. Patent Publications 2002/0072784,
2004/0166140, and 2005/0149000; for discussion of microchip-based
delivery systems). Microchips can be implanted at any desired
location in the CNS (as described above). Depending on the size of
the device, it can also be implanted at or adjacent to a nerve,
nerve tract, ganglion, etc., of the PNS. For example, microchips
can be implanted within or internal to the epineurium or
perineurium of a nerve.
Methods for Focal Delivery
[0202] In certain embodiments of the invention, compositions
comprising a plasticity-modifying agent and optionally a
proteolysis enhancing agent are administered to a subject by focal
delivery. Focal delivery may be accomplished in a number of
different ways. Implantation of a polymer-based drug delivery
device or microchip such as those described above at a site within
the central nervous system or within or adjacent to a nerve, nerve
tract, or ganglion within the peripheral nervous system is a
suitable method to achieve focal delivery.
[0203] Internal (implantable) or external pumps can be employed for
administering a substantially fluid composition of the invention.
Such pumps typically include a drug reservoir from which continuous
or intermittent release occurs into the target tissue or in the
vicinity thereof via a catheter. In certain embodiments of the
invention, treatment is carried out using an implantable pump and a
catheter having a proximal end coupled to the pump and having a
discharge portion for infusing therapeutic dosages of one or more
agents described herein into a predetermined infusion site in brain
tissue or into the spinal canal (intrathecal delivery).
[0204] Infusion (which term is used to refer to administration of a
substantially fluid material to a location in the body by means
other than injection) may be carried out in a continuous or nearly
continuous manner, or may be intermittent. The pump may be
programmed to release predetermined amounts of the agent at
predetermined time intervals. U.S. Pat. No. 4,692,147 (assigned to
Medtronic, Inc., Minneapolis, Minn.) describes a suitable pump. In
certain embodiments one or more of the infusion systems known as
the Synchromed.RTM. Infusion System (manufactured by Medtronic,
Inc., Minneapolis, Minn.; see web site having URL
www.medtronic.com) is used. However, it will be appreciated that
the pump may take the form of any device used for moving fluid from
a reservoir. Mechanical, pressure-based, osmotic, or electrokinetic
means may be used.
[0205] In order to deliver an agent to the brain parenchyma, a
catheter attached to the pump may be implanted so that the
discharge portion lies in the brain parenchyma (see, e.g., U.S.
Pat. No. 6,263,237 for description of a variety of suitable systems
and methods for implanting them into the body of a subject and
directing the administration of an active agent to a desired
location in the brain). Continuous ICM is a relatively new
technique of regional delivery of therapeutic agents directly into
brain parenchyma, which establishes a bulk flow current that has
the potential to homogeneously distribute even large molecules
(see, e.g., Laske, 1997 for an example of administration of an
agent to a region within the brain).
[0206] In certain embodiments of the invention, the agent is
delivered to one or more of the CSF-containing cavities or chambers
of the central nervous system, e.g., the ventricles or cisterna
magna, which is located at the bottom of the skull. As is well
known in the art, there are two lateral ventricles and midline
third and fourth ventricles within the brain. To deliver an agent
to a ventricle or the cisterna magna using an infusion pump, the
catheter may be implanted so that the discharge portion lies in the
ventricle or the cisterna. The agent diffuses out of the ventricle
or cisterna magna. Delivery to these locations therefore allows
delivery of the agent to a relatively wide area of the brain rather
than localizing it more closely to a specific site.
Intraventricular or intracisternal administration is considered to
be administration to the nervous system. In certain embodiments of
the invention delivery to a CSF-containing space, e.g., a
ventricle, is accomplished by surgically implanting a catheter
through the skull so that the tip has access to the space. The
other end of the catheter is then connected to a reservoir (e.g.,
an Ommaya reservoir), which is placed beneath the scalp (i.e.,
subcutaneously). This method is in use for delivery of
chemotherapeutic agents (see, e.g., Ommaya and Punjab, 1963;
Galicich and Guido, 1974; Machado, 1985; Obbens, 1985; and
Al-Anazi, 2000).
[0207] If the subject suffers from damage to the spinal cord, the
catheter is implanted so that the discharge portion lies in an
intrathecal space of the spinal cord while the other end is
connected to the pump reservoir. Methods for administering agents
to the spinal fluid (i.e., intrathecally) are well known in the
art. Such methods are commonly used in the treatment of chronic
pain, and are routinely used to deliver analgesic agents over a
period of months. Similar methods are of use in the present
invention (see, e.g., Lamer, 1994; Paice, 1996; Winkemuller, 1996;
Tutak, 1996; and Roberts, 2001 for descriptions of the use of
implantable pumps for delivery of a variety of different
therapeutic agents for treatment of a number of different
conditions).
[0208] For delivery to the PNS, suitable methods include injection
or infiltration into a nerve or nerve trunk, e.g., adjacent to a
site of nerve damage, and implantation of a polymer-based delivery
device or microchip either adjacent to a site of nerve damage.
Methods for administering anesthetic agents to diverse nerves,
nerve bundles, etc., within the PNS are well known in the art, and
any of these methods are applicable in the context of the present
invention.
[0209] In certain embodiments of the invention, a solution
comprising a polymer, a plasticity-modifying agent, and optionally
one or more additional active agents is administered by injection
or infusion using any of the means described above. The polymer
assembles to form a gel upon administration, e.g., following
contact with physiological fluids. Such assembly may, for example,
be triggered by exposure to monovalent or divalent cations. For
example, U.S. Publication 2002/0160471 describes self-assembling
peptides that form hydrogels. U.S. Pat. No. 6,129,761 describes a
variety of different self-assembling polymers and polymers that
require a polymerizing agent or cross-linking agent to facilitate
assembly. Certain of these polymers assemble to form hydrogel
structures upon contact with physiological fluids following
administration to a subject. In another embodiment a collagen-based
system is used (see, e.g., PCT Publication WO 00/47130, which
describes injectable collagen-based systems for delivery of cells
or therapeutic agents).
Delivery Location, Timing, Duration of Treatment, and Dose
[0210] The plasticity-modifying agent(s) can be administered using
any route of administration, e.g., oral, intravenous,
intraperitoneal, intramuscular, intradermal, transdermal,
subcutaneous, pulmonary (e.g., by inhalation into the lungs),
nasal, etc. The route and dose will be selected so as to achieve
effective concentrations in the nervous system without undue side
effects.
[0211] The location at which a composition of the invention is to
be administered or implanted may be selected with relation to the
particular condition being treated. For example, if the subject has
suffered an injury or damage to the brain, e.g., as a result of
stroke, trauma, etc., the composition may be delivered to the brain
parenchyma or to one or more of the ventricles of the brain or to
the cisterna magna. If the subject has suffered an injury or damage
to the spinal cord, a composition of the invention may be delivered
to the spinal cord, e.g., by implanting or administering a
composition within the spinal canal. If the plasticity-modifying
agent or an inventive composition crosses the blood-brain barrier,
it can be delivered systemically, e.g., by oral, intravenous,
intraperitoneal, intramuscular, intradermal, transdermal,
subcutaneous, pulmonary (e.g., by inhalation into the lungs),
nasal, etc. administration.
[0212] The area to which the agent is to be administered may be,
for example, an area that has been damaged (e.g., an ischemic
lesion) or an area adjacent to an area that has been damaged. The
agent(s) may be administered to any region, nucleus, or functional
area within the brain including, but not limited to, any of the
major subdivisions of the brain (cortex, hippocampus, cerebellum,
thalamus, midbrain, brain stem), which include motor cortex,
sensory cortex including visual cortex, auditory cortex, and
somatosensory cortex, language areas of cortex, frontal cortex,
internal capsule, basal ganglia, thalamus, and/or other area noted
above, etc. As noted above, numerous specific areas within the
brain have been defined based on anatomical and histological
considerations. In addition, areas in the brain that are
responsible for performing various tasks have been defined on
functional grounds and are well known in the art (see, e.g.,
Kandel, supra; and Victor and Ropper, supra).
[0213] In certain embodiments of the invention, the area that has
been damaged is identified. The area that has been damaged can be
identified using a variety of different imaging techniques known in
the art. For example, and without limitation, suitable methods
include imaging techniques such as magnetic resonance imaging
(MRI), optionally imaging features associated with blood flow such
as perfusion, diffusion, or both, computed tomography (CT),
positron emission tomography (PET), ultrasound, etc. Imaging
techniques that image structure and/or function are available.
Functional studies can be performed, e.g., using labeled substrates
such as glucose to identify regions of the brain that are
metabolically inactive and/or that do not respond to stimulation,
suggesting that they are functionally inactive (see, e.g., Grossman
and Yousem, supra).
[0214] Clinical diagnosis can be used instead of, or in addition
to, imaging techniques. For example, the area to which damage has
occurred can be identified by performing a neurological
examination. Deficits noted on the neurological examination can be
correlated with damage to particular areas of the central and/or
peripheral nervous system (Kandel, supra; and Victor and Ropper,
supra). In certain conditions, such as neuropsychiatric disorders
of developmental or adult origin; a genetic test may be used in
addition to a clinical diagnosis.
[0215] Any of the foregoing methods can be utilized acutely (e.g.,
within hours to a few days of a damaging event such as stroke or
injury) or at later times (e.g., several days to weeks, months, or
years following the event). The characteristic evolution of the
appearance of nervous system lesions is well known in the art, so
the practitioner can readily identify the location of damaged
tissue at any desired time point relative to the time at which the
event causing the damage occurred.
[0216] In certain embodiments of the invention, the agent is
delivered at or adjacent to a site where tissue necrosis and/or
scar tissue formation has occurred in the CNS. Areas of necrosis
can be identified using various imaging techniques such as those
mentioned above. Symptoms may also be used to guide selection of an
appropriate location at which to implant the matrix. For example,
if a subject experiences impairment of a particular function such
as movement, sensation, speech, etc., then the portion of the brain
that is normally responsible for control or achievement of that
function, or the corresponding area on the contralateral side of
the subject's body, may be selected as a suitable site for
implantation of a drug delivery device of the invention. Standard
surgical techniques can be used.
[0217] In some embodiments of the invention the agent is
administered to an area adjacent to a region that has been damaged
by an infarct, e.g., to the peri-infarct area. Without wishing to
be bound by any theory, peri-infarct regions are likely to be sites
of clinically relevant cortical remodeling following stroke. For
example, the agent may be administered to a site that is located up
to approximately 0.5 cm from the edge of an infarcted area, up to
1.0 cm from the edge of an infarcted area, or up to 2 cm from the
edge of an infarcted area. In some embodiments the agent is
administered to a site immediately adjacent to an infarcted area,
e.g., up to 0.5 cm from the edge of the infarcted area. In some
embodiments of the invention the agent is administered to the
ischemic penumbra adjacent to an area of severe ischemia following
stroke (see, e.g., Furlan et al., 1996). The ischemic penumbra is a
region of brain tissue that experiences mild to moderate ischemia
but remains viable for a period of time following a stroke (e.g.,
up to several hours or longer) and may be salvageable if perfusion
is re-established and/or through the use of neuroprotective agents.
The ischemic penumbra may be operationally defined using, e.g.,
diffusion and perfusion MRI (Schlaug et al., 1999; and Kidwell et
al. 2003). One of ordinary skill in the art will be able to select
an appropriate definition and measurement technique.
[0218] In some embodiments of the invention, the agent is
administered to a location on the opposite side of the brain from
the side where damage has occurred. The site of administration may
be substantially symmetrically located with respect to the region
that has been damaged. Without wishing to be bound by any theory,
it is possible that following damage to a particular region of the
brain, the contralaterally located region reorganizes so as to
assume responsibility for functions that were previously performed
by the damaged region. For example, a portion Of the brain that
normally (e.g. prior to injury) generates movement commands for the
left hand only may reorganize so as to generate commands to both
hands following damage to a portion of the brain that previously
commanded the right hand.
[0219] As mentioned above, delivery by injection or infusion pump
is suitable for compositions in which an agent of the invention is
dissolved in a liquid and for compositions comprising
microparticles of suitable dimensions. The polymer-based drug
delivery devices of the invention will typically be implanted into
the subject in an appropriate location in the nervous system so
that they will release the active agent at a desired location. For
example, they may be implanted into the brain parenchyma. They may
also be implanted into a ventricle or into the spinal canal in
various embodiments of the invention. The location for implantation
is selected so as to achieve an effective concentration of the
active agent at a desired location in the nervous system, i.e.,
typically reasonably close to the location at which it is desired
to achieve the effective concentration. Care is taken to avoid
disrupting undamaged portions of the nervous system to the extent
possible. Imaging may be used to guide administration or
implantation of the compositions and drug delivery devices of the
invention, e.g., they may be administered or implanted under
stereotactic guidance.
[0220] The agent(s) can be administered in a continuous or
intermittent fashion. Intermittent or pulsatile delivery may be
performed at times selected in accordance with the active half-life
of the agent in order to maintain a therapeutically useful dose
and/or may be performed in accordance with physiological patterns
such as circadian rhythms, or during periods when the subject
either is or is not engaged in particular activities. If the agent
is administered using an implanted device such as a pump or
microchip, an external controller may be used to trigger release at
a desired time, or the device can be programmed to release the
agent at particular times or intervals.
[0221] In some embodiments, compositions of the invention may be
administered to a subject following an event that damages the brain
or spinal cord or following diagnosis of a neuropsychiatric or
neurodevelopmental disorder for a finite period of time. For
example, compositions of the invention may be administered to a
subject for up to 1 week, up to 4 weeks, up to 2 months, up to 6
months, up to 12 months, up to 18 months, up to 2 years, up to 5
years, up to 10 years, up to 20 years, or even longer. In some
embodiments, compositions of the invention may be administered to a
subject following an event that damages the brain or spinal cord or
following diagnosis of a neuropsychiatric or neurodevelopmental
disorder for the rest of the subject's life.
[0222] In some embodiments, compositions of the invention are not
administered immediately after an event that damages the brain or
spinal cord or following diagnosis of a neuropsychiatric or
neurodevelopmental disorder. To give but a few examples,
administration may be initiated after certain other therapeutic
strategies (e.g. behavioral therapies) have been performed; after
the subject has reached a desired level of health; after the
subject has reached a desired age; etc. In some embodiments,
compositions of the invention are administered at least 1 week, at
least 4 weeks, at least 2 months, at least 6 months, at least 12
months, at least 18 months, at least 2 years, at least 5 years, at
least 10 years, at least 20 years, or even longer, after an event
that damages the brain or spinal cord or following diagnosis of a
neuropsychiatric or neurodevelopmental disorder.
[0223] In some embodiments, compositions of the invention may be
administered for a period of time and may then be discontinued. For
example, administration may be discontinued when the subject
responds to the administration (e.g. if symptoms improve, if damage
is reversed, if plasticity has been modified, if function has been
restored to the nervous system, if neural development has been
stimulated, etc.). To give another example, administration may be
discontinued when the subject has reached at least one desired
endpoint or treatment milestone. In some embodiments, compositions
of the invention may be administered to a subject for up to 1 week,
up to 4 weeks, up to 2 months, up to 6 months, up to 12 months, up
to 18 months, up to 2 years, up to 5 years, up to 10 years, up to
20 years, or even longer, before being discontinued. In some
embodiments, administration of compositions of the invention that
has been discontinued may be resumed at any point in time after
discontinuing the administration. To give but one hypothetical
example, (i) a plasticity-modifying agent may be administered to a
subject following diagnosis with a neurodevelopmental disorder;
(ii) the subject's symptoms may disappear; (iii) administration of
the plasticity-modifying agent may be discontinued; (iv) the
symptoms may return; and (v) administration of the
plasticity-modifying agent may be resumed. In some embodiments,
administration may be discontinued for up to 4 weeks, up to 2
months, up to 6 months, up to 12 months, up to 18 months, up to 2
years, up to 5 years, up to 10 years, up to 20 years, or even
longer, before administration is resumed.
[0224] In certain embodiments of the invention, the compositions of
the invention are administered at times varying from immediately
after to considerably after, e.g., least 3 hours after, the onset
or occurrence of a damaging event such as a stroke or injury. For
example, the initial administration may be a few minutes to hours,
e.g., at least 6, 12, 24, 36, or 48 hours after the onset or
occurrence of a damaging event. In certain embodiments of the
invention the initial administration is between 24 hours and 1 week
after the onset or occurrence of a damaging event, between 1 week
and 1 month after the onset or occurrence of a damaging event, or
between 1 and 3 months, 3 and 6 months, 6 and 12 months after the
onset or occurrence of a damaging event, etc. The initial
administration may occur at times greater than 1 year following the
onset or occurrence of a specific damaging event, e.g., between 1-5
years, etc. In some embodiments of the invention the initial
administration occurs after the subject has reached a plateau of
functional recovery. For example, the subject may have failed to
display improvement on one or more standardized tests, or may have
failed to experience subjective improvement during the preceding
1-3 months, 3-6 months, or longer. For treatment of
neuropsychiatric disorders, neurodegenerative diseases, nutrient
deprivation, neoplastic diseases, and other conditions for which
there is no specific identifiable damaging event, administration
can occur at any time following diagnosis of the disease.
[0225] The total time period during which treatment occurs, and the
number of treatments within such time period, can vary. The total
duration of treatment (i.e., the time interval between the first
and the last treatment) can range from days to weeks, months, or
years. For example, the total duration may be 1 day; 1 week; 4
weeks; 1, 3, 6, 9, or 12 months, between 1 and 2 years; 2 and 5
years; 2 and 10 years; 2 and 20 years; etc. If the agent is
administered in discrete doses in addition to or instead of being
administered continuously, subjects may receive anywhere from a
single dose to dozens or even hundreds or thousands of doses. The
time interval between doses can be varied. It may, for example, be
desirable to administer the agent for a defined time period each
day, e.g., 10 minutes/day, 1 hr/day, etc.
[0226] The dose of the plasticity-modifying agent will be selected
taking into account the particular agent, the condition being
treated, the route of administration, and other relevant factors.
The dose (or doses) may be, e.g., an amount effective to promote
growth or sprouting of axons, promote structural reorganization of
synaptic connections, increase formation of new synaptic
connections, increase dendritic spine motility, inhibit structural
or functional degeneration (e.g., degeneration that would otherwise
be expected to take place) or any combination of the foregoing. The
dose may range from about 0.001 to 100 mg/kg body weight, e.g. from
about 0.01 to 25 mg/kg body weight. The dose may, for example,
range between 1 .mu.g/kg and 100 mg/kg, e.g., between 10 .mu.g/kg
and 10 mg/kg. Exemplary doses range from 0.1 to 20 mg/kg body
weight, e.g., about 1 to 10 mg/kg.
[0227] The dose of the proteolysis-enhancing agent will be selected
to enhance the effect of the plasticity-modifying agent. Typically
the dose for each administration of the proteolysis-enhancing agent
will be significantly lower than the dose that would be required to
cause lysis of a significant blood clot when administered to the
vascular system. Exemplary, non-limiting doses ranges for a
proteolysis-enhancing agent, e.g., tPA, include one or more of the
following: (i) a dose sufficient to achieve a concentration of
between 10 and 100,000 IU/ml or between 100 and 10,000 IU/ml or
between 100 and 1,000 IU/ml in the extracellular fluid or in a
CSF-containing cavity such as a ventricle or the spinal canal; a
dose between 1 .mu.g/day and 10 mg/day; a dose between 1 .mu.g/day
and 1 mg/day; a dose 5 .mu.g/day and 500 .mu.g/day; a dose between
10 .mu.g/day and 100 .mu.g/day, etc.
[0228] Various dosing regimens may be used. For example, it may be
desirable to give a relatively large "loading dose" initially and
then administer smaller doses either continuously or intermittently
so as to maintain an effective concentration in the region of the
nervous system being treated. It will also be appreciated that, in
general, the more focally directed the delivery, the lesser the
total dose that may be required. Thus direct administration via a
catheter to a specific brain region may require a lower total dose
than delivery to a ventricle. Furthermore, the larger the area of
damage and/or the greater the amount of reorganization and/or
recovery required, the larger might be the dose.
[0229] If desired, the concentration of the plasticity-modifying
agent (or any other agent whose administration is contemplated in
the present invention) can be monitored, e.g., in the CSF of the
subject. The dose can be adjusted accordingly to obtain a desired
concentration.
[0230] In certain embodiments of the invention the agent(s) is/are
administered, e.g., released, in a defined temporal relation to
rehabilitative therapy, e.g., during, prior to, or following
engagement of the subject in one or more rehabilitative activities.
The agent(s) may, for example, be administered up to 5 minutes to
12 hours prior to the activity, up to 5 minutes to 12 hours after
the activity, during the activity, or immediately prior to or
immediately following the start of a therapy session, e.g., up to 5
minutes prior to the beginning of a therapy session or up to 5
minutes following the start of a therapy session. By "therapy
session" is meant any period of time in which the subject is
engaged in performing activities that have been suggested or
prescribed by a health care provider for purposes of assisting the
functional recovery of the subject following damage to the CNS or
PNS or for improving the functioning of a subject suffering from a
neurodevelopmental disorder. The health care provider need not be
present during the therapy session, e.g., the subject may perform
the activities independently or with the assistance of personnel
other than a health care provider.
Administration of Additional Active Agent(s), Cells, and Gene
Therapy
[0231] In various embodiments of the invention, one or more
additional active agents is administered to the subject in
conjunction with administration of the plasticity-modifying agent
and, optionally, the proteolysis-enhancing agent. The additional
active agents may be administered concurrently or sequentially. The
additional active agent may be delivered focally but may
alternatively be administered systemically using any suitable route
of administration (e.g., oral, intravenous, intramuscular,
subcutaneous, transdermal, pulmonary, nasal, etc.). The additional
active agent may be delivered in the same solution or dosage form
as the proteolysis-enhancing agent. The additional active agent may
be incorporated into a polymeric matrix together with the
proteolysis-enhancing agent and delivered via a polymer-based drug
delivery device or delivered using a pump or any other delivery
system disclosed herein.
[0232] In some embodiments of the invention an agent other than a
proteolytic agent is administered, wherein the agent cleaves one or
more components of the extracellular matrix at a bond other than a
peptide bond. For example, the agent may cleave a polysaccharide
portion of an ECM component such as a proteoglycan or
glycosaminoglycan. Examples of suitable agents include
chondroitinases (which cleave chondroitin sulfate and hyaluronic
acid), hyaluronidases, heparinases (which cleave heparin),
heparanase (which cleaves heparan sulfate), etc.
[0233] In certain embodiments of the invention, the additional
active agent is a neural growth enhancing agent. A neural growth
enhancing agent is any molecule or cell that promotes, enhances,
increases, etc., one or more aspects of the growth or regeneration
of neural tissue. For example, the molecule or cell may promote
axon growth. A neural growth enhancing agent, as used herein, can
be a neurally active growth factor, neurotransmitter or
neurotransmitter analog, neurally active metal, modulator of a
synaptic signaling molecule, or cell. It will be understood that
typically "cell," as used in this context, refers to multiple
cells. The term "neurally active" means that the agent exerts a
biological effect on neural tissue. For example, the agent may
exert an effect that enhances structural and/or functional nervous
system reorganization or recovery.
[0234] The invention therefore provides compositions comprising a
plasticity-modifying agent, a neural growth enhancing agent, and,
optionally a proteolysis-enhancing agent. The invention provides
drug delivery devices comprising the composition. The drug delivery
device can be, for example, any of the drug delivery devices
described herein.
[0235] The invention further provides methods for promoting
recovery or reorganization in the nervous system of a subject
comprising the step of: administering a plasticity-modifying agent,
a neural growth enhancing agent, and, optionally a
proteolysis-enhancing agent to a subject in need of enhancement of
recovery or reorganization of the nervous system. The subject is
typically in need of recovery or reorganization of the nervous
system as a result of ischemic, hemorrhagic, neoplastic,
degenerative, traumatic, and/or neurodevelopmental damage to the
nervous system. The invention provides methods of treating a
subject in need of enhancement of recovery or reorganization in the
nervous system comprising the step of: administering a
plasticity-modifying agent, a neural growth enhancing agent, and,
optionally a proteolysis-enhancing agent to the subject. The
subject is typically in need of enhancement of recovery or
reorganization of the nervous system as a result of ischemic,
hemorrhagic, neoplastic, degenerative, traumatic, and/or
neurodevelopmental damage to the nervous system. Any of the agents
in the aforementioned methods can be administered focally to the
central or peripheral nervous system either individually or in
combination using any of the methods described herein. Either or
both of the agents can be administered by any alternate route of
administration. Certain features of this aspect of the invention,
e.g., dose ranges, adjunct therapy, etc., can be similar to those
described for other aspects of the invention.
[0236] Neurally active growth factors include, but are not limited
to, nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin-1 (NT-3), neurotrophin-4/5 (NT-4/5), ciliary
neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial
cell derived growth factor (GDNF), neurturin, artemin, persephin,
acidic or basic fibroblast growth factor (aFGF, bFGF), osteogenic
protein-1 (OP-1), vascular endothelial growth factor (VEGF),
erythropoietin (EPO), and granulocyte colony stimulating factor
(G-CSF).
[0237] "Synaptic signaling molecules" refer to endogenous molecules
that are activated downstream of calcium entry into cells through
synaptic activation or following release of calcium from
intracellular stores and that transduce electrical activity into
structural changes in neurons. These include a variety of kinases
such as calcium/calmodulin-dependent protein kinase II and IV,
protein kinase C (PKC), protein kinase A (PKA), extracellular
signal regulated kinase (ERK), cyclic AMP (cAMP) dependent kinase,
along with molecules such as cyclic AMP response element binding
protein (CREB), activity regulated cytoskeletal associated protein
(arc), troponin C; and Rac and Rho pathways and their associated
kinases. G protein coupled receptors transduce information from the
extracellular space to intracellular signals (among other
activities) and are also considered to be synaptic signaling
molecules. Modulators (Le., agents that activate or inhibit) of a
number of these signaling molecules are known in the art and are of
use in the present invention. Molecules that can bind to G protein
coupled receptors importantly include those that can activate or
inhibit (a) PKA and cAMP; (b) cyclic GMP, and (c) PKC. Pathways
downstream of GPCR activation importantly regulate CREB, BDNF,
actin, reorganization of the dendritic and axonal cytoskeleton,
etc. By way of example, activators of CAMP include Sp-cAMPS
(Sigma), which may to be delivered into the brain at a typical dose
of 0.02-0.5 .mu.g/kg/day, and Rolipram.RTM. (Sigma), which can be
given intramuscularly at a dose of 1-100 .mu.g/kg/day (Ramos et
al., Neuron 2003). Rolipram is a phosphodiesterase inhibitor, which
prevents breakdown of cAMP. Inhibition of cAMP can also, under
certain conditions, have a stimulatory effect on synapses and is of
use in certain-embodiments of the invention. Inhibitors of cAMP
include Rp-cAMPS (Sigma), which can be delivered into the brain at
a typical dose of 0.02-0.5 .mu.g/kg/day (Ramos et al., 2003).
[0238] An activator of cGMP is 8-Br-cGMP; an inhibitor is Rp-cGMPs.
Both are typically delivered focally. Effective doses on neurite
growth and dynamics in brain slices are about 10-100 .mu.M
(Nishiyama et al., 2003). Another inhibitor is ODQ; an effective
dose for influencing axon growth is about 10 .mu.M (Leamey et al.,
2001). Activators of PKC include diacylglycerol and
phosphatidylserine. An inhibitor is a drug called GF109203X (GFX).
Effective doses in slices are approximately 10-100 .mu.M (Nishiyama
et al., 2003).
[0239] It is noted that doses presented here should in no way be
considered limiting. In general, the invention encompasses doses at
least 10 to 100 fold lower than those described here, and doses up
to the maximum tolerated dose of the agent, as consistent with
sound medical judgment. Furthermore, dosage routes for specific
agents are mentioned here by way of example and are not intended to
be limiting. In general, any suitable route of administration can
be used. In particular, any of these agents may be administered
using the methods for focal administration described herein.
[0240] Neurally active small molecules include a number of the
modulators and neurotransmitters described above as well as diverse
compounds known in the art to influence nervous system function
(see, e.g., Goodman and Gilman, supra; and Kandel, supra).
[0241] Neurotransmitters are naturally occurring compounds that
generally fall into the categories of small molecules (e.g.,
catecholamines) and peptides. A neurotransmitter for use in the
present invention can be excitatory or inhibitory. Exemplary
neurotransmitters include, but are not limited to, acetylcholine,
dopamine, serotonin, glycine, glutamate, epinephrine,
norepinephrine, and gamma aminobutyric acid (GABA). A
neurotransmitter analog as used herein is a compound other than a
naturally occurring neurotransmitter that exerts an excitatory or
inhibitory effect on a neurotransmitter receptor. The analog will
typically bear a structural resemblance to a naturally occurring
neurotransmitter and will compete with it for binding to its
receptor.
[0242] Neurally active metals include magnesium and zinc. The
magnesium and/or zinc can be provided in any suitable form.
Typically the metal will be provided in the form of a salt that
contains a metal cation and an anion that serves as a counterion.
The counterion can be an organic or inorganic substance. For
example, the counterion can be phosphate, carbonate, gluconate,
citrate, sulfate, acetate, maltonate, oxalate, or any other
pharmaceutically acceptable ion such as those mentioned below. In
some embodiments the metal cation is provided as a chelate, in
which the metal cation is complexed with an organic molecule such
as a heterocyclic ring.
[0243] Gene therapy methods may be used to increase expression of
genes that encode products, e.g., plasticity-enhancing agents,
proteolysis-enhancing agents, and/or agents that promote nervous
system functional and/or structural reorganization and/or recovery.
Gene therapy encompasses delivery of nucleic acids comprising
templates for synthesis of a molecule of interest to a cell of
interest. The nucleic acid (or a nucleic acid derived from the
nucleic acid as, for example, by reverse transcription) may be
incorporated into the genome of the cell or remain permanently in
the cell as an episome. Gene therapy also encompasses delivery of
nucleic acids that do not integrate or remain permanently in the
cell to which they are delivered. Such approaches permit temporary
or transient synthesis of a molecule of interest. Methods and
materials for performing gene therapy are well known in the art and
will not be extensively reviewed here (see, e.g., Berry, 2001; Han,
2000; and Thomas and Klibanov, 2003).
[0244] Vectors and delivery vehicles (e.g., polymeric matrices)
that provide nucleic acids comprising templates for synthesis of
polypeptides may be incorporated into a composition of the
invention or administered separately. Typically, the nucleic acid
includes a coding sequence for a gene to be expressed in a cell of
interest and also includes appropriate expression signals, e.g.,
promoters, terminators, etc., to ensure proper expression.
[0245] In general, either viral or non-viral vectors may be used.
For example, herpes virus, adenovirus, adeno-associated virus,
retroviruses, or lentiviruses may be used. It may be desirable to
avoid the use of intact viruses in delivering templates to cells.
Thus it may be desirable to deliver DNA vectors or linear DNA
molecules. These vectors may, but need not, include viral sequences
such as long terminal repeats, etc. Any of a wide variety of agents
useful for transfection may be used to enhance uptake of nucleic
acids by cells. Vectors are taken up by cells in the nervous
system, and the polypeptide of interest is expressed and, usually
secreted.
[0246] In some embodiments of the invention, cells are administered
to a subject. In some embodiments of the invention, cells serve as
a source for a plasticity-enhancing agent. For example, the cells
may secrete IGF1 into the extracellular space. In certain
embodiments of the invention, cells are genetically modified prior
to their administration to increase their synthesis of a
plasticity-enhancing agent. For example, cells may be stably
transformed with a vector that comprises a template for
transcription of an RNA that encodes the agent. Cells may be
sequestered in a non-biodegradable reservoir or compartment that
retains them at a particular location and prevents their
integration with cells at the site of administration or their wider
dispersal.
[0247] In some embodiments of the invention, cells are administered
to a subject who may receive a composition comprising a
plasticity-modifying agent and optionally a proteolysis-enhancing
agent. In some embodiments cells contribute to structural and/or
functional recovery of the nervous system. Cells can be neurons,
glia, or non-neural cells. Suitable cells include, but are not
limited to, Schwann cells and olfactory ensheathing glia (Bunge,
2003). Cells can be of a single cell type, or combinations of
different cell types can be administered. Cells may replace or
supplement neural tissue that has been irreversibly damaged and/or
provide supportive functions. In some embodiments, neural stem
cells are administered. Multipotent neural stem cells, capable of
giving rise to both neurons and glia, line the cerebral ventricles
of all adult animals, including humans. Distinct populations of
nominally glial progenitor cells, which also have the capacity to
generate several cell types, are dispersed throughout the
subcortical white matter and cortex (Goldman 2005). In some
embodiments, adult or embryonic stem cells are administered. Such
cells can be derived from a location outside the nervous system,
e.g., the bone marrow, liver, umbilical cord, etc. Cells of any
type can be used. Cells can be autologous or non-autologous. In
certain embodiments, cells are from the same species as the
subject.
[0248] In certain embodiments of the invention the cells are
administered in a polymeric scaffold, made of certain of the
materials such as those described above that provide a hospitable
environment to maintain cell viability. The polymer material may be
biodegradable. The matrix or scaffold may be formed prior to
implantation into the nervous system of a subject or may form
following administration, e.g., upon contact with physiological
fluids. Encapsulation of cells in a variety of different polymeric
matrices or scaffolds is well known in the art (see, e.g., U.S.
Pat. Nos. 6,129,761 and 6,858,229; U.S. Patent Publication
2002/0160471; and Teng, 2002).
[0249] In addition to or instead of the various active agents
described above, which are selected primarily based on their useful
properties for enhancing structural or functional recovery or
reorganization in the nervous system, various other substances can
be administered. Such substances include, but are not limited to,
antibiotics or antifungal agents to treat or reduce the risk of
infection, chemotherapeutic agents to treat tumors, etc.
[0250] It is to be understood that the invention explicitly
includes compositions comprising each specific combination of any
of the proteolysis-enhancing agents described herein, optionally in
combination with any of the proteolysis-enhancing agents described
herein and/or any of the additional active agents described herein.
Because it would not be practical to list each and every
combination, only a few examples are provided here. For example,
the invention includes a composition comprising IFN.gamma. and tPA.
The composition may further include a neurally active growth factor
(e.g., BDNF). The invention also includes a composition comprising
tPA and a modulator of a synaptic signaling molecule (e.g., tPA and
Rolipram); a composition comprising tPA and a neurotransmitter
(e.g., tPA and serotonin); a composition comprising tPA and a
neurally active metal (e.g., tPA and magnesium); a composition
comprising tPA and a neurally active small molecule; a composition
comprising tPA and a cell (e.g., tPA and a neural stein cell), etc.
Similarly, the invention includes compositions comprising (i)
plasmin and (ii) a neurally active growth factor, a synaptic
signaling molecule, a neurotransmitter, a neurally active metal,
and/or a cell. Compositions comprising 3, 4, 5, or more of the
proteolysis-enhancing agents and/or additional agents are
encompassed. The invention provides a polymer-based drug delivery
device comprising any of these compositions and an implantable
microchip comprising any of these compositions or designed to
administer the agents individually.
[0251] The invention encompasses administration of one or more of
any of the proteolysis-enhancing agents described herein in
conjunction with one or more of any of the additional agents
described herein to a subject in need of reorganization and/or
recovery of the nervous system. The subject has typically
experienced ischemic, hemorrhagic, neoplastic, traumatic,
degenerative, and/or neurodevelopmental damage to the central or
peripheral nervous system. Agents can be administered together or
separately. In some embodiments both the proteolysis-enhancing
agent(s) and the additional agent(s) are administered focally. In
some embodiments, the proteolysis-enhancing agent(s) are
administered focally to the nervous system and the additional
agent(s) are administered by an alternate route (e.g.,
intravenously or orally).
Therapeutic Applications and Adjunct Therapy
[0252] The compositions and methods of the invention are of use in
treating subjects who have experienced events such as stroke or
injury (e.g., due to accident or surgery). The compositions and
methods of the invention find use for treating subjects suffering
from a variety of other diseases and conditions including, but not
limited to, neurodegenerative diseases such as multiple sclerosis,
amyotrophic lateral sclerosis, subacute sclerosing panencephalitis,
Parkinson's disease, Huntington's disease, muscular dystrophy, and
conditions caused by nutrient deprivation or toxins (e.g.,
neurotoxins, drugs of abuse). Certain of the compositions and
methods are of use for treating neurodevelopmental diseases such as
autism or dyslexia, i.e., diseases in which at least a portion of
the nervous system fails to develop normal structure and/or
function. Certain of the compositions and methods are of use for
treating neuropsychiatric diseases such as schizophrenia and
bipolar disorders, i.e., diseases in which at least a portion of
the nervous system fails to achieve its typical level of cognitive
function. Certain of the compositions and methods are of use for
providing cognitive enhancement and/or for treating cognitive
decline, e.g., "benign senescent forgetfulness," "age-associated
memory impairment," "age-associated cognitive decline," etc.
(Petersen 2001; Burns 2002). These terms are intended to reflect
the extremes associated with normal aging rather than a precursor
to pathologic forms of memory impairment. Thus these conditions are
distinct from Alzheimer's disease. Certain of the compositions and
methods are of use for treating Alzheimer's disease. In certain
embodiments of the invention, the subject does not have, e.g., has
not been diagnosed with, Alzheimer's disease. In certain
embodiments of the invention the subject is not suspected of having
Alzheimer's disease. In certain embodiments of the invention the
subject has not been identified as having an increased risk for
developing Alzheimer's disease. Methods for treating or preventing
Alzheimer's disease, to the extent that any such methods are
described and/or enabled in PCT Publication WO 01/58476 are
explicitly excluded from certain embodiments of the instant
invention.
[0253] Any of a wide variety of functional impairments may be
treated using the compositions and methods of the invention. In
some embodiments, compositions are used to promote restoration of
respiratory function after spinal cord injury (SCI). For this
purpose, compositions are typically administered to the spinal
cord, e.g., intrathecally. If desired, administration can be
localized to the region of the spinal cord injury, e.g., the
cervical region of the spinal cord. Respiratory disorders are the
leading cause of morbidity and mortality after SCI, affecting
nearly half of all patients with a neurological deficit after SCI.
Respiratory impairments resulting from cervical SCI, the most
common clinical case, frequently render survivors chronically or
permanently ventilator dependent, a sequelae which can dramatically
compromise quality of life. There are no drug treatments for
breathing disorders associated with SCI. Studies have established
that the breathing system possesses a highly dynamic system of
neuroplasticity which manifests both at the developmental stage as
well as at the adulthood. Work in the laboratory of one of the
inventors has demonstrated that even with nearly 50% phrenic
respiratory motor region loss in the adult rat spinal cord,
respiratory function can recover spontaneously in 5-6 weeks after a
mid-cervical spinal cord injury. While the ultimate outcome from
this neuroplasticity-mediated event is encouraging, the required
lengthy period imposes serious life or death challenges to SCI
patients. The present invention may significantly stimulate
post-SCI respiratory neural circuit reorganization, and thus may
quickly restore respiratory function after incomplete spinal cord
transection, which is a frequent clinical occurrence.
[0254] Surgery for various conditions can sometimes result in
damage to nerves. In some embodiments of the invention, the
compositions and methods are used to regenerate, repair or
otherwise restore function after nerves of the PNS supplying
muscles, organs, or other parts of the body, or carrying
information from a part of the body, have been necessarily or
accidentally disconnected or damaged during surgery. In some
embodiments, the present invention is used to regenerate, repair or
prevent degeneration of nerves, e.g., nerves supplied by the spinal
cord to the muscles, organs, or other parts of the body, or that
enter the spinal cord from sensory receptors from the body. Some
embodiments include regeneration or repair of damaged or
degenerated nerves in the CNS, for example the optic nerve or the
auditory nerve, or prevention of degeneration of axon tracts or
fiber bundles in the CNS due to diseases, disorders, and/or damage.
These embodiments include, but are not limited to, the regrowth,
recovery, repair or prevention of degeneration of ascending or
descending fiber tracts and connections in the spinal cord, and of
fiber tracts and connections in other structural and functional
subdivisions of the CNS. Some embodiments include rewiring or
reorganizing brain pathways so as to elicit novel functions from
existing brain regions. An example of this embodiment is
enhancement of brain function, particularly when coupled with
practice regimens that engage specific brain regions.
[0255] In certain embodiments of the invention, the subject to whom
a composition of the invention is administered is engaged in a
program of rehabilitative therapy or training. Such programs
typically ensue after injury or stroke, but also include programs
of remediation and training in a variety of disorders of
developmental or adult onset. Such programs are commonly employed
in disorders such as dyslexia, autism, Asperger's Syndrome,
Pervasive Developmental Disorders--Not Otherwise Specified,
Tourette's Syndrome, Personality Disorders, Schizophrenia and
related disorders (see, e.g., Diagnostic and Statistical Manual of
Mental Disorders, 4th Ed., DSM-IV, American Psychiatric
Association, 1994, Diagnostic and Statistical Manual, Am.
Psychiatric Assoc., Washington, D.C. for discussion of these
disorders). Numerous rehabilitation programs for victims of stroke,
spinal cord injury, and/or other forms of nervous system damage are
known to those skilled in the art, and the subject can be engaged
in any such program (see, e.g., Gillen and Burkhardt, supra, for a
discussion of suitable programs for victims of stroke). Similar
programs may be used for victims of other forms of damage to the
brain (see, e.g., Somers, supra, for a discussion of suitable
programs for victims of spinal cord damage). Suitable programs for
individuals suffering from damage to the PNS are also known in the
art. A rehabilitation program is typically designed and recommended
by a health care provider with knowledge in the area of
rehabilitative therapy. Therapy sessions may involve the
participation of a health care provider. However, the subject may
also engage in sessions or tasks associated with the program
without the assistance or supervision of the health care
provider.
[0256] The subject can be engaged in the program in a defined
temporal relation with respect to the administration of the agent.
For example, the subject can be engaged in the program during a
time period in which the agent is being administered and/or during
which the agent is present in effective amounts in the nervous
system. In some embodiments, a dose of the agent is administered
within a defined time period prior to engagement of the subject in
a particular rehabilitative session or task. For example, the agent
may be administered and/or may be present in an effective amount at
any time up to 24 hours, 48 hours, or up to 1 week prior to the
time at which the subject will be engaged in the session or task,
or the agent may be administered and/or may be present in an
effective amount at any time up to 24 hours, 48 hours, or up to 1
week following completion of the session or task. Typically the
subject will be engaged in the program over a period of weeks,
months, or years, i.e., the subject will participate in multiple
therapy sessions over a period of time. The subject's participation
in such sessions can be coordinated with administration of the
agent so as to achieve an optimal effect. The beneficial effects of
rehabilitative therapy may at least in part be due to structural
and/or functional reorganization that occurs as a result of such
therapy. Without wishing to be bound by any theory, the inventors
propose that the proteolysis-enhancing activities and/or synaptic
plasticity activities of the agents disclosed herein may facilitate
this process. Thus an at least additive and potentially synergistic
effect may result.
[0257] The methods and compositions of the invention may be tested
using any of a variety of animal models for injury to the nervous
system. Models that may be used include, but are not limited to,
rodent, rabbit, cat, dog, or primate models for thromboembolic
stroke (Krueger and Busch, 2001; Gupta, 2004), models for spinal
cord injury (Webb et al., 2004), etc. (see Examples 6 and 7 and
references in Schmidt and Leach, 2003). The methods and
compositions may also be tested in humans.
[0258] A variety of different methods, including standardized tests
and scoring systems, are available for assessing recovery of motor,
sensory, behavioral, and/or cognitive function in animals and
humans. Any suitable method can be used. To give but one example,
the American Spinal Injury Association score, which has become the
principal instrument for measuring the recovery of sensory function
in humans, could be used (see, e.g., Martinez-Arizala A., 2004;
Thomas and Noga, 2004; Kesslak J P and Keirstead H S, 2003; for
examples of various scoring systems and methods).
[0259] Desirable dose ranges for use in humans may be established
by testing the agent(s) in tissue culture systems and in animal
models taking into account the efficacy of the agent(s) and also
any observed toxicity.
Pharmaceutical Compositions
[0260] Suitable preparations, e.g., substantially pure preparations
of the proteolysis-enhancing agents, optionally together with one
or more additional active agents, may be combined with
pharmaceutically acceptable carriers, diluents, solvents, etc., to
produce an appropriate pharmaceutical composition. In general,
methods and ingredients for producing pharmaceutical compositions
known to one of skill in the art are used. The description herein
is for exemplary purposes and is not intended to be limiting. It is
to be understood that the pharmaceutical compositions of the
invention, when administered to a subject, are typically
administered for a time and in an amount sufficient to treat the
disease or condition for whose treatment they are administered.
Suitable modes of administration and formulations are described
herein.
[0261] Further provided are pharmaceutically acceptable
compositions comprising a pharmaceutically acceptable derivative
(e.g., a prodrug) of any of the agents of the invention, by which
is meant any non-toxic salt, ester, salt of an ester or other
derivative of an agent of this invention that, upon administration
to a recipient, is capable of providing, either directly or
indirectly, an agent of this invention or an active metabolite or
residue thereof. As used herein, the term "active metabolite or
residue thereof" means that a metabolite or residue thereof also
possesses similar activity to the parent agent. For example, rather
than administering an active polypeptide, a zymogen (i.e., an
inactive or less active enzyme precursor that requires a
biochemical change, such as a hydrolysis reaction revealing the
active site, for it to become an active enzyme) could be
administered.
[0262] The term "pharmaceutically acceptable carrier, adjuvant, or
vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that
does not destroy the pharmacological activity of the agent with
which it is formulated. Furthermore, it is recognized that
preparation methods for the pharmaceutical compositions are
typically selected so as to not substantially reduce the activity
of the agent with which they are formulated.
[0263] Pharmaceutically acceptable salts of certain of the agents
of this invention include those derived from pharmaceutically
acceptable inorganic and organic acids and bases. Examples of
suitable acid salts include acetate, adipate, alginate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, citrate,
camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other
acids, such as oxalic, while not in themselves pharmaceutically
acceptable, may be employed in the preparation of salts useful as
intermediates. Salts derived from appropriate bases include alkali
metal (e.g., sodium and potassium), alkaline earth metal (e.g.,
magnesium), ammonium and N+(C1-4 alkyl)4 salts. This invention also
envisions the quaternization of any basic nitrogen-containing
groups of the compounds disclosed herein. Water or oil-soluble or
dispersible products may be obtained by such quaternization.
[0264] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Pharmaceutical
compositions suitable for injection or infusion typically include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. Suitable carriers include
physiological saline, bacteriostatic water, water for injection,
dextrose solutions, phosphate buffered saline (PBS), or Ringer's
solution. Antibacterial and/or antifungal agents; chelating agents,
such as ethylenediaminetetraacetic acid; buffers, such as acetates,
citrates, or phosphates; and agents for the adjustment of tonicity,
such as sodium chloride or dextrose, can be included. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. It may be advantageous to formulate the compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active
agent(s) calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
preparation can, for example, be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0265] Sterile injectable or infusable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent, optionally with one or a combination of
ingredients enumerated above, followed by filtered sterilization.
Typically solutions are free of endotoxin. Generally, dispersions
are prepared by incorporating the active compound into a sterile
vehicle which contains a basic dispersion medium and optionally
other ingredients. In the case of sterile powders for the
preparation of sterile solutions, the usual methods of preparation
are vacuum drying and freeze-drying (e.g., lyophilization) which
yields a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
EXAMPLES
Example 1
Identification and Analysis of Genes that are Differentially
Regulated Under Visual Deprivation Paradigms
Materials and Methods
[0266] RNA Preparation and Microarray Analysis
[0267] Studies were performed in mice (129/SvEv) at the peak of the
critical period.sup.28, postnatal day (P) 27. All animal protocols
were approved by MIT's Committee on the Care and Use of Animals and
followed NIH guidelines. For monocular deprivation (MD), animals
were anesthetized with avertin (0.016 ml/g) and the eyelids of one
eye sutured (at P11-12 for 15-16 days for microarray analyses). For
dark-reared (DR) animals (aged P27-30), the procedure was the same
described above, with the exception that the animals were
anesthetized in darkness and not exposed to light until deeply
anaesthetized; in these mice only the binocular response was
evaluated and compared to that in control animals.
[0268] In a first set of experiments we extracted total RNA from V1
of normally reared P27 mice (control, n=3 samples), from V1 of P27
mice born and reared in darkness (DR, n=3 samples), and from V1
contralateral to the deprived eye of P27 mice in which monocular
deprivation was started at P11-12, before eye-opening (MD, n=6
samples; three samples were done with deprivation of the right eye
and 3 with deprivation of the left eye; these 6 samples were
considered as a group because no significant differences were
observed between right and left eye deprivation). For each sample,
animals came from different litters and the tissue was derived from
V1 of at least two different animals. In both groups of animals,
monocular and binocular portions were included for analysis.
[0269] Mice were anesthetized with Nembutal (100 mg/kg),
decapitated and the skull opened. A micro blade was used to remove
a small core of tissue from the visual cortex of the appropriate
hemisphere. Total RNA was extracted and purified, according to the
instructions in the "Eukaryotic Target Preparation" manual
available on the Affymetrix website. Fragmented, biotinylated cRNA
was hybridized to the Affymetrix mouse genome U74v2 GeneChip set,
which contains oligonucleotides that correspond to a total of
36,902 probes targeting genes and expressed sequence tags (ESTs)
(Affymetrix). Array processing (hybridization, washing, staining
and scanning) was performed by the Biopolymer Laboratory at MIT
following standard Affymetrix protocols. A global scaling algorithm
was used to normalize the expression level data from all
samples.
[0270] In additional experiments in which the effects of short-term
(4 days from P23-27) MD were investigated, as well the effects of
IGF1 infusion concurrent with MD, a total of four experimental
groups were analyzed: a new group of control animals (3 samples),
the ipsilateral and the contralateral cortex of mice monocularly
deprived for four days (3 samples for the ipsilateral and 3 samples
for the contralateral cortex), the contralateral cortex of mice
that were monocularly deprived for four days and were injected IP
daily with IGF1 solution (3 samples). Tissue was removed and the
RNA extracted as described above, and labeled RNA was hybridized to
the Affymetrix mouse genome 430.2 chip, which contains
oligonucleotides that correspond to a total of 42,000 probes
targeting genes and ESTs.
[0271] Data Analysis
[0272] Significance Analysis of Microarrays
[0273] A method for the Significance Analysis of Microarrays to
assess changes in gene expression was used.sup.31, and the method
was implemented in MATLAB (The Mathworks, Natick, Mass.). The
method allows the comparison of the expression level of each gene
under two conditions (e.g., MD vs. control; or DR vs. control).
Under the null hypothesis that there are no changes in expression,
the output is a probability of observing the given differences by
chance (obtained by shuffling the data from the two conditions).
Results of this analysis were compared against those obtained by
setting a fixed threshold on the minimum intensity of each gene and
a minimum ratio of expression between the two conditions.
Correlations between replicates were calculated as correlation
coefficients (c.c.) for all conditions: control
(c.c.=0.99.+-.0.002), MD 16 days (c.c.=0.9.+-.0.05), MD 4 days
contralateral (c.c.=0.99.+-.0.001), MD 4 days ipsilateral
(0.99.+-.0.005), MD 4 days contralateral plus IGF1
(c.c.=0.99.+-.0.004).
[0274] GO Annotations
[0275] For the first set of experiments, Gene Ontology (GO)
annotations were retrieved for each of the genes
(http://www.geneontology.org/). Mapping of each Affymetrix probe to
gene names was done using the annotations from Affymetrix
(http://www.affymetrix.com/). GO provides information about the
molecular function of a given gene (e.g. nucleic acid binding, ion
transporter activity, etc.), the biological processes in which is
involved (e.g. cell growth, cell communication), and the cellular
location (e.g. nucleus, cytoplasm, etc.). For each of these
organizing principles, GO provides a list of different categories
to which each gene may be assigned. FatiGO.sup.32 was used to
identify categories for biological functions that are over- or
under-represented in the different protocols of visual input
deprivation.
[0276] Semi-Quantitative RT-PCR
[0277] RNA was extracted as described above and cDNA was obtained
with the Superscript First-Strand Synthesis System for RT-PCR
(Invitrogen). PCR was performed according to the Invitrogen
instruction manual. For each sample, PCR was run for the selected
molecules and for Glycerol Phosphate Dehydrogenase (GPDH) as a
control. PCR products were stained with ethidium bromide and run on
an agarose gel. The intensity of each band was evaluated with
ImageJ software (http://rsb.info.nih.gov/ij/) and normalized by the
level of GPDH expression.
Results
[0278] DNA microarrays were used to examine large scale changes in
gene expression in the V1 region of the cortex following
dark-rearing (DR) and monocular deprivation (MD), using
quantitative analyses of single genes as well as computational
analyses of gene network activation (FIG. 1A). Mice used for
microarray analyses of long-term visual deprivation were: (a) DR
animals reared in complete darkness from birth till P27, the peak
of the critical period for ocular dominance plasticity in
mice.sup.28, (b) MD animals which had one eyelid sutured from
before eye-opening (at P11-12) through P27, and (c) P27 control
animals, reared in standard conditions (FIG. 1A). The time course
of the deprivation protocols was chosen to ensure as comparable
periods of deprivation as possible in the DR and MD
conditions--that is, starting at birth and continuing till P27. V1
was identified by stereotaxic coordinates and its location
confirmed with both optical imaging of intrinsic signals.sup.29 and
by retrograde labeling of cells in the lateral geniculate nucleus
(LGN) from injections of Alexa-CTB made in cortex.sup.30. RNA was
extracted from V1 and hybridized to microarrays (Affymetrix).
First, the expression level of gene transcripts was compared
between control and deprived animals using a procedure for the
Significance Analysis of Microarrays.sup.31 (FIG. 1B, C). Two lists
of genes were obtained for each deprivation protocol: those that
were up-regulated in the deprived conditions versus control (1930
genes: 1730 genes up-regulated after DR and 200 genes up-regulated
after MD), and those which were down-regulated in the deprived
conditions versus control (1381 genes: 950 genes down-regulated
after DR and 431 genes down-regulated after MD; FIG. 1D). The
complete list of significantly (P.ltoreq.0.01) up- and
down-regulated genes is reported in tables for each experiment at
(http://ramonycajal.mit.edu/kreiman/resources/v1plasticity/) and in
Tables 4-9 herein (presented in the Appendix).
[0279] The Gene Ontology (GO) database.sup.32,33 was used to group
differentially expressed genes according to the biological
processes in which they are involved. Of the 3311 differentially
expressed genes in visually deprived groups, 1227 have known
functions and have been reported in GO categories (level 3) for
general biological processes. This analysis showed that some
biological processes are common to both deprivation conditions,
whereas others are differentially, or even exclusively, represented
in one condition or the other.
[0280] For instance, genes implicated in "metabolism" and "cell
communication" were upregulated in both conditions, with a stronger
representation in DR cortex. At the same time, genes implicated in
"cell motility" and "cell growth and maintenance" were primarily
upregulated after DR. On the other hand, genes comprising "cellular
physiological processes" and "organismal physiological processes"
were primarily upregulated after MD. This overview suggested that
while some similar mechanisms underlie the two forms of
deprivation, distinct cellular processes may also be implicated in
the two conditions.
[0281] To analyze the distinction further, a more detailed
examination of genes encoding glutamatergic and GABA receptors was
performed, including subunits of NMDA, AMPA and metabotropic
glutamate receptors and subunits of GABA-A and GABA-B receptors.
Table 1 shows changes in the expression of different subunits of
GABA and glutamate receptors in MD and DR. "+" indicates a
significant (two tailed t test P.ltoreq.0.05) increase in the mRNA
level in the deprived condition relative to control; "=" indicates
no significant change. No gene was downregulated after deprivation
relative to control.
TABLE-US-00001 Receptor MD DR GluR1 = + GluR2 + + GluR3 + + NMDA1 =
+ NMDA2A = + NMDA2B = + NMDA2C = = NMDA2D = = mGluR3 = = mGluR5 = =
mGluR8 = = GABAA.alpha.1 = + GABAA.alpha.2 + + GABAA.alpha.3 + +
GABAA.alpha.4 = + GABAA.alpha.6 + = GABAA.beta.1 + + GABAA.beta.2 =
+ GABAA.beta.3 + + GABAA.gamma.1 = = GABAA.gamma.2 = +
GABAA.gamma.3 = = GABAA.delta. = = GABAA.epsilon. = = GABAB1 = =
GABAC.rho.1 = = GABAC.rho.2 = =
[0282] This comparison of the main forms of excitatory and
inhibitory transmission in the cortex showed that a substantial set
of excitatory and inhibitory receptor genes was upregulated after
DR. MD also upregulated both sets, but a smaller subset than DR
(FIG. 2A). None of these receptor genes was downregulated after
either form of deprivation. Thus, expression of both excitatory and
inhibitory receptor genes is broadly upregulated in response to
visual deprivation, but the response is stronger in the case of DR,
where there is complete absence of light, than in the case of MD,
where there is still visual stimulation through the closed eyelid
though not in patterned form.sup.34.
[0283] Several studies have reported that DR induces a delay in the
maturation of inhibition.sup.11,35,36. No change in GAD65
expression was observed after DR or MD, but an increase in GAD67
expression was observed after DR (FIG. 2B). More generally, a
reduction was observed in expression of only one gene associated
with cortical inhibitory neurons: all the probes associated with
parvalbumin were downregulated after DR, whereas probes associated
with other markers of inhibitory neurons.sup.37,38, including
calbindin, somatostatin, calretinin, cholecystokinin and
neuropeptide Y, were either upregulated or did not change after DR
(FIG. 2B). There was no change in any of these markers after MD
(see also below, and FIG. 9). Thus, the functional reduction of
inhibition and of inhibitory neurons after DR.sup.36 is possibly
mediated specifically by a reduction in the number of neurons
expressing parvalbumin.
[0284] Next, the microarray expression levels of a subset of genes
(FIG. 3A) were compared to an independent measure of gene
expression using semi-quantitative RT-PCR performed on independent
samples from those used for microarrays. The genes selected were
significantly up-regulated (two-tailed t test P<0.05) in DR or
MD cortex versus control, with at least a 1.5-fold greater
expression after one or other form of deprivation. Furthermore,
selected genes were in the top 5% in a list of probes rank-ordered
by change in expression after DR or MD, based on calculation of the
signal to noise ratio of each gene (from the mean microarray
expression levels and standard deviations in deprived and control
conditions). Analysis of representative genes that were upregulated
after DR alone, after MD alone, or after both, is shown in FIG.
3B,C. Genes upregulated after DR (but not MD) in the microarray
data included molecules associated with synaptic structure and
function, such as those involved in synapse formation (Neurexin1
and Synapsin 2), synaptic transmission mechanisms such as
exocytosis (Synaptotagmin 1), neurotransmitter receptors (GluR1),
and calcium-activated signaling (CaMKII.alpha. and CREB). Changes
observed with RT-PCR were consistent with the observations from the
microarray data. That is, an increase in the expression of these
molecules in the DR cortex was observed, and there was a greater
increase in the DR condition compared to MD for each of them.
[0285] Fewer genes were up-regulated after MD (but not DR) compared
to control, and they included molecules that are usually implicated
in cellular pathology, including carcinogenesis (the DEAD-box RNA
helicase DDX6.sup.39) and degeneration (Signal Transducers and
Activators of Transcription 1, STAT1--see below), or are activated
by seizure (CaMKII.delta..sup.40). These genes also showed greater
expression in the RT-PCR analysis. Finally, genes that were
upregulated after both DR and MD included molecules associated with
synaptic activity (GluR3 and GABA-A.alpha.2), as well as molecules
associated with neuronal growth and reorganization of connections
(Insulin-like Growth Factor Binding Protein 5, IGFBP5--see below),
and aspects of brain development (Nuclear Factor IB,
NfiB.sup.41-43). In all of these instances, relative expression
levels measured with RT-PCR were consistent with the microarray
expression levels. Overall, these data suggest increased activation
of a wide range of synaptic and neuronal mechanisms in V1 of DR
animals, and to a lesser extent in MD animals, compared to control
animals. Conversely, they suggest an increased activation of
neuronal growth and degeneration mechanisms in MD animals, and to a
lesser extent in DR animals, compared to control animals.
[0286] While the effects of MD are pronounced in the long term,
they are also significant in the short term.sup.14-17. To examine
similarities and differences with the long (16 day) period of MD, a
microarray analysis of a short (4 day) period of MD, from P23-27,
was performed. Short-term MD led to changes in the expression of
many more genes than long-term. MD. About 50% of the genes that
were up- or down-regulated after long-term MD were also altered in
expression after short-term MD; the upregulated genes included
DDX6, IGFBP5 and NFiB. Genes upregulated by long-term MD but not
short-term MD included STAT1 and CaMKII.delta.. While some genes
associated with synaptic transmission (such as GluR1, GluR3 and
GABA-A.alpha.2) did not change after short-term MD, more
transmission-related genes (such as Synapsin 2 and Synaptotagmin 1)
were up- or down-regulated after short-term compared to long-term
MD.
Example 2
Identification of Gene Sets and Pathways Enriched in Genes that are
Differentially Regulated in Visual Deprivation Paradigms
Materials and Methods
[0287] Gene Set Enrichment Analysis (GSEA) considers even small
variations in all the mRNA probes of a group of genes, thereby
assessing the enrichment of the whole gene set, and is relevant for
detecting modest but coordinated changes in the expression of
groups of functionally related genes. Such an analysis has
particular value when an increase in the activity of several genes
in a set could be more important than the strong activation of a
single gene in a molecular cascade. Furthermore, the genes in the
set typically share some functional or structural properties.
Different gene sets have different sizes (for example, the gene set
"Channel-passive-transporter" has 238 probes, while the "IGF1
pathway" has 46 probes), and all the probes corresponding to a
single gene are reported in each gene set. A recent description of
the method.sup.44 was followed here; a more detailed description
has now appeared.sup.85.
[0288] Let .sub.S.mu..sub.i denote the mean expression level across
samples of probe i (i=1, . . . , N where N is the total number of
probes) in condition S (where S=DR, MD or control) and let
.sub.S.sigma..sub.i denote the standard deviation across samples.
For a given probe i, the signal to noise ratio (SNR) of the
deprivation condition is defined with respect to the control. For
example, for dark rearing, the SNR was defined as
SNR i DR = .mu. i DR - .mu. i control .sigma. i DR - .sigma. i
control . ##EQU00001##
Probes were ranked according to the SNR value yielding an ordered
list L={g.sub.1, . . . , g.sub.N).
[0289] Given a set G containing N.sub.G probes it can be assessed
whether the set of probes is significantly over- or
under-represented in one of the deprivation conditions with respect
to the control condition (irrespective of whether the expression of
the individual probes changed significantly or not). A
representative example illustrating the algorithm is shown in FIG.
4A. The following two cumulative distribution functions are
defined: P.sub.hit(i)=proportion of genes in the set G that show a
rank less than
i ( P hit ( i ) = # [ g ( j .ltoreq. i ) .di-elect cons. G ] N G )
##EQU00002##
and P.sub.miss(i)=proportion of genes outside the set G that show a
rank less than
i ( P miss ( i ) = # [ g ( j .ltoreq. i ) G ] N - N G ) .
##EQU00003##
The running enrichment score is defined as
RES(i)=P.sub.hit(i)-P.sub.miss(i) (FIG. 4A, top) and is derived
from the position or rank of the genes in the set (FIG. 4A,
bottom). The enrichment score ES is the maximum deviation from 0 of
RES(i). If the genes in the set are highly enriched in the
deprivation condition and appear first in the ordered list L, then
P.sub.hit will grow faster with 1 than P.sub.miss for initial
values of i and this will lead to a high positive ES value.
Conversely, if the genes in the set are under-expressed in the
deprivation condition and do not appear at the beginning of the
list L, then P.sub.miss will grow faster with i than P.sub.hit and
this will lead to a high negative ES score. If the genes in the set
are randomly distributed, then the ES will show a value close to 0.
The statistical significance of a particular value of ES is
assessed by comparing it with the null distribution obtained by
randomly shuffling the condition labels (deprivation and control)
for each probe (using 1,000 permutations).
[0290] The procedure just described was repeated for each gene set,
obtaining an enrichment score and an enrichment probability value
for each set. It is possible to define a set of genes based on
several different criteria. Here, sets of genes defined by common
functional or structural properties in 3 specific biological
databases were studied: BioCarta (http://www.biocarta.com/),
GenMapp (http://www.genmapp.org/), and GO
(http://www.geneontology.org/). When a large number of gene sets is
considered as in the present case, care should be taken because of
the multiple comparisons involved and therefore the increased
likelihood that one comparison will yield a significant result by
chance. The multiple comparisons question was addressed here by
controlling the Family Wise Error Rate.sup.6. To compare enrichment
scores across gene sets, the enrichment scores are normalized by
centering and scaling the ES using the mean and variance of each
data, gene set pair. Throughout the text and in Tables 4 and 5, the
normalized enrichment scores (NES) is shown for the gene sets
enriched in dark rearing or monocular deprivation relative to
control, or vice versa.
Results
[0291] Apart from the expression of individual genes, sets of genes
that are linked together in specific functional pathways may be
differentially expressed in DR and long-term MD and thereby lead to
different cellular and molecular responses following the two forms
of deprivation. To examine this possibility, a computational tool
was used--Gene Set Enrichment Analysis (GSEA)--that considers the
activation of sets of genes (such as cellular pathways,
co-expressed genes, or genes in the same genomic locus) rather than
the expression of a single transcript.sup.44,45. Thus, the extent
to which a set of genes or a pathway is enriched in the deprivation
paradigms was able to be measured with respect to control (or vice
versa). 1374 pathways and gene sets taken from the following
databases were considered: BioCarta, GenMapp, and GO. An example of
the computation of the running and normalized enrichment score
(NES) is shown in FIG. 4A for the ADP Ribosylation Factor (ARF)
Pathway. The expression levels for the 19 probes in this pathway
are shown in FIG. 4B. Qualitatively, FIG. 4B shows that most of
these probes were more highly expressed after MD than in control.
Quantitatively, FIG. 4A shows that many of these probes were highly
ranked in the rank-ordered set of MD probes, leading to a high
running enrichment score for the ARF pathway. The gene sets with
the highest scores in the deprived conditions versus control are
listed in Table 2, which is a representation of the top Gene Sets
enriched in DR (left column) and MD (right column) versus control.
The Gene Sets are ranked according to their Normalized Enrichment
Score. Gene Sets that are enriched in both conditions are shown
with light shading. A star indicates that at least one probe of the
correspondent Gene Set has been confirmed with RT-PCR. The gene
sets with the highest scores in the control versus deprived
conditions (i.e., are downregulated after deprivation) are listed
in Table 3. The Gene Sets are ranked according to their Normalized
Enrichment Score.
TABLE-US-00002 TABLE 3 C > DR NES C > MD NES 1
Neuropeptide_hormone -17.0 20S_core_proteasome.sub.-- -5.3 complex
2 Gas_exchange -14.3 Ribosome -4.6 3 Scavenger_receptor -13.1
Circulation -4.0 4 Serine_type.sub.-- -12.8 NADH_dehydrogenase -4.0
endopeptidase 5 Enzyme_binding.sub.-- -12.6
NADH_dehydrogenase.sub.-- -3.8 activity ubiquinone_activity 6
Spliceosomal_subunit -10.1 Endopeptidase_activity -3.6 7 chr4q21
-9.1 Structural_constituent.sub.-- -3.2 of_ribosome
[0292] These pathways were all significantly enriched (permutation
test, P<0.0001) within the data set, based on a statistical
comparison of enrichment scores obtained with 1000 randomly
permutated gene sets. The GSEA method revealed quantitatively that
different gene sets were preferentially activated after DR and MD.
For example, the top enriched gene sets after DR included those
involved in cellular activity, encompassing both metabolism related
pathways (such as "metabolism" and "growth hormone pathway"), and
synaptic activity related networks (such as "channel passive
transporter," "vesicle-coat-protein," and "secretory vesicles").
After MD, however, the majority of the top enriched gene sets
corresponded to pathways activated by growth factors ("epidermal
growth factor," "insulin-like growth factor 1," and "platelet
derived growth factor") and neuronal remodeling and degeneration
("nuclear factor of activated T cells," "JAK-STAT cascade," and
"embryogenesis and morphogenesis"). Several gene sets were enriched
in both conditions but were ranked in a different order confirming
that common processes are also shared between the two
conditions.
TABLE-US-00003 TABLE 2 DR > C NES MD > C NES 1
Channel_passive_transporter .star-solid. 27.3 egfPathway
.star-solid. 16.4 2 Metabolism 25.6 igf1Pathway .star-solid. 9.7 3
mapkPathway .star-solid. 22.6 EGF_receptor_signaling_pathway 9.5 4
Vesicle_coat_protein 21.6 pdgfPathway .star-solid. 8.7 5 chr14q31
21.0 Embryogenesis_and_morphogenesis 8.0 6 ghPathway 20.0
Helicase_activity .star-solid. 7.9 7 chr8p12 18.8 tpoPathway
.star-solid. 7.6 8 Secretory_vesicles .star-solid. 18.6 nfatPathway
.star-solid. 7.5 9 chr20p12 17.8 Monocyte_AD_pathway 7.0 10
Apoptosis_regulator_activity 17.6 arfPathway 6.8 11
Protein_amino_acid_phosphorylation 17.4 JAK_STAT_cascade
.star-solid. 6.7 12 chr4q12 17.3 Differentiation_in_PC12
.star-solid. 6.6 13 rarrxrPathway 17.1 Channel_passive_transporter
.star-solid. 6.4 14 ATPase_activity 17.0 tcrPathway .star-solid.
6.2 15 chr5q33 .star-solid. 16.8 Transmembrane_RPTP 6.0 16
insulinPathway 16.8 ghPathway .star-solid. 5.8 17
Neurotransmitter_secretion .star-solid. 16.6
Inositolphosphatidylinositol_kinase_activity 5.6 18 edg1Pathway
16.6 keratinocytePathway 5.6 19 egfPathway 16.5 at1rPathway
.star-solid. 5.6 20 RAS_protein_signal_transduction 16.5
gleevecPathway .star-solid. 5.6 21
Telomerase_dependent_telomere_maintenance 16.4 ngfPathway 5.5 22
Endoplasmic_reticulum .star-solid. 16.0 il2rbPathway 5.5 23
par1Pathway 15.6 Cancer_related_testis .star-solid. 5.5 24
ngfPathway 15.4 Adrenergic 5.4 25 at1rPathway .star-solid. 15.3
il7Pathway 5.3 26 Cancer_related_testis 15.3 il2Pathway
.star-solid. 5.3 27 erk5Pathway .star-solid. 15.2 Dag1 5.3 28
JNK_MAPK_pathway 15.1 G_alpha_5_pathway .star-solid. 5.2 29
chr15q22 15.0 PTEN_pathway 5.2 30 Ngvm_c8 15.0 cblPathway 5.1 31
arenrf2Pathway .star-solid. 14.9 B_cell_receptor_complexes 5.0 32
Microtubule_binding_activity 14.9 p53_signalling 5.0 33 arfPathway
14.7 arenrf2Pathway .star-solid. 4.9 34 Potassium_ion_transport
.star-solid. 14.5 chr20p12 4.8 35 mtorPathway 14.4 pitx2Pathway 4.8
36 crebPathway .star-solid. 14.3 igf1rPathway 4.8 37 gleevecPathway
14.3 hdacPathway .star-solid. 4.7 38
Protein_amino_acid_dephosphorylation 14.3 ccr5Pathway .star-solid.
4.7 39 myosinPathway 14.3 Insoluble_fraction 4.6 40 pdgfPathway
14.1 Granule_cell_survival .star-solid. 4.4 41 Ngvm_c32
.star-solid. 14.0 35_cyclic_nucleotide_phosphodiesterase_activity
4.4 42 Microtubule_associated_complex 14.0 hivnefPathway 4.3 43
Neuronal_transmission .star-solid. 13.9
GPI_anchored_membrane_bound_receptor 4.2 44 erkPathway 13.6
Positive_regulation_of_transcription 4.2 45 CD40_pathway_map
.star-solid. 13.6 tnfr1Pathway 4.2 46 Wnt_Signaling 13.6
Neuronal_transmission .star-solid. 4.2 47 Ion_transporter_activity
13.5 Transmembrane_RTK_signalling 4.1 48
Calmodulin_binding_activity .star-solid. 13.3 Synaptic_transmission
.star-solid. 4.1 49 GPCR_pathway 13.1 spryPathway 4.1 50 chr2p22
13.1 Golgi 4.0
[0293] The genes previously identified with RT-PCR as highly
expressed after DR or MD were also present in specific gene sets
with high NES values (corresponding gene sets are marked),
indicating that highly expressed genes together enrich specific
pathways or networks of activation. The distribution of positive
NES values for the DR versus control comparison is shown in FIG.
4C, which also shows the running enrichment scores for two pathways
containing the molecules Creb and GluR1, respectively. The NES
distribution for the MD versus control comparison is shown in FIG.
4D, together with the running enrichment scores for two pathways
containing the molecules STAT1 and IGFBP5/IGF1, respectively. Each
of these genes appears early in the rank-ordered set of DR or MD
genes (i.e., is one of the top enriched genes in the set and
contributes significantly to the running enrichment score shown in
FIG. 4C, D). Indeed, individual pathways often contain a number of
genes that are implicated in DR or ma Conversely, individual genes
are often included in multiple pathways enriched after DR or MD.
Many genes are common between the two deprivation conditions, as
expected, but several are different (cf. FIG. 3). Considering the
100 most enriched gene sets in deprivation conditions, 1928 probes
are present in DR but not MD gene sets, 1590 probes are present in
MD but not DR gene sets, and 2361 probes are present in both MD and
DR gene sets.
Example 3
Expression of Selected Proteins Encoded by Differentially Expressed
Genes
Materials and Methods
[0294] Immunohistochemistry
[0295] Mice were anesthetized and transcardially perfused with a
solution of 4% paraformaldehyde. The appropriate brain hemispheres
were removed and equilibrated in 30% sucrose in PBS. Coronal
sections containing visual cortex were cut using a freezing
microtome. Immunohistochemistry for GluR1 (1:500, Upstate), IGFBP5
(1:500, USBiological), CaMK2alpha (1:500, Sigma), PhosphoCREB
(1:500, Cell Signaling), activated Stat1 (1:500, Abeam),
parvalbumin (1:1000, Chemicon), calretinin (1:500, Chemicon),
somatostatin (1:300, Chemicon), neuropeptideY (1:400, Chemicon),
synapsin 1 (1:500, Chemicon), IGF1 (1:250, Chemicon), GAD 67
(1:400, Chemicon), IGF1R (1:500, Upstate), PI3K--catalytic subunit
110 (1:400, Upstate), phosphorylated-Akt (1:250, Cell Signaling),
was carried out as described elsewhere.sup.82,83. For each
staining, analysis was repeated in parallel for control and
deprived animals. Experiments were carried out at least on two
animals for each group and repeated twice. The intensity of
staining in sections from control and deprived animals was
evaluated with ImageJ software (http://rsb.info.nih.gov/ij/).
Counts of parvalbumin, calretinin, somatostatin and NPY-positive
cells were performed as described elsewhere.sup.29.
Results
[0296] The results described thus far represent information at the
mRNA level. Given that multiple control mechanisms can exert their
actions after the transcriptional stage, analysis of protein
expression is can be used to confirm the functional activation of a
pathway beyond RNA analyses. To further examine the regulation of
the genes described above and their associated pathways, the
expression of their proteins was analyzed using
immunohistochemistry.
[0297] First, markers were examined for selected classes of
interneurons. Since all the microarray probes for parvalbumin were
downregulated after DR (FIG. 2B) while other interneuron markers
remained unchanged or increased, it was determined whether a
similar pattern were reflected in the number of neurons that were
immuno-positive for these markers. A significant decrease (by 40%,
p<0.01) in the number of parvalbumin-positive neurons in DR
relative to control animals (FIG. 5A) was observed, while
calretinin-positive neurons remained unaltered and the number of
neurons positive for somatostatin and neuropeptide Y increased
(P<0.05). For all the antibodies examined, there was no effect
of MD on the number of stained neurons. Thus, the reported effect
of DR as delaying inhibition is likely due to a delay in the
development of neurons that express parvalbumin.
[0298] Following up the highly enriched gene sets after DR, the
expression of GluR1 (FIG. 5B) phospho-CREB (FIG. 5C), and
CaMKII.alpha. were examined, present in the "CREB pathway" gene
set. Each of these molecules was over-expressed in V1 of DR animals
compared to control, consistent with previous reports of the
involvement of CaMKII.alpha. in DR.sup.46, of GluR1 as a substrate
for CaMKII.alpha. expression.sup.47, and of CREB-mediated gene
expression as related to the maturation of the visual
cortex.sup.48. Similarly, following MD, two novel proteins were
examined, activated STAT1 and IGFBP5, which are constituents of
highly enriched gene sets, though neither has been previously
implicated in the cortical effects of MD or any form of visual
deprivation. STAT proteins are phosphorylated by Janus Kinases
(JAK); the JAK-STAT cascade is usually activated in response to
cytokine signaling, but is also upregulated in response to nerve
injury and ischemia.sup.49-51. Immunostaining for the
phosphorylated form of STAT1, indicating activation of the JAK-STAT
cascade, showed that the molecule was significantly upregulated in
V1 after MD (FIG. 5D). IGFBP5 is widely expressed in the
brain.sup.52 and binds IGF1, a peptide that is genetically related
to insulin.sup.5354,55. IGFBP5 expression was significantly
upregulated in V1 after long-term MD (FIG. 5E).
Example 4
Administration of IGF1 Counteracts Effects of Monocular
Deprivation
Materials and Methods
[0299] Monocular Deprivation
[0300] For monocular deprivation, animals were anesthetized with
avertin (0.016 ml/g) and the eyelids of one eye were sutured (at
P20-22 for 7 days for imaging experiments). Before imaging, the
suture was removed and the deprived eye re-opened. Only animals in
which the deprivation sutures were intact and the condition of the
deprived eye appeared healthy were used for the imaging session.
For DR animals (aged P27-30), the procedure was the same described
above, with the exception that the animals were anesthetized in
darkness and not exposed to light until deeply anaesthetized; in
these mice only the binocular response was evaluated and compared
to that in control animals.
[0301] Optical Imaging of V1
[0302] Mice (129/SvEv and C57Bl/6) aged P26-30 were anesthetized
with urethane (1.5 g/Kg) and chlorprothixene (0.2 mg), as
described.sup.84. Skin was excised and the skull exposed over V1. A
custom-made attachment was used to fix the head and minimize
movements. The cortex was covered with agarose solution (1.5%) and
a glass cover slip. During the imaging session the animal's body
temperature was kept constant with a heating blanket and the EKG
monitored constantly. Eyes were periodically treated with silicone
oil and the animal allowed to breathe pure oxygen. Red light (630
nm) was used to illuminate the cortical surface, and the change of
luminance was captured by a CCD camera (Cascade 512B, Roper
Scientific) during the presentation of visual stimuli (STIM,
Optical Imaging). Custom software was developed to control the
image acquisition and synchronization between the camera and
stimuli. An elongated horizontal or vertical white bar
(9.degree..times.72.degree.) over a uniformly gray background was
drifted continuously through the up-down or peripheral-central
dimension of the visual field. After moving to the last position,
the bar would jump back to the initial position and start another
cycle of movement--thus, the chosen region of visual space
(72.degree..times.72.degree.) was stimulated in periodic fashion (9
sec/cycle). Images of visual cortex were continuously captured at
the rate of 15 frames/sec during each stimulus session of 25
minutes. Four sets of stimuli (upward, downward, leftward,
rightward) were randomly presented to either eye monocularly or
both eyes simultaneously.
[0303] A temporal high pass filter (135 frames) was employed to
remove slow noise components, after which the temporal Fast Fourier
Transform (FFT) component at the stimulus frequency (9 sec.sup.-1)
was calculated pixel by pixel from the whole set of images. No
spatial averaging was done. The amplitude of the FFT component was
used to measure the strength of visually driven response for each
eye, and the ocular dominance index was derived from each eye's
response (R) at each pixel as ODI=(Rcontra-Ripsi)/(Rcontra+Ripsi).
The binocular zone was defined as the region with equivalent
driving from both eyes.
[0304] IGF1 Treatment
[0305] For IGF1 treatment, a solution containing GPE, the
functional peptide of IGF1, was prepared as described.sup.56: 300
.mu.g of GPE was injected intra-peritoneally daily for the entire
period of deprivation. This peptide is referred to as "IGF1" in the
Results below.
Results
[0306] IGFBP5 is one of the most upregulated genes after MD, with
one of the highest mRNA expression levels after RT-PCR, and the
highest differential level of protein expression after MD or DR.
Furthermore, the IGF1 pathway is one of the top enriched pathways
after MD in the GSEA, and both IGFBP5 and IGF1 are constituents of
several highly enriched pathways after MD. The present invention
encompasses the recognition that the upregulation of IGFBP5
following MD could imply a competitive role for IGF1 in mediating
ocular dominance plasticity after MD, and that exogenous
application of IGF1 could then prevent the effect of MD (see, for
example, ref. 56). The possible functional involvement of the
IGF1/IGFBP5 system in experience-dependent plasticity in visual or
any cortex has not been examined to date. Thus, the physiological
effects of IGF1 administration on ocular dominance plasticity in V1
were determined in vivo (FIG. 6).
[0307] IGF1 is able to cross the blood brain barrier.sup.56, thus,
intra-peritoneal administration of IGF1 prevents the effects of
ischemia in the CNS.sup.57. Optical imaging of intrinsic signals
was used to evaluate the strength of signals from each eye in the
physiologically identified binocular portion of V1 (FIG. 6A).
Imaging was performed on three age-matched groups of mice during
the critical period: control animals (n=3), animals monocularly
deprived for 7 days (n=4), and MD animals with IGF1 delivered
intraperitoneally during the period of deprivation (n=3). FIG. 6B
shows the ocular dominance distribution of pixels within the
binocular zone in individual control, MD and MD+IGF1 animals. The
pixel distribution in control mice favored the contralateral eye,
as described previously with single unit recordings.sup.28 and
visual evoked potentials.sup.58. Suturing the contralateral eye
caused the ocular dominance distribution to shift towards the open,
ipsilateral, eye. Simultaneous administration of IGF1 prevented the
ocular dominance shift towards the open eye. A comparison of the
mean ocular dominance index across the population of animals (FIG.
6C) showed that deprivation of the contralateral eye shifted the
index significantly relative to control animals (P<0.05,
treating each animal as a single datum), whereas MD combined with
administration of IGF1 prevented the shift (P>0.2).
[0308] The mechanisms of IGF1/IGFBP5 action were investigated by
asking if specific cell types and proteins were associated with the
pathway. To clarify whether IGFBP5 is expressed in excitatory or
inhibitory neurons, a double immunostaining for IGFBP5 and GAD67
was performed, and IGFBP5 was shown to be expressed in a range of
neurons--not exclusively in inhibitory interneurons (FIG. 7A). Next
the expression in VI of several molecules involved in IGF1
signaling.sup.53,59 was assayed by immunostaining after MD alone
and after MD with concurrent delivery of IGF1 (FIG. 7B). IGFBP5
immunostaining showed a significant increase after short-term MD,
and no change from normal levels in short-term MD animals that also
received IGF1 during the deprivation period (MD+IGF1). Expression
of the IGF1 receptor (IGF1R), on the other hand, was significantly
down-regulated after MD, and expression was partially restored in
MD+IGF1 animals. Phosphatidylinositol 3-Kinase (PI3K), which is
activated by IGF1, was significantly diminished in expression after
MD but was fully restored after MD+IGF1 treatment (P<0.05 for
both comparisons; FIG. 7B).
[0309] Expression of one of the substrates of PI3K,
phosphorylated-Akt, was significantly reduced by MD and restored by
addition of IGF1. Because IGF1 and PI3K signaling have been related
to neuronal transmission.sup.60-62, changes in synaptic activity
were screened for by immunostaining for synapsin 1. The level of
synapsin expression did not change significantly in MD animals
versus control, but MD+IGF1 animals showed a significant increase
(P<0.05). Finally, a microarray analysis of MD+IGF1 animals was
performed for comparison with MD animals, to examine genes that
might be differentially regulated by IGF1 and hence be associated
specifically with IGF1 mechanisms. Expression of only a small
fraction of genes was significantly altered in MD+IGF1 animals
compared to MD animals (see Tables 10 and 11). Adding IGF1
significantly downregulated IGFBP5 and upregulated PI3K compared to
MD alone (P<0.01). Thus, PI3K appears to be an important signal
downstream of IGF1 in mediating ocular dominance plasticity.
Example 5
Release of a Plasticity-Modifying Agent from Hydrogel Discs
[0310] In order to demonstrate the release of a
plasticity-modifying agent over time from a hydrogel matrix
suitable for drug delivery, hydrogel discs containing various
amounts of IGF1 are fabricated and subjected to incubation in a PBS
solution, during which release of IGF1 is measured over time.
[0311] The hydrogel consists of a poly(ethylene glycol) (PEG) core
with poly(lactic acid) (PLA) linkages (i.e., it contains
hPLA-b-PEG-PLA macromers) and has been previously described
(Sawhney, et al., 1993; and Burdick, et al., 2002). In order to
fabricate discs, the hydro gel macromer is combined with IFN.gamma.
and the photoinitiator
2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone,
(Ciba-Geigy) in a PBS solution. The solution (50 .mu.l) is placed
into a mold of the desired dimensions and then crosslinked under UV
light for 10 minutes to cause polymerization, thereby resulting in
discs of hydrogel with dimensions of approximately 5 mm by 1
mm.
[0312] The hydrogel discs are placed in 0.5 ml of PBS solution and
release is monitored over 14 days using an ELISA kit according to
the manufacturer's directions. Three hydrogel discs are tested for
each of the conditions (2 different loading doses each for
single-chain and two-chain tPA), and the amount of tPA released was
averaged at each time point. Data are analyzed to determine the
relationship between IGF1 release and the amount of IGF1 present in
the disc. The relationship allows for the control of the amount of
IGF1 released by changing the amount of IGF1 initially loaded into
the gel. The total amount of IGF1 released can be calculated from
the concentrations and the fact that the discs are incubated in 0.5
ml PBS solution. This information can be used to determine the
amount of IGF1 and the amount of hydrogel needed to deliver a
desired dose over time.
Example 6
Effect of IGF1 on Recovery from Spinal Cord Injury
Materials and Methods
[0313] In a first set of experiments, 6 female Sprague-Dawley rats
were anesthetized and spinal cord injury (SCI) was induced at T10
by using the New York University impactor with a 10 gm weight and a
12.5 mm weight drop. Behavioral tests were conducted on the first
post-operative day and then weekly. The BBB (Basso, Beattie,
Bresnahan) behavioral test was used to examine hind limb reflexes
as well as coordinated use of the hind limbs (Basso et al., 1995;
and Basso, et al., 1996). This "BBB" scale has been adopted by the
Multicenter Animal Spinal Cord Injury Study and by other workers in
the field. Therefore, use of the BBB as an outcome measure after
experimental SCI supports easier interlaboratory comparison of
results.
[0314] A second operation is conducted three days post-operatively
at T8-T9 for a bolus micro-injection of 10 tag of IGF1 or GPE and,
in some experiments, also 10 .mu.g of tPA (human two-chain tissue
plasminogen activator; American Diagnostic a, Inc.) reconstituted
from lyophilized powder to 10 .mu.g/10 .mu.L) into three of the six
rats. Following the bolus injection, an osmotic minipump (Alzet
Model 2002: 14 day pump; Durect Corp., Cupertino, Calif.) loaded
with IGF1 or GPE and, in some experiments, also tPA (200 .mu.L
total volume, delivering 0.5 .mu.l/hour, 10 .mu.g IGF1 or GPE, and,
in some experiments, 10 .mu.g tPA/day) is implanted at the side of
injury and delivered tPA for 10 consecutive days. At the 6.sup.th
post-operative week, BDA and Fluorogold injections are made in
cortex to assess the extent of corticospinal tract regrowth and
reconnection, and at the 10.sup.th post-operative week, animals are
perfused and their spinal cords were removed for histological
analysis. Implanted minipumps are saved for analysis of IFG1
activity (and in some experiments tPA activity) in the remaining
solution.
[0315] A second set of experiments is performed on a larger group
of animals using the same techniques as the first except that Alzet
Model 1007B:7 day pumps holding a total volume of 90 .mu.l,
infusing 0.5 .mu.l/hour are used, and delivery continues for 7 days
rather than 10.
[0316] In a third set of experiments, GPE is administered
intraperitoneally at a range of different doses (10 .mu.g-1 mg)
daily.
[0317] In a fourth set of experiments, GPE is administered
intraperitoneally at a range of different doses (10 .mu.g-1 mg)
daily and a pump delivering tPA is implanted as described
above.
[0318] In all experiments, the extent of corticospinal tract
regrowth and reconnection is evaluated and histology is performed.
Anatomical analysis with hematoxylin and eosin staining is
performed to evaluate the contusion site. Sections are stained with
solvent blue [SB]/hematoxylin and eosin as described in Teng and
Wrathall, 1997. The integrity of the residual white matter is
assessed. For example, high quality myelin stain in the spared
white matter demonstrates existence of myelinated axons.
[0319] Functional parameters are assessed. Pre-operatively, animals
performance on the BBB test is expected to have a baseline value of
21. On the first post-operative day, all animals are expected to be
significantly impaired on the BBB test, and their scores reduced to
0. After 10 weeks of recovery, control animals typically achieve a
final score of about 2.5 on the BBB test while treated animals are
expected to achieve a higher score, e.g., a final score close to 9,
which is considered significant improvement.
Example 7
Effect of IGF1 with or without tPA in an Animal Model of Stroke
[0320] Thirty rats are trained on a battery of behavioral tasks
until they achieved an asymptotic level of competence. Rats then
receive occlusion of the middle cerebral artery (MCAO) according to
standard procedures. After recovery from surgery, the rats are
significantly impaired on all of the behavioral tasks. At the time
of MCAO surgery, 20 of the 30 rats are also implanted with an
osmotic mini-pump (Alzet model 2001: 7 day pump with 90 .mu.l total
volume and 1.0 .mu.l/hour infusion) for intraventricular infusion
contralateral to the site of the MCAO. For 10 of the 20 rats, the
mini-pumps are filled with IGF1 at 10 .mu.g/day. For the other 10
rats the mini-pumps are filled with IGF1 at 10 .mu.g/day and human
two-chain tissue plasminogen activator (tPA; American Diagnostica,
Inc.) at 10 .mu.g/day. The other 10 rats receive daily
intraperitoneal injections of GPE at a dose ranging from 10 .mu.g
to 10 mg, e.g., 300 .mu.g.
[0321] Treatment is initiated 2 days following MCAO and maintained
for 7 days. Control and treated rats are subsequently tested weekly
for behavioral recovery.
EQUIVALENTS AND SCOPE
[0322] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention, described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims.
[0323] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims.
[0324] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Thus, for example, reference to "a
nanoparticle" includes a plurality of such nanoparticle, and
reference to "the cell" includes reference to one or more cells
known to those skilled in the art, and so forth. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context. The invention includes
embodiments in which exactly one member of the group is present in,
employed in, or otherwise relevant to a given product or process.
The invention includes embodiments in which more than one, or all
of the group members are present in, employed in, or otherwise
relevant to a given product or process. Furthermore, it is to be
understood that the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, descriptive terms, etc., from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Furthermore, where the claims recite a
composition, it is to be understood that methods of using the
composition for any of the purposes disclosed herein are included,
and methods of making the composition according to any of the
methods of making disclosed herein or other methods known in the
art are included, unless otherwise indicated or unless it would be
evident to one of ordinary skill in the art that a contradiction or
inconsistency would arise.
[0325] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that each subgroup of the
elements is also disclosed, and any element(s) can be removed from
the group. It should it be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not been specifically set forth
in haec verba herein. It is noted that the term "comprising" is
intended to be open and permits the inclusion of additional
elements or steps.
[0326] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0327] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., plasticity-modifying
condition, any plasticity-modifying agent, any
proteolysis-enhancing agent, any active agent, any drug delivery
system, any mode of administration, any dosage regimen, any
therapeutic application, etc.) can be excluded from any one or more
claims, for any reason, whether or not related to the existence of
prior art.
[0328] The publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
REFERENCES
[0329] 1. Katz, L. C. & Shatz, C. J. Synaptic activity and the
construction of cortical circuits. Science 274, 1133-8 (1996).
[0330] 2. Sur, M. & Leamey, C. A. Development and plasticity of
cortical areas and networks. Nat Rev Neurosci 2, 251-62 (2001).
[0331] 3. Berardi, N., Pizzorusso, T., Ratto, G. M. & Maffei,
L. Molecular basis of plasticity in the visual cortex. Trends
Neurosci 26, 369-78 (2003). [0332] 4. Hensch, T. K. Critical period
regulation. Annu Rev Neurosci 27, 549-79 (2004). [0333] 5. Desai,
N. S., Cudmore, R. H., Nelson, S. B. & Turrigiano, G. G.
Critical periods for experience-dependent synaptic scaling in
visual cortex. Nat Neurosci 5, 783-9 (2002). [0334] 6. Wallace, W.
& Bear, M. F. A morphological correlate of synaptic scaling in
visual cortex. J Neurosci 24, 6928-38 (2004). [0335] 7. Kirkwood,
A., Rioult, M. C. & Bear, M. F. Experience-dependent
modification of synaptic plasticity in visual cortex. Nature 381,
526-8 (1996). [0336] 8. Philpot, B. D., Espinosa, J. S. & Bear,
M. F. Evidence for altered NMDA receptor function as a basis for
metaplasticity in visual cortex. J Neurosci 23, 5583-8 (2003).
[0337] 9. Fagiolini, M., Pizzorusso, T., Berardi, N., Domenici, L.
& Maffei, L. Functional postnatal development of the rat
primary visual cortex and the role of visual experience: dark
rearing and monocular deprivation. Vision Res 34, 709-20 (1994).
[0338] 10. Morales, B., Choi, S. Y. & Kirkwood, A. Dark rearing
alters the development of GABAergic transmission in visual cortex.
J Neurosci 22, 8084-90 (2002). [0339] 11. Iwai, Y., Fagiolini, M.,
Obata, K. & Hensch, T. K. Rapid critical period induction by
tonic inhibition in visual cortex. J Neurosci 23, 6695-702 (2003).
[0340] 12. Turrigiano, G. G. & Nelson, S. B. Homeostatic
plasticity in the developing nervous system. Nat Rev Neurosci 5,
97-107 (2004). [0341] 13. Wiesel, T. N. & Hubei, D. H.
Single-Cell Responses in Striate Cortex of Kittens Deprived of
Vision in One Eye. J Neurophysiol 26, 1003-17 (1963). [0342] 14.
Trachtenberg, J. T., Trepel, C. & Stryker, M. P. Rapid
extragranular plasticity in the absence of thalamocortical
plasticity in the developing primary visual cortex. Science 287,
2029-32 (2000). [0343] 15. Trachtenberg, J. T. & Stryker, M. P.
Rapid anatomical plasticity of horizontal connections in the
developing visual cortex. J Neurosci 21, 3476-82 (2001). [0344] 16.
Oray, S., Majewska, A. & Sur, M. Dendritic spine dynamics are
regulated by monocular deprivation and extracellular matrix
degradation. Neuron 44, 1021-30 [0345] (2004). [0346] 17. Mataga,
N., Mizuguchi, Y. & Hensch, T. K. Experience-dependent pruning
of dendritic spines in visual cortex by tissue plasminogen
activator. Neuron 44, 1031-41 [0347] (2004). [0348] 18. Shatz, C.
J. & Stryker, M. P. Ocular dominance in layer IV of the cat's
visual cortex and the effects of monocular deprivation. J Physiol
281, 267-83 (1978). [0349] 19. Antonini, A. & Stryker, M. P.
Rapid remodeling of axonal arbors in the visual cortex. Science
260, 1819-21 (1993). [0350] 20. Crowley, J. C. & Katz, L. C.
Development of ocular dominance columns in the absence of retinal
input. Nat Neurosci 2, 1125-30 (1999). [0351] 21. Crowley, J. C.
& Katz, L. C. Early development of ocular dominance columns.
Science 290, 1321-4 (2000). [0352] 22. Crair, M. C., Gillespie, D.
C. & Stryker, M. P. The role of visual experience in the
development of columns in cat visual cortex. Science 279, 566-70
(1998). [0353] 23. Tagawa, Y., Kanold, P. O., Majdan, M. &
Shatz, C. J. Multiple periods of functional ocular dominance
plasticity in mouse visual cortex. Nat Neurosci 8, 380-8 (2005).
[0354] 24. Yang, C. B., Zheng, Y. T., Li, G. Y. & Mower, G. D.
Identification of Munc13-3 as a candidate gene for critical-period
neuroplasticity in visual cortex. J Neurosci 22, 8614-8 (2002).
[0355] 25. Prasad, S. S. et al. Gene expression patterns during
enhanced periods of visual cortex plasticity. Neuroscience 111,
35-45 (2002), [0356] 26. Ossipow, V., Pellissier, F., Schaad, O.
& Ballivet, M. Gene expression analysis of the critical period
in the visual cortex. Mol Cell Neurosci 27, 70-83 (2004). [0357]
27. Lachance, P. E. & Chaudhuri, A. Microarray analysis of
developmental plasticity in monkey primary visual cortex. J
Neurochem 88, 1455-69 (2004). [0358] 28. Gordon, J. A. &
Stryker, M. P. Experience-dependent plasticity of binocular
responses in the primary visual cortex of the mouse. J Neurosci 16,
3274-86 (1996). [0359] 29. Newton, J. R., Ellsworth, C., Miyakawa,
T., Tonegawa, S. & Sur, M. Acceleration of visually cued
conditioned fear through the auditory pathway. Nat Neurosci 7,
968-73 (2004). [0360] 30. Majewska, A. & Sur, M. Motility of
dendritic spines in visual cortex in vivo: changes during the
critical period and effects of visual deprivation. Proc Natl Acad
Sci USA 100, 16024-9 (2003). [0361] 31. Tusher, V. G., Tibshirani,
R. & Chu, G. Significance analysis of microarrays applied to
the ionizing radiation response. Proc Natl Acad Sci USA 98, 5116-21
(2001). [0362] 32. Al-Shahrour, F., Diaz-Uriarte, R. & Dopazo,
J. FatiGO: a web tool for finding significant associations of Gene
Ontology terms with groups of genes. Bioinformatics 20, 578-80
(2004). [0363] Ashburner, M. & Lewis, S. On ontologies for
biologists: the Gene Ontology--untangling the web. Novartis Found
Symp 247, 66-80; discussion 80-3, 84-90, 244-52 (2002). [0364] 34.
Akerman, C. J., Smyth, D. & Thompson, I. D. Visual experience
before eye-opening and the development of the retinogeniculate
pathway. Neuron 36, 869-79 (2002). [0365] 35. Papadopoulos, G. C.,
Cavanagh, M. E., Antonopoulos, J., Michaloudi, H. & Parnavelas,
J. G. Postnatal development of somatostatin-containing neurons in
the visual cortex of normal and dark-reared rats. Exp Brain Res 92,
473-8 (1993). [0366] 36. Benevento, L. A., Bakkum, B. W. &
Cohen, R. S. gamma-Aminobutyric acid and somatostatin
immunoreactivity in the visual cortex of normal and dark-reared
rats. Brain Res 689, 172-82 (1995). [0367] 37. Lund, J. S. &
Lewis, D. A. Local circuit neurons of developing and mature macaque
prefrontal cortex: Golgi and immunocytochemical characteristics. J
Comp Neurol 328, 282-312 (1993). [0368] 38. Flames, N. & Marin,
O. Developmental mechanisms underlying the generation of cortical
inter-neuron diversity. Neuron 46, 377-81 (2005). [0369] 39.
Abdelhaleem, M. Do human RNA helicases have a role in cancer?
Biochim Biophys Acta 1704, 37-46 (2004). [0370] 40. Murray, K. D.,
Isackson, P. J. & Jones, E. G. N-methyl-D-aspartate receptor
dependent transcriptional regulation of two
calcium/calmodulin-dependent protein kinase type II isoforms in
rodent cerebral cortex. Neuroscience 122, 407-20 (2003). [0371] 41.
das Neves, L. et at Disruption of the murine nuclear factor I-A
gene (Nfia) results in perinatal lethality, hydrocephalus, and
agenesis of the corpus callosum. Proc Natl Acad Sci USA 96,
11946-51 (1999). [0372] 42. Shu, T., Butz, K. G., Plachez, C.,
Gronostajski, R. M. & Richards, L. J. Abnormal development of
forebrain midline glia and commissural projections in Nfia
knock-out mice. J Neurosci 23, 203-12 (2003). [0373] 43.
Steele-Perkins, G. et al. The transcription factor gene Nfib is
essential for both lung maturation and brain development. Mol Cell
Biol 25, 685-98 (2005). [0374] 44. Mootha, V. K. et al.
PGC-1alpha-responsive genes involved in oxidative phosphorylation
are coordinately downregulated in human diabetes. Nat Genet 34,
267-73 (2003). [0375] 45. Sweet-Cordero, A. et al. An oncogenic
KRAS2 expression signature identified by cross-species
gene-expression analysis. Nat Genet 37, 48-55 (2005). [0376] 46.
Neve, R. L. & Bear, M. F. Visual experience regulates gene
expression in the developing striate cortex. Proc Natl Acad Sci USA
86, 4781-4 (1989). [0377] 47. Xue, J., Li, G., Laabich, A. &
Cooper, N. G. Visual-mediated regulation of retinal CaMKII and its
GluR1 substrate is age-dependent. Brain Res Mol Brain Res 93,
95-104 (2001). [0378] 48. Pham, T. A., Impey, S., Storm, D. R.
& Stryker, M. P. CRE-mediated gene transcription in neocortical
neuronal plasticity during the developmental critical period.
Neuron 22, 63-72 (1999). [0379] 49. Yao, G. L., Kato, H., Khalil,
M., Kiryu, S. & Kiyama, H. Selective upregulation of cytokine
receptor subchain and their intracellular signalling molecules
after peripheral nerve injury. Eur J Neurosci 9, 1047-54 (1997).
[0380] 50. Schwaiger, F. W. et al. Peripheral but not central
axotomy induces changes in Janus kinases (JAK) and signal
transducers and activators of transcription (STAT). Eur J Neurosci
12, 1165-76 (2000). [0381] 51. Justicia, C., Gabriel, C. 86 Planas,
A. M. Activation of the JAK/STAT pathway following transient focal
cerebral ischemia: signaling through Jak 1 and Stat3 in astrocytes.
Glia 30, 253-70 (2000). [0382] 52. Iwadate, H., Sugisaki, T., Kudo,
M. & Kizuki, K. Actions of insulin-like growth factor binding
protein-5 (IGFBP-5) are potentially regulated by tissue kallikrein
in rat brains. Life Sci 73, 3149-58 (2003). [0383] 53. Bondy, C. A.
& Cheng, C. M. Signaling by insulin-like growth factor 1 in
brain. Eur J Pharmacol 490, 25-31 (2004). [0384] 54. Zheng, W. H.
& Quirion, R. Comparative signaling pathways of insulin-like
growth factor-1 and brain-derived neurotrophic factor in
hippocampal neurons and the role of the PI3 kinase pathway in cell
survival. J Neurochem 89, 844-52 (2004). [0385] 55. Obata, S.,
Obata, J., Das, A. & Gilbert, C. D. Molecular correlates of
topographic reorganization in primary visual cortex following
retinal lesions. Cereb Cortex 9, 238-48 (1999). [0386] 56.
Sizonenko, S. V., Sirimanne, E. S., Williams, C. E. & Gluckman,
P. D. Neuroprotective effects of the N-terminal tripeptide of IGF1,
glycine-proline-glutamate, in the immature rat brain after
hypoxic-ischemic injury. Brain Res 922, 42-50 (2001). [0387] 57.
Guan, J., Bennet, L., Gluckman, P. D. & Gunn, A. J.
Insulin-like growth factor-1 and post-ischemic brain injury. Prog
Neurobiol 70, 443-62 (2003). [0388] 58. Porciatti, V., Pizzorusso,
T. & Maffei, L. The visual physiology of the wild type mouse
determined with pattern VEPs. Vision Res 39, 3071-81 (1999). [0389]
59. Laurino, L. et al. PI3K activation by IGF1 is essential for the
regulation of membrane expansion at the nerve growth cone. J Cell
Sci 118, 3653-62 (2005). [0390] 60. Liou, J. C., Tsai, F. Z. &
Ho, S. Y. Potentiation of quantal secretion by insulin-like growth
factor-1 at developing motoneurons in Xenopus cell culture. J
Physiol 553, 719-28 (2003). [0391] 61. Seto, D. et al. Insulin-like
growth factor-I inhibits endogenous acetylcholine release from the
rat hippocampal formation: possible involvement of GABA in
mediating the effects. Neuroscience 115, 603-12 (2002). [0392] 62.
Blair, L. A. & Marshall, J. IGF1 modulates N and L calcium
channels in a PI 3-kinase-dependent manner. Neuron 19, 421-9
(1997). [0393] 63. Lodovichi, C., Berardi, N., Pizzorusso, T. &
Maffei, L. Effects of neurotrophins on cortical plasticity: same or
different? J Neurosci 20, 2155-65 (2000). [0394] 64. Bear, M. F.,
Kleinschmidt, A., Gu, Q. A. & Singer, W. Disruption of
experience-dependent synaptic modifications in striate cortex by
infusion of an NMDA receptor antagonist. J Neurosci 10, 909-25
(1990). [0395] 65. Roberts, E. B., Meredith, M. A. & Rarnoa, A.
S. Suppression of NMDA receptor function using antisense DNA block
ocular dominance plasticity while preserving visual responses. J
Neurophysiol 80, 1021-32 (1998). [0396] 66. Hensch, T. K. et al.
Local GABA circuit control of experience-dependent plasticity in
developing visual cortex. Science 282, 1504-8 (1998). [0397] 67.
Hensch, T. K. & Stryker, M. P. Columnar architecture sculpted
by GABA circuits in developing cat visual cortex. Science 303,
1678-81 (2004). [0398] 68. Pizzorusso, T. et al. Reactivation of
ocular dominance plasticity in the adult visual cortex. Science
298, 1248-51 (2002). [0399] 69. Mataga, N., Nagai, N. & Hensch,
T. K. Permissive proteolytic activity for visual cortical
plasticity. Proc Natl Acad Sci USA 99, 7717-21 (2002). [0400] 70.
Huang, Z. J. et al. BDNF regulates the maturation of inhibition and
the critical period of plasticity in mouse visual cortex. Cell 98,
739-55 (1999). [0401] 71. Fagiolini, M. et al. Specific GABAA
circuits for visual cortical plasticity. Science 303, 1681-3
(2004). [0402] 72. White, L. E., Coppola, D. M. & Fitzpatrick,
D. The contribution of sensory experience to the maturation of
orientation selectivity in ferret visual cortex. Nature 411,
1049-52 (2001). [0403] 73. Corriveau, R. A., Huh, G. S. &
Shatz, C. J. Regulation of class I MHC gene expression in the
developing and mature CNS by neural activity. Neuron 21, 505-20
(1998). [0404] 74. Xu, W., Nair, J. S., Malhotra, A. & Zhang,
J. J. B cell antigen receptor signaling enhances IFN-gamma-induced
Stat1 target gene expression through calcium mobilization and
activation of multiple serine kinase pathways. J Interferon
Cytokine Res 25, 113-24 (2005). [0405] 75. Tonner, E. et al.
Insulin-like growth factor binding protein-5 (IGFBP-5) potentially
regulates programmed cell death and plasminogen activation in the
mammary gland. Adv Exp Med Biol 480, 45-53 (2000). [0406] 76.
McGee, A. W., Yang, Y., Fischer, Q. S., Daw, N. W. &
Strittmatter, S. M. Experience-driven plasticity of visual cortex
limited by myelin and Nogo receptor. Science 309, 2222-6 (2005).
[0407] 77. Wang, W. F., Kiyosawa, M., Ishiwata, K. & Mochizuki,
M. Glucose metabolism in the visual structures of rat monocularly
deprived by eyelid suture after postnatal eye opening. Jpn J
Ophthalmol 49, 6-11 (2005). [0408] 78. Bondy, C. A. & Cheng, C.
M. Insulin-like growth factor-1 promotes neuronal glucose
utilization during brain development and repair processes. Int Rev
Neurobiol 51, 189-217 (2002). [0409] 79. Maffei, L., Berardi, N.,
Domenici, L., Parisi, V. & Pizzorusso, T. Nerve growth factor
(NGF) prevents the shift in ocular dominance distribution of visual
cortical neurons in monocularly deprived rats. J Neurosci 12,
4651-62 (1992). [0410] 80. Polleux, F., Whitford, K. L.,
Dijkhuizen, P. A., Vitalis, T. & Ghosh, A. Control of cortical
interneuron migration by neurotrophins and PI3-kinase signaling.
Development 129, 3147-60 (2002). [0411] 81. Righi, M., Tongiorgi,
E. & Cattaneo, A. Brain-derived neurotrophic factor (BDNF)
induces dendritic targeting of BDNF and tyrosine kinase B mRNAs in
hippocampal neurons through a phosphatidylinositol-3
kinase-dependent pathway. J Neurosci 20, 3165-74 (2000). [0412] 82.
Tropea, D., Capsoni, S., Covaceuszach, S., Domenici, L. &
Cattaneo, A. Rat visual cortical neurones express TrkA NGF
receptor. Neuroreport 13, 1369-73 (2002). [0413] 83. Tropea, D.,
Caleo, M. & Maffei, L. Synergistic effects of brain-derived
neurotrophic factor and chondroitinase ABC on retinal fiber
sprouting after denervation of the superior colliculus in adult
rats. J Neurosci 23, 7034-44 (2003). [0414] 84. Kalatsky, V. A.
& Stryker, M. P. New paradigm for optical imaging: temporally
encoded maps of intrinsic signal. Neuron 38, 529-45 (2003). [0415]
85. Subramanian, A. et al. Gene set enrichment analysis: a
knowledge-based approach for interpreting genome-wide expression
profiles. Proc Natl Acad Sci USA 102, 15545-50 (2005). [0416] 86.
Storey, J. D. & Tibshirani, R. Statistical significance for
genomewide studies. Proc Natl Acad Sci USA 100, 9440-5 (2003).
[0417] 87. Al-Anazi A, Bernstein M (2000). Modified stereotactic
insertion of the Ommaya reservoir. J Neurosurg, 92:1050-1052.
[0418] 88. Antonini, A., Fagiolini, M., and Stryker, M. P. (1999).
Anatomical correlates of functional plasticity in mouse visual
cortex. Journal of Neuroscience 19, 4388-4406. [0419] 89. Antonini,
A., and Stryker, M. P. (1993). Rapid remodeling of axonal arbors in
the visual cortex. Science 260, 1819-1821. [0420] 90. Russo, V. C.,
et al., Endocrine Rev., 26(7): 916-943 (2005). [0421] 91. Foster,
F., et al., J. Cell Sci. 116:3037-3040 (2003). [0422] 92. Paez, J.
and Sellers, W., Cancer Treat Res. 115:145-67 (2003). [0423] 93.
Kinney, J., et al., J. Neurosci., 26(5): 1604 (2006). [0424] 94.
Asselbergs, et al., (1995) J. Biotechnol., 42(3):221-233. [0425]
95. Baranes, D., Lederfein, D., Huang, Y. Y., Chen, M., Bailey, C.
H., and Kandel, E. R. (1998). Tissue plasminogen activator
contributes to the late phase of LTP and to synaptic growth in the
hippocampal mossy fiber pathway. Neuron 21, 813-825. [0426] 96.
Basso, D M, et al., (1995). A sensitive and reliable locomotor
rating scale for open field testing in rats. J. Neurotrauma,
12(1):1-21. [0427] 97. Basso, D M., et al. (1996). Graded
histological and locomotor outcomes after spinal cord contusion
using the NYU weight-drop device versus transection. Exp. Neural.,
139(2): 244-256. [0428] 98. Benita et al. (1984) J. Pharm. Sci.
73:1721-1724. [0429] 99. Berry, M., et al., (2001) Gene therapy for
central nervous system repair, Curr. Opin. Mol. Ther. 3: 338-49.
[0430] 100. Biernaskie, J. and Corbett J. (2001) Enriched
Rehabilitative Training Promotes Improved Forelimb Motor Function
and Enhanced Dendritic Growth after Focal Ischemic Injury, The
Journal of Neuroscience, 21(14):5272-5280. [0431] 101. Bizik, J.,
et al. (1990) Cell Regul.; 1(12): 895-905. [0432] 102. Blue, M. E.,
and Parnavelas, J. G. (1983). The formation and maturation of
synapses in the visual cortex of the rat. II. Quantitative
analysis. J Neurocytol 12, 697-712. [0433] 103. Bonhoeffer, T., and
Yuste, R. (2002). Spine Motility: Phenomenology, Mechanisms, and
Function. Neuron 35, 1019-1027. [0434] 104. Brody E N, Gold L.
(2000) J Biotechnol., 74(1):5-13. [0435] 105. Brummelkamp, T. R.,
et al. (2002) A system for stable expression of short interfering
RNAs in mammalian cells. Science 296:550-553. [0436] 106. Bunge, M
B and Pearse, D D (2003) J Rehabil Res Dev. 40(4 Suppl 0:55-62.
Burns, A. & Zaudig, M (2002). Mild cognitive impairment in
older people. The Lancet 360, 1963-1965. [0437] 107. Callaway, E.
M., and Katz, L. C. (1990). Emergence and refinement of clustered
horizontal connections in cat striate cortex. J Neurosci 10,
1134-1153. [0438] 108. Callaway, E. M., and Katz, L. C. (1991).
Effects of binocular deprivation on the development of clustered
horizontal connections in cat striate cortex. Proc Natl Acad Sci
USA 88, 745-749. [0439] 109. Chen, R., et al. (2002) Neuroscience,
"Nervous System Reorganization Following Injury", 111(4): 761-773.
[0440] 110. Cho, I H, et al., (2004) Purification and
characterization of six fibrinolytic serine-proteases from
earthworm Lumbricus rubellus. J Biochem Mol Biol. 2004 Mar. 31;
37(2):199-205. [0441] 111. Cotten and Birnstiel, (1989) EMBO J.
8:3861-3866. [0442] 112. Cramer, S., et al. (1997) A functional MRI
study of subjects recovered from hemiparetic stroke, Stroke, 28:
2518-2527. [0443] 113. Dang W, Daviau T, Brem H (1996).
Morphological characterization of polyanhydride biodegradable
implant gliadel during in vitro and in vivo erosion using scanning
electron microscopy. Pharm Res, 13:683:91. [0444] 114. De Felipe,
J., Marco, P., Fairen, A., and Jones, E. G. (1997). Inhibitory
synaptogenesis in mouse somatosensory cortex. Cereb Cortex 7,
619-634. [0445] 115. DeVivo, M. J., Epidemiology of traumatic
spinal cord injury, in Kischblum, S., Campagnolo, D. I., DeLlisa,
J. A. (eds.) Spinal Cord Medicine, 2002. Lippincott Williams &
Wilkins, Philadelphia, pp. 69-81. [0446] 116. Dityatev, A., and
Schachner, M. (2003). Extracellular matrix molecules and synaptic
plasticity. Nat Rev Neurosci 4, 456-468. [0447] 117. Dunaevsky, A.,
Tashiro, A., Majewska, A., Mason, C., and Yuste, R. (1999).
Developmental regulation of spine motility in the mammalian central
nervous system. Proc Natl Acad Sci USA 96, 13438-13443. [0448] 118.
Elbashir, S M, et al., (2001) Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells. Nature. 24;
411(6836):494-8. [0449] 119. Elokdah H, et al. (2004) Tiplaxtinin,
a novel, orally efficacious inhibitor of plasminogen activator
inhibitor-1: design, synthesis, and preclinical characterization. J
Med Chem. 47(14):3491-4. [0450] 120. Emptage, N., Bliss, T. V., and
Fine, A. (1999). Single synaptic events evoke NMDA
receptor-mediated release of calcium from internal stores in
hippocampal dendritic spines. Neuron 22, 115-124. [0451] 121.
Engert, F., and Bonhoeffer, T; (1999). Dendritic spine changes
associated with hippocampal long-term synaptic plasticity. Nature
399, 66-70. [0452] 122. Fagiolini, M., Fritschy, J. M., Low, K.,
Mohler, H., Rudolph, U., and Hensch, T. K. (2004). Specific GABAA
circuits for visual cortical plasticity. Science 303, 1681-1683.
[0453] 123. Fagiolini, M., and Hensch, T. K. (2000). Inhibitory
threshold for critical-period activation in primary visual cortex.
Nature 404, 183-186. [0454] 124. Fawcett, J W and Asher, R A (1999)
The glial scar and central nervous system repair. Brain Res Bull.
49(6):377-91. [0455] 125. Feng, G., Mellor, R. H., Bernstein, M.,
Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M.,
Lichtman, J. W., and Sanes, J. R. (2000). Imaging neuronal subsets
in transgenic mice expressing multiple spectral variants of GFP.
Neuron 28, 41-51. [0456] 126. Fischer, M., Kaech, S., Knutti, D.,
and Matus, A. (1998). Rapid actin-based plasticity in dendritic
spines. Neuron 20, 847-854. [0457] 127. Fischer, M., Kaech, S.,
Wagner, U., Brinkhaus, H., and Matus, A. (2000). Glutamate
receptors regulate actin-based plasticity in dendritic spines. Nat
Neurosci 3, 887-894. [0458] 128. Fiumelli, H., Jabaudon, D.,
Magistretti, P. J., and Martin, J. L. (1999). BDNF stimulates
expression, activity and release of tissue-type plasminogen
activator in mouse cortical neurons. Eur J Neurosci 11; 1639-1646.
[0459] 129. Fleming A B, Saltzman W M (2002). Pharmacokinetics of
the carmustine implant. Clin Pharmackinet, 41:403-19. [0460] 130.
Fukazawa, Y., Saitoh, Y., Ozawa, F., Ohta, Y., Mizuno, K., and
Inokuchi, K. (2003). Hippocampal LTP Is Accompanied by Enhanced
F-Actin Content within the Dendritic Spine that Is Essential for
Late LTP Maintenance In Vivo. Neuron 38, 447-460. [0461] 131.
Furlan, M., et al., (1996) Spontaneous neurological recovery after
stroke and the fate of the ischemic penumbra", Ann. Neurol.,
40:216-226. [0462] 132. Gale K, Kerasidis H, Wrathall J R (1985)
Spinal cord contusion in the rat: behavioral analysis of functional
neurological impairment. Exp. Neurol 88:123-134. [0463] 133.
Galicich J H, Guido L J (1974). Ommaya device in carcinomatous and
leukemic meningitis. Surgical experience in 45 cases. Surg Clin
North Am 54:915-922. [0464] 134. Ge, T., et al., (2005) Cloning of
thrombolytic enzyme (lumbrokinase) from earthworm and its
expression in the yeast Pichia pastoris. Protein Expr Purif. 2005
July; 42(1):20-8. [0465] 135. Oils, A., et al. (2002)
Characterization and comparative evaluation of a novel PAI-1
inhibitor. Thromb Haemost. 88(1):137-43. [0466] 136. Goldman S.
(2005) Stem and progenitor cell-based therapy of the human central
nervous system. Nat Biotechnol. 23(7):862-71. [0467] 137. Gordon,
J. A., and Stryker, M. P. (1996). Experience-dependent plasticity
of binocular responses in the primary visual cortex of the mouse.
Journal of Neuroscience 16, 3274-3286. [0468] 138. Gray, E. (1959).
Electron microscopy of synaptic contacts on dendritic spines of the
cerebral cortex. Nature 183, 1592-1593. [0469] 139. Gualandris, A.,
Jones, T. E., Strickland, S., and Tsirka, S. E. (1996). Membrane
depolarization induces calcium-dependent secretion of tissue
plasminogen activator. J Neurosci 16, 2220-2225. [0470] 140. Guo, J
T, et al., Nucleic Acids Res. 32 (Web Server issue):W522-5, Jul. 1,
2004). [0471] 141. Gupta, Y K. and Briyal, S., (2004) Animal models
of cerebral ischemia for evaluation of drugs. Indian J Physiol
Pharmacol. 48(4):379-94. [0472] 142. Hall, A. (1998). Rho GTPases
and the actin cytoskeleton. Science 279, 509-514. [0473] 143. Han,
S.- O., R. I. Mahato, Y. K. Sung, and S. W. Kim. (2000) Development
of Biomaterials for gene therapy. Mol. Therapy 2:302-317. [0474]
144. Harenberg, (1998), Med. Res. Rev., 18:1-20. [0475] 145.
Heinemann U., et al., (2001); Curr Opin Biotechnol. 12(4):348-54.
[0476] 146. Herman J K (2005) Evaluation of PAI-039
[{1-Benzyl-5-[4-(trifluoromethoxy)phenyl]-1H-indol-3-yl}(oxo)acet-
ic Acid], a Novel Plasminogen Activator Inhibitor-1 Inhibitor, in a
Canine Model of Coronary Artery Thrombosis. Pharmacol Exp Ther.
314(2):710-6. [0477] 147. Hensch, T. K., Fagiolini, M., Malaga, N.,
Stryker, M. P., Baekkeskov, S., and Kash, S. F. (1998). Local GABA
circuit control of experience-dependent plasticity in developing
visual cortex. Science 282, 1504-1508. [0478] 148. Hering, H., and
Sheng, M. (2001). Dendritic spines: structure, dynamics and
regulation. Nat Rev Neurosci 2, 880-888. [0479] 149. Heynen, A. J.,
Yoon, B. J., Liu, C. H., Chung, H. J., Huganir, R. L., and Bear, M.
F. (2003). Molecular mechanism for loss of visual cortical
responsiveness following brief monocular deprivation. Nat Neurosci
6, 854-862. [0480] 150. Higgins D L and Bennett W F, (1990) Tissue
Plasminogen Activator: The Biochemistry and Pharmacology of
Variants Produced by Mutagenesis. Annual Review of Pharmacology and
Toxicology Vol. 30: 91-121. [0481] 151. Huang, Z. J., Kirkwood, A.,
Pizzorusso, T., Porciatti, V., Morales, B., Bear, M. F., Maffei,
L., and Tonegawa, S. (1999). BDNF regulates the maturation of
inhibition and the critical period of plasticity in mouse visual
cortex. Cell 98, 739-755. [0482] 152. Hubel, D. H., and Wiesel, T.
N. (1970). The period of susceptibility to the physiological
effects of unilateral eye closure in kittens. J Physiol 206,
419-436. [0483] 153. Johannson, B. (2000) "Brain Plasticity and
Stroke Rehabilitation", Stroke, 31:223-230. [0484] Kanematsu, A.,
et al. (2004) Collagenous matrices as release carriers of exogenous
growth factors. Biomaterials. 25(18):4513-20. [0485] 154. Kesslak J
P, Keirstead H S. (2003) Assessment of behavior in animal models of
spinal cord injury. J Spinal Cord Med. 26(4):323-8. [0486] 155.
Koester, H. J., and Sakmann, B. (1998). Calcium dynamics in single
spines during coincident pre- and postsynaptic activity depend on
relative timing of back-propagating action potentials and
subthreshold excitatory postsynaptic potentials. Proc Natl Acad Sci
USA 95, 9596-9601. [0487] 156. Krueger K, Busch E. Protocol of a
thromboembolic stroke model in the rat: review of the experimental
procedure and comparison of models. Invest Radiol. 2002.
37(11):600-8. [0488] 157. Krystosek, A., and Seeds, N. W. (1981).
Plasminogen activator release at the neuronal growth cone. Science
213, 1532-1534. [0489] 158. Lamer T J (1994). Treatment of
cancer-related pain: when orally administered medications fail.
Mayo Clin Proc, 69:473-80. [0490] 159. Laske, D W, et al., 1997
Nat. Med. Tumor regression with regional distribution of the
targeted toxin TF-CRM107 in patients with malignant brain tumors.
3(12):1362-8. [0491] 160. Leamey C A, et al., (2001) Disruption of
retinogeniculate pattern formation by inhibition of soluble
guanylyl cyclase. J Neurosci. 21(11):3871-80. [0492] 161. Lendvai,
B., Stem, E. A., Chen, B., and Svoboda, K. (2000).
Experience-dependent plasticity of dendritic spines in the
developing rat barrel cortex in vivo. Nature 404, 876-881. [0493]
162. Liang, A., et al., (2005) Characterization of a small molecule
PAI-1 inhibitor, ZK4044. Thromb Res. 115(4):341-50. Epub 2004 Nov.
13. [0494] 163. Liberatore G T, et al., Vampire bat salivary
plasminogen activator (desmoteplase): a unique fibrinolytic enzyme
that does not promote neurodegeneration. Stroke. 2003 February;
34(2):537-43. [0495] 164. Liepert, J., et al. (2000)
Treatment-Induced Cortical Reorganization After Stroke in Humans,
Stroke, 31:1210-1216. [0496] 165. Machado M, Salcman M, Kaplan R S,
Montgomery E (1985). Expanded role of the cerebrospinal fluid
reservoir in neuroongocology: indications, causes of revision, and
complications. Neurosurgery 17:600-603. [0497] 166. Majewska, A.,
Brown, E., Ross, J., and Yuste, R. (2000a). Mechanisms of calcium
decay kinetics in hippocampal spines: role of spine calcium pumps
and calcium diffusion through the spine neck in biochemical
compartmentalization. J Neurosci 20, 1722-1734. [0498] 167.
Majewska, A., and Sur, M. (2003). Motility of dendritic spines in
visual cortex in vivo: Changes during the critical period and
effects of visual deprivation. Proc Natl Acad Sci USA 100,
16024-16029. [0499] 168. Majewska, A., Tashiro, A., and Yuste, R.
(2000b). Regulation of spine calcium dynamics by rapid spine
motility. J Neurosci 20, 8262-8268. [0500] 169. Maletic-Savatic,
M., Malinow, R., and Svoboda, K. (1999). Rapid dendritic
morphogenesis in CA1 hippocampal dendrites induced by synaptic
activity. Science 283, 1923-1927. [0501] 170. Martinez-Arizala A.
(2003) Methods to measure sensory function in humans versus
animals. J Rehabil Res Dev. 40(4 Suppl 1):35-9. [0502] 171. Mataga
N, Mizuguchi Y, Hensch T K (2004) Experience-dependent pruning of
dendritic spines in visual cortex by tissue plasminogen activator.
Neuron 44:1031-1041. [0503] 172. Mataga, N., Nagai, N., and Hensch,
T. K. (2002). Permissive proteolytic activity for visual cortical
plasticity. Proc Natl Acad Sci USA 99, 7717-7721. [0504] 173.
Matus, A., Ackermann, M., Pehling, G., Byers, H. R., and Fujiwara,
K. (1982). High actin concentrations in brain dendritic spines and
postsynaptic densities. Proc Natl Acad Sci USA 79, 7590-7594.
[0505] 174. Mathiowitz and Langer (1987) J. Controlled Release
5:13-22. [0506] 175. Mathiowitz et al. (1987) Reactive Polymers
6:275-283. [0507] 176. Mathiowitz et al. (1988) J. Appl. Polymer
Sci. 35:755-774. [0508] 177. Mathiowitz et al. (1990) Scanning
Microscopy 4:329-340; [0509] 178. Mathiowitz et al. (1992) J. Appl.
Polymer Sci., 45:125-134. [0510] 179. McKinney, R. A., Capogna, M.,
Diirr, R., and Gahwiler, B. H. (1999). Miniature synaptic events
maintain dendritic spines via AMPA receptor activation. Nature
Neuroscience 2, 44-49.
[0511] 180. McManus, M. T., and P. A. Sharp. (2002) Gene silencing
in mammals by short interfering RNAs. Nature Rev. Genetics.
3:737-747. [0512] 181. Milwidsky, et al. (1991), Thrombo.
Haemostat., 65:389-393. [0513] 182. Muller, C. M., and Griesinger,
C. B. (1998). Tissue plasminogen activator mediates reverse
occlusion plasticity in visual: cortex. Nat Neurosci 1, 47-53.
[0514] 183. Nelles, G., et al. (1999) "Reorganization of sensory
and motor systems in hemiplegic stroke patients. A positron
emission study.", Stroke 30:1510-1516. [0515] 184. Nishiyama M, et
al., (2003) Cyclic AMP/GMP-dependent modulation of Ca2+ channels
sets the polarity of nerve growth-cone turning. Nature.
423(6943):990-5. [0516] 185. Noble L J, Wrathall J R (1985) Spinal
cord contusion in the rat: morphometric analyses of alterations in
the spinal cord. Exp Neurol 88:135-149. [0517] 186. Noble L J,
Wrathall J R (1989a) Correlative analysis of lesion development and
functional status after graded spinal cord contusive injuries in
the rat. Exp Neurol 103:34-40. [0518] 187. Noble L J, Wrathall J R
(1989b) Distribution and time course of protein extravasation in
the spinal cord after contusive injury. Brain Res 482:57-66. [0519]
188. Obbens E A M T, Leavents M E, Beal J W, Lee Y Y (1985). Ommaya
reservoirs in 387 cancer patients: a 15-year experience. Neurology
35:1274-1278. [0520] 189. Ohtani A, Inhibitory effect of a new
butadiene derivative on the production of plasminogen activator
inhibitor-1 in cultured bovine endothelial cells. J Biochem
(Tokyo). 1996 December; 120(6):1203-8. Related Articles, Links
[0521] 190. Olson, C. R., and Freeman, R. D. (1975). Progressive
changes in kitten striate cortex during monocular vision. J
Neurophysiol 38, 26-32. [0522] 191. Ommaya A K, Punjab M B (1963).
Subcutaneous reservoir and pump for sterile access to ventricular
cerebrospinal fluid. Lancet, 2:983-984. [0523] 192. Oray S,
Majewska A, Sur M (in press) Effects of synaptic activity on
dendritic spine motility of developing cortical layer 5 pyramidal
neurons. Cerebral Cortex. [0524] 193. Paice J A, Penn R D, Shott S
(1996). Intraspinal morphine for chronic pain: a retrospective,
multicenter study. J Pain Symptom Manage, 11:71-80. [0525] 194.
Panjabi M, Wrathall J R (1988) Biomechanical analysis of spinal
cord injury and functional loss. Spine 13:1365-1370. [0526] 195.
Parkinnen (1993), J. Biol. Chem. 268: 19726-19738. [0527] 196.
Petersen, R. C., et al., (2001). Current Concepts in Mild Cognitive
Impairment. Arch. Neural. 58, 1985-1992. [0528] 197. Pizzorusso,
T., Medini, P., Berardi, N., Chierzi, S., W. Fawcett, J., and
Maffei, L. (2002). Reactivation of ocular dominance plasticity in
the adult visual cortex. Science 298, 1248-1251. [0529] 198. Qian,
Z., Gilbert, M. E., Colicos, M. A., Kandel, E. R., and Kuhl, D.
(1993). Tissue-plasminogen activator is induced as an
immediate-early gene during seizure, kindling and long-term
potentiation. Nature 361, 453-457. [0530] 199. Raines A, Dretc.hen
K L, Marx K, Wrathall J R (1988) Spinal cord contusion in the rat:
somatosensory evoked potentials as a function of graded injury. J
Neurotrauma 5:151-160. [0531] 200. Ramos B P, et al., Dysregulation
of protein kinase a signaling in the aged prefrontal cortex: new
strategy for treating age-related cognitive decline. Neuron,
40(4):835-45. [0532] 201. Rijken, D. C. and Collen, D. (1981)
Purification and characterization of the plasminogen activator
secreted by human melanoma cells in culture. J. Biol. Chem., 256,
7035-7042. [0533] 202. Roberts L J, Finch P M, Goucke C R, Price L
M (2001). Outcome of intrathecal opioids in chronic non-cancer
pain. Eur J Pain, 5:353-61. [0534] 203. Sakata, et al. (1999), Am.
Heart J., 137:1094-1099. [0535] 204. Sali, A. and Blundell, T L,
(1993) J. Mol. Biol., 234, 779-815. [0536] 205. Santini, J T, et
al. (2000) Microchips as Controlled Drug-delivery Devices
Angewandte Chemie, International Edition, Vol. 39, pp. 2396-2407.
[0537] 206. Sawtell, N. B., Frenkel, M. Y., Philpot, B. D.,
Nakazawa, K., Tonegawa, S., and Bear, M. F. (2003). NMDA
Receptor-Dependent Ocular Dominance Plasticity in Adult Visual
Cortex. Neuron 38, 977-985. [0538] 207. Schlaug, G., et al. (1999)
The ischemic penumbra: operationally defined by diffusion and
perfusion MRI. Neurology. 53(7):1528-37. [0539] 208. Schlott, et
al. (1997), J. Biol. Chem. 272: 6067-6072. Schmidt, C. E. and
Leach, J. B., Neural tissue engineering: strategies for repair and
regeneration. Annu. Rev. Biomed. Eng., 2003. 5: 293-347. [0540]
209. Shatz, C. J., and Stryker, M. P. (1978). Ocular dominance in
layer IV of the cat's visual cortex and the effects of monocular
deprivation. J Physiol 281, 267-283. [0541] 210. Siconolfi, L. B.,
and Seeds, N. W. (2001). Induction of the plasminogen activator
system accompanies peripheral nerve regeneration after sciatic
nerve crush. J Neurosci 21, 4336-4347. [0542] 211. Sprengers, E. D.
and Kluft, C. (1987). Plasminogen activator inhibitors. Blood 69,
381-387. [0543] 212. Star, E. N., Kwiatkowski, D. J., and Murthy,
V. N. (2002). Rapid turnover of actin in dendritic spines and its
regulation by activity. Nat Neurosci 5, 239-246. [0544] 213. Sun,
et al., (2000) Pharmacol. Rev., 52:325. [0545] 214. Takenaga, M.,
et al., (2004) Optimum formulation for sustained-release insulin.
Int J Pharm. 271(1-2):85-94. [0546] 215. Teng Y D, Wrathall J R
(1996) Evaluation of cardiorespiratory parameters in rats after
spinal cord trauma and treatment with NBQX, an antagonist of
excitatory amino acid receptors. Neurosci Lett 209:5-8. [0547] 216.
Teng, Y D and Wrathall, J R (1997) J. Neuroscience; 17(11), pp.
4359-4366. [0548] 217. Teng, Y. D., et al. (2002) Functional
recovery following traumatic spinal cord injury mediated by a
unique polymer scaffold seeded with neural stem cells. Proc Natl.
Acad Sci USA, 99(5): p. 3024-9. [0549] 218. Teng, Y. D., et al.
(2004). Proc. Natl. Acad. Sci. 101(9), pp. 3071-3076. [0550] 219.
Thomas C K, Noga B R. (2003) Physiological methods to measure motor
function in humans and animals with spinal cord injury. J Rehabil
Res Dev. 40(4 Suppl 1):25-33. [0551] 220. Thomas, N., and Klibanov,
A. M. (2003) Non-viral gene therapy: polycation-mediated DNA
delivery. Appl. Microbiol. Biotechnol. 62:27-34. [0552] 221. Toombs
C F. (2001) Alfimeprase: pharmacology of a novel fibrinolytic
metalloproteinase for thrombolysis. Haemostasis. 31(3-6):141-7.
[0553] 222. Trachtenberg, J. T., and Stryker, M. P. (2001). Rapid
anatomical plasticity of horizontal connections in the developing
visual cortex. J Neurosci 21, 3476-3482. [0554] 223. Trachtenberg,
J. T., Trepel, C., and Stryker, M. P. (2000). Rapid extragranular
plasticity in the absence of thalamocortical plasticity in the
developing primary visual cortex. Science 287, 2029-2032. [0555]
224. Tutak U, Doleys D M (1996). Intrathecal infusion systems for
treatment of chronic low back and leg pain of noncancer origin.
South Med J, 89:295-300. [0556] 225. Usman, et al., (1996) Curr.
Opin. Struct. Biol., 1:527. [0557] 226. Webb, A A, et al. (2004)
Behavioural analysis of the efficacy of treatments for injuries to
the spinal cord in animals. Vet Rec. 155(8):225-30. [0558] 227.
Werb, Z. (1997). ECM and cell surface proteolysis: regulating
cellular ecology. Cell 91, 439-442. [0559] 228. Westphal M, Hild D
C, Bortey E, Delavault P, Olivares R, Warnke P C, Whittle I R,
Jaaskelainen J, Ram Z (2003). A phase 3 trial of local chemotherapy
with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in
patients with primary malignant glioma. Neuro-oncol 5:79-88. [0560]
229. White (1998), J. Am. Coll. Cardiol., 31: 487-496. [0561] 230.
Wiesel, T. N., and Hubel, D. H. (1963). Single-Cell Responses in
Striate Cortex of Kittens Deprived of Vision in One Eye. J
Neurophysiol 26, 1003-1017. [0562] 231. Winkemuller M, Winkemuller
W (1996). Long-term effects of continuous intrathecal opioid
treatment in chronic pain of nonmalignant etiology. J Neurosurg,
85:458-467. [0563] 232. Wishart D., (2005) Curr Pharm Biotechnol.,
6(2):105-20. [0564] 233. Wrathall J R, Pettegrew R, Harvey F (1985)
Spinal cord contusion in the rat: production of graded,
reproducible injury groups. Exp Neurol 88:108-122. [0565] 234. Wu M
P, Tamada J A, Brem H, Langer R (1994). In vivo versus in vitro
degradation of controlled release polymers for intracranial
surgical therapy. J Biomed Mater Res, 28:387-95. [0566] 235. Xerri,
C. et al. (1998) Plasticity of primary somatosensory motor cortex
paralleling sensorimotor skill recovery from stroke in adult
monkeys, J. Neurophysiol., 79:2119-2148. [0567] 236. Ye, B., et
al., (2004) Synthesis and biological evaluation of piperazine-based
derivatives as inhibitors of plasminogen activator inhibitor-1
(PAI-1). Bioorg Med Chem Lett. 14(3):761-5. [0568] 237. Yelverton,
E., et al., (1983) Cloning and expression of human tissue-type
plasminogen activator cDNA in E. coli., Nature, 301(5897):214-21
[0569] 238. Yuste, R., and Denk, W. (1995). Dendritic spines as
basic functional units of neuronal integration. Nature 375,
682-684. [0570] 239. Zavalova, L., (1996) Genes from the medicinal
leech (Hirudo medicinalis) coding for unusual enzymes that
specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds
and help to dissolve blood clots. Mol Gen Genet. 253(1-2):20-5.
[0571] 240. Zavalova L, et al., (2002) Fibrinogen-fibrin system
regulators from bloodsuckers. Biochemistry (Mose).
67(1):135-42.
TABLE-US-00004 [0571] Lengthy table referenced here
US20150174212A1-20150625-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00005 Lengthy table referenced here
US20150174212A1-20150625-T00002 Please refer to the end of the
specification for access instructions.
TABLE-US-00006 Lengthy table referenced here
US20150174212A1-20150625-T00003 Please refer to the end of the
specification for access instructions.
TABLE-US-00007 Lengthy table referenced here
US20150174212A1-20150625-T00004 Please refer to the end of the
specification for access instructions.
TABLE-US-00008 Lengthy table referenced here
US20150174212A1-20150625-T00005 Please refer to the end of the
specification for access instructions.
TABLE-US-00009 Lengthy table referenced here
US20150174212A1-20150625-T00006 Please refer to the end of the
specification for access instructions.
TABLE-US-00010 Lengthy table referenced here
US20150174212A1-20150625-T00007 Please refer to the end of the
specification for access instructions.
TABLE-US-00011 Lengthy table referenced here
US20150174212A1-20150625-T00008 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150174212A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
* * * * *
References