U.S. patent application number 12/960607 was filed with the patent office on 2012-01-12 for self-assembling peptides for regeneration and repair of neural tissue.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Rutledge Ellis-Behnke, Gerald Schneider, Shuguang Zhang.
Application Number | 20120010140 12/960607 |
Document ID | / |
Family ID | 35506061 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120010140 |
Kind Code |
A1 |
Ellis-Behnke; Rutledge ; et
al. |
January 12, 2012 |
SELF-ASSEMBLING PEPTIDES FOR REGENERATION AND REPAIR OF NEURAL
TISSUE
Abstract
The present invention provides methods and compositions for
enhancing regeneration and/or repair of neural tissue. One method
include providing a nanoscale structured material at the site of
injury, wherein the nanoscale structured material provides an
environment that is permissive for regeneration of neural tissue
and allows axon growth from a location on one side of a site of
injury or barrier to a location on the other side of the site of
injury or barrier. A second method includes introducing a
composition comprising self-assembling peptides into the subject at
the site of injury, wherein the peptides are amphiphilic peptides
that comprise substantially equal proportions of hydrophobic and
hydrophilic amino acids and are complementary and structurally
compatible. A variety of compositions comprising a nanoscale
structured material or precursor thereof, and an additional
substance such as a regeneration promoting factor, are also
provided. In certain embodiments of the invention the nanoscale
structured material or precursor thereof comprises self-assembling
peptides. The invention further provides compositions and methods
for repair of an intervertebral disc, including nucleus pulpusos
repair.
Inventors: |
Ellis-Behnke; Rutledge;
(Canton, MA) ; Schneider; Gerald; (Somerville,
MA) ; Zhang; Shuguang; (Lexington, MA) |
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
|
Family ID: |
35506061 |
Appl. No.: |
12/960607 |
Filed: |
December 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10968790 |
Oct 18, 2004 |
7846891 |
|
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12960607 |
|
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60512510 |
Oct 17, 2003 |
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Current U.S.
Class: |
514/8.4 ;
514/17.7; 514/7.6; 514/8.3 |
Current CPC
Class: |
A61K 38/10 20130101;
A61K 38/17 20130101; A61P 25/02 20180101; A61P 25/00 20180101 |
Class at
Publication: |
514/8.4 ;
514/17.7; 514/7.6; 514/8.3 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61K 38/18 20060101 A61K038/18; A61P 25/02 20060101
A61P025/02; A61K 38/08 20060101 A61K038/08; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The United States Government has provided grant support
utilized in the development of the present invention. In
particular, National Institutes of Health grant number EY00126 has
supported development of this invention. The United States
Government may have certain rights in the invention.
Claims
1-5. (canceled)
6. A method for treating a subject with a neuronal injury
comprising: introducing to the subject, at the site of the neuronal
injury, a composition comprising a plurality of peptides, the
peptides comprising between 8 and 16 amino acids, wherein the
peptides are amphiphilic and comprise substantially equal
proportions of alternating hydrophobic and hydrophilic amino acids
and are complementary and structurally compatible.
7-55. (canceled)
56. A method for treating a subject with spinal dysfunction or
damage comprising: introducing to the subject, at the site of the
spinal dysfunction, a composition comprising a plurality of
peptides, the peptides comprising between 8 and 16 amino acids,
wherein the peptides are amphiphilic and comprise substantially
equal proportions of alternating hydrophobic and hydrophilic amino
acids that are complementary and structurally compatible.
72. The method of claim 6, wherein each peptide has an amino acid
sequence that includes at least one instance of RADA.
73. The method of claim 6, wherein each peptide has an amino acid
sequence that includes at least one instance of RADA16 (SEQ ID NO.
1).
74. The method of claim 6, further comprising the step of
assembling the peptides in situ.
75. The method of claim 74, further comprising the step of adding
ions to the composition to induce assembly.
76. The method of claim 6, wherein the peptides are self-assembled
prior to the introducing step.
77. The method of claim 6, wherein the peptides are self-assembled
during the introducing step.
78. The method of claim 6, wherein the composition is in
solution.
79. The method of claim 78, wherein the composition has a peptide
concentration between about 0.1 percent (1 mg/ml) and about 10
percent (100 mg/ml).
80. The method of claim 79, wherein the composition has a peptide
concentration between about 0.5 percent (5 mg/ml) and about 5
percent (50 mg/ml).
81. The method of claim 79, wherein the composition has a peptide
concentration of about 1 mg/ml, about 5 mg/ml, about 10 mg/ml,
about 15 mg/ml, about 20 mg/ml or about 25 mg/ml.
82. The method of claim 6, wherein the introducing step is by
injection.
83. The method of claim 6, wherein the site of the neuronal injury
is in the central nervous system.
84. The method of claim 6, wherein the site of the neuronal injury
is in the peripheral nervous system.
85. The method of claim 6, wherein the composition comprises a
regeneration promoting factor.
86. The method of claim 85, wherein the regeneration promoting
factor is selected from the group consisting of: nerve growth
factor (NGF), brain derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), neurotrophin-4/5 (Nt-4/5), ciliary
neurotrophic factor (CNTF), glial cell derived growth factor
(GDNF), neurturin (NTN), persephin (PSP), artemin (ART), acidic
fibroblast growth factor (aFGF), basic fibroblast growth factor
(bFGF), growth-associated protein 43 (GAP-43),
cytoskeleton-associated protein 23 (CAP-23), B-cell lymphoma 2
(Bcl-2), L1, neural cell adhesion molecule (NCAM), N-cadherin,
agrin, laminin, acetylcholine receptor inducing activity protein
(ARIA), a semaphorin, a slit protein, a netrin, and an ephrin.
87. The method of claim 1, wherein the composition comprises an
siRNA, shRNA, or a template for synthesis of an siRNA or shRNA that
is targeted to a transcript that encodes the molecule that inhibits
regeneration or repair.
88. A method for preventing the formation of scar tissue in nerve
tissue in a subject comprising: introducing to the subject, at the
site of the nerve tissue, a composition comprising a plurality of
peptides, the peptides comprising between 8 and 16 amino acids,
wherein the peptides are amphiphilic and comprise substantially
equal proportions of alternating hydrophobic and hydrophilic amino
acids that are complementary and structurally compatible.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application 60/512,510, filed Oct. 17, 2003, which is incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0003] While soft tissues (e.g., muscle and skin) and bone possess
considerable capacity for recovery after injury, inadequate nerve
repair frequently limits the extent to which normal function is
regained. In the peripheral nervous system (PNS), nerves are often
able to regenerate on their own, if the injury is small enough.
Larger injuries may be effectively treated surgically, either by
direct reconnection of damaged nerve ends with the nerve sheath the
axons previously used to reach their destination or with grafts
harvested from elsewhere in the body. However, clinical functional
recovery rates generally approach only 80% following nerve graft,
and the procedure has the additional disadvantage of requiring two
surgeries. An alternative approach to nerve repair in the PNS is to
provide an artificial conduit, such as the NeuraGen.TM. Nerve Guide
(Integra LifeSciences, Plainsboro, N.J.), a collagen tube providing
a conduit for axonal growth across a nerve gap [83]. However, this
treatment is typically reserved for small defects (e.g., several
millimeters).
[0004] Clinical treatment of injuries to the central nervous system
(CNS) is considerably less successful. Unlike nerves in the PNS,
axons in the CNS do not undergo significant regeneration in their
native environment. Thus initial therapy is usually limited to
removal of bone fragments to prevent secondary injury and
administration of drugs such as corticosteroids to reduce swelling.
Currently there is no effective treatment available to completely
restore nerve function in the CNS. Rehabilitation, in which
patients train remaining nerves to compensate for loss due to
injury, remains the mainstay of therapy.
[0005] Despite the relatively bleak outlook for regeneration of
injured nerves in the CNS, advances in other areas of medical care
have greatly improved the rate of survival of patients with
traumatic CNS injuries, e.g., traumatic injury to the spinal cord.
Nearly 94% of patients with spinal cord injuries survive the first
year following injury, and of these, 93% are able to be discharged
back into the community [84]. Approximately 10,000-12,000
individuals suffer spinal cord injuries each year in the United
States, bringing the projected prevalence rate in the United States
to nearly 280,000 by the year 2014 [84].
[0006] The number of patients with traumatic spinal cord or brain
injury is dwarfed by the number of persons who experience damage to
the CNS due to diseases and stroke [85], or as a consequence of
conditions such as primary brain tumors, brain metastastes from
tumors elsewhere in the body, or surgery for these conditions.
Survivors of stroke and survivors of brain lesions due to tumors or
surgical damage frequently experience permanent deficits in
function due to loss of brain tissue either as a direct consequence
of injury or secondary to events such as swelling and/or release of
neurotoxic substances from necrotic tissue. While emerging
therapies for these patients may offer the potential to limit such
damage, prospects for restoring function lost due to death of brain
cells and disruption of brain architecture remain poor, and
therapeutic efforts focus on rehabilitation.
[0007] It is therefore evident that a significant need in the art
exists for improved treatments that would enhance repair and
regeneration in the CNS. In addition, there remains a need in the
art for improved treatments that would enhance nerve repair and
regeneration in the PNS since current treatments, while frequently
effective, have a number of disadvantages.
SUMMARY OF THE INVENTION
[0008] The present invention addresses these needs, among others.
In one aspect, the invention provides a method for enhancing repair
or regeneration of neural tissue at a site of injury in a mammalian
subject comprising: providing a nanoscale structured material, or a
precursor thereof, at the site of injury, wherein the nanoscale
structured material provides an environment that is permissive for
regeneration of neural tissue and allows axon growth from one side
of a site of injury or barrier to the other side of the site of
injury or barrier. In certain embodiments of the invention the step
of providing comprises introducing a composition comprising a
precursor of the nanoscale structured material at or in the
vicinity of the site of injury, wherein the precursor assembles in
situ to form the nanscale structured material.
[0009] In another aspect, the invention provides a method for
enhancing repair or regeneration of neural tissue at a site of
injury in a mammalian subject comprising: introducing a composition
comprising self-assembling peptides (SAPs), also referred to as
sapeptides, into the subject at or in the vicinity of the site of
injury, wherein the peptides are amphiphilic peptides that comprise
substantially equal proportions of hydrophobic and hydrophilic
amino acids and are complementary and structurally compatible. In
certain embodiments of the invention the peptides self-assemble
into a macroscopic structure, e.g., a beta-sheet macroscopic
structure. "In the vicinity" of a site of injury refers to a
location close enough to the site of injury that the composition
can reach the site of injury in a therapeutically effective
amount.
[0010] In another aspect, the invention provides a composition for
enhancing regeneration or repair of neural tissue comprising: (i) a
nanoscale structured material, or a precursor thereof, wherein the
nanoscale structured material provides an environment that is
permissive for regeneration of neural tissue and allows axon growth
from one side of a site of injury or barrier to the other side of
the site of injury or barrier; and (ii) a substance selected from
the group consisting of: regeneration promoting factors, substances
that counteract a molecule that inhibits regeneration or growth of
neural tissue, nutrients, and templates for synthesis of a
regeneration enhancing protein.
[0011] The invention further provides a composition for enhancing
regeneration or repair of neural tissue comprising: (i)
self-assembling peptides, wherein the peptides are amphiphilic
peptides that comprise substantially equal proportions of
hydrophobic and hydrophilic amino acids and are complementary and
structurally compatible; and (ii) a substance selected from the
group consisting of: regeneration promoting factors, substances
that counteract a molecule that inhibits regeneration or growth of
neural tissue, nutrients, and templates for synthesis of a
regeneration enhancing protein. In certain embodiments of the
invention the peptides self-assemble into a macroscopic structure,
e.g., a beta-sheet macroscopic structure.
[0012] The invention further provides compositions and methods for
replacement, repair, and/or regeneration of spinal tissue. In
particular, the compositions and methods are of use for
replacement, repair, and/or regeneration of intervertebral disc
tissue, e.g., nucleus pulposus tissue. The intervertebral disc
tissue to be replaced, repaired, or regenerated may be tissue that
is damaged in an accident, during surgery, etc., or may be tissue
that has degenerated or herniated for some other reason. The
invention provides an intervertebral disc replacement. The
invention further provides a nucleus pulposus replacement.
[0013] 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: 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. In addition, the references listed
in [86] are incorporated herein by reference. In the event of a
conflict between the instant specification and any of the
incorporated references, the specification will control.
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1A-1F shows scanning electron microscope (SEM) images
of a peptide hydrogel. At low magnifications the scaffold resembles
felt (a-c). At high magnifications, the interwoven nanofibers in
the structure can be seen (d-f).
[0015] FIG. 1G shows the proposed molecular model of the
self-assembly of peptides (in this case a peptide containing RADA
(SEQ ID NO: 31) sequences). The alanines form an overlapping
hydrophobic interaction on one side of the peptide. The arginines
and aspartates form complementary ionic bonds on the other side.
These ionic self-complementary can undergo molecular
self-assembly.
[0016] FIG. 2 is a parasagittal view of an adult hamster brain
showing a schematic representation of the lesion that transected
the optic tract in the middle of the superior colliculus. Scale bar
500 microns.
[0017] FIG. 3 is a dorsal view reconstruction of the hamster brain
with cortex removed. Rostral is to the right and caudal is to the
left. The straight black line is the location of the transection of
the optic tract made at postnatal day 2 (P2). Other areas of the
brain shown include the superior colliculus (SC), pretectal area
(PT), lateral posterior nucleus (LP), medial geniculate body (MGB),
and inferior colliculus (IC).
[0018] FIG. 4A is a schematic illustration of a parasagittal
section of the dorsal midbrain of a hamster; rectangle is the
approximate location of images 4A and 4B. Scale bar 500
microns.
[0019] FIG. 4B is an image showing a parasagittal section from the
brain of a 1 month old hamster with a saline-injected lesion
inflicted at day P2. Arrows show the path and extent of the knife
cut.
[0020] FIG. 4C is an image showing a part of a section similar to
that shown in FIG. 4B, from a 1 month hamster which had a lesion
inflicted at day P2 that was injected with 10 microliters of 1% SAP
RADA16-I. Retinal projections are shown in green. Scale bar 100
microns. The inset shows a SAP scaffold seen in much higher
magnification (scale bar 1 micron), taken with a scanning electron
microscope.
[0021] FIG. 4D shows an enlarged view of area indicated with the
box in FIG. 4C.; Regrown axons are seen in white. Scale bar 100
microns.
[0022] FIGS. 5A and 5B show images of parasaggital sections from
animals sacrificed at 24 and 72 hours postlesion and injection of
SAP. FIG. 5A shows 24 hour survival cases. FIG. 5B shows 72 hour
survival cases. In each panel, the image on the left shows lesions
injected with SAP and Congo Red (similar to saline injected
controls), while the image on the right shows lesions injected with
SAP alone.
[0023] FIGS. 6A and 6B show images of parasaggital sections from
animals one month postlesion and injection of SAP. FIG. 6A shows
images from two animals injected with SAP and Congo Red (similar to
saline controls). FIG. 6B shows images from two animals injected
with SAP alone.
[0024] FIG. 7A shows a dark-field photo (composite) of a lesion
site 2 months after infliction of the lesion and injection of SAP.
FIG. 7B is a corresponding bright-field image. Scale bars 100
microns.
[0025] FIG. 8 shows frames from a videotape of an adult animal
whose ability to respond to a visual stimulus following is being
tested and quantified. The figure shows an animal that had been
subjected to transection of the optic tract. Following transection
of the optic tract, peripheral nerve (PN) bridges were surgically
implanted to promote neural tissue regeneration. The animal turns
toward the stimulus in the affected left visual field in small
steps, prolonged here by movements of the stimulus away from him.
Each frame is taken from a single turning movement, at times 0.00,
0.27, 0.53, and 0.80 sec from movement initiation. The animal
reached the stimulus just after the last frame. This is about 0.20
sec slower than most turns by a normal animal and shows a return of
functional vision after treatment. Similar results were obtained
following treatment with SAP.
[0026] FIG. 9 is a dorsal view reconstruction of the adult hamster
brain depicting the lesion of the brachium of the SC (red). The
lesion site also indicate the location of the SAP injection.
IC=inferior colliculus, LGB=lateral geniculate body, LP=lateral
posterior nucleus, MGB=medial geniculate body, PT=pretectal
area.
[0027] FIG. 10 is a fluorescence microscopy image of a parasagittal
section of the dorsal midbrain of a hamster. Rostral is left and
caudual is right. The section is from an 8 month old hamster and
was taken 2 months following injection of 10 .mu.l of 1% SAP
RAD16-I into a lesion that transected the brachium of the SC. The
bright yellow in the middle of the picture that extends from the
lower left to the upper right is in the middle of the lesion site.
Retinal projections are in green.
[0028] FIG. 11 is a parasagittal section of the dorsal midbrain of
a hamster. Rostral is left and caudual is right. The section is
from an 8 month old hamster and was taken 2 months following
injection of 10 .mu.l of 1% SAP RAD16-I into a lesion that
transected the brachium of the SC. The lesion site extends from top
of the picture to the bottom in the middle of the picture. White
arrows indicate the middle of the lesion. Retinal projections are
in green. This figure was taken at lower magnification than FIG. 11
and thus shows healing of the lesion over a larger scale. The site
of the cut is essentially indistinguishable from the surrounding
unlesioned tissue.
[0029] FIG. 12 shows behavioral data from six animals from
behavioral tests that assessed the response of animals to a visual
stimulus. The animals had been subjected to a lesion that
transected the brachium of the SC at 2 months of age. The lesion
was injected with 10 .mu.l of 1% SAP RAD 16-I at the time of
surgery, and the animals were allowed to recover. Animals were
tested by assessing their response when presented with a stimulus
(a seed) that was brought into the visual field from the side. The
x axis show the dates tested. The y-axis represents the number of
times the animal turned toward the stimulus during testing,
expressed as a percentage of the number of times the stimulus was
presented on the side of the animal that was rendered blind by the
lesion. An upward trend signifies improving vision while a downward
trend would signify worsening vision. The dark blue line is the
behavioral results of an adult animal the anatomical results for
which are shown in FIG. 9. The aqua line is believed to represent
an animal that experienced a rapid return of vision, which was
complete prior to the start of behavioral testing.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE
INVENTION
I. Definitions
[0030] The following definitions are of use in understanding the
invention.
[0031] "Antibody" refers to an immunoglobulin, which may be natural
or wholly or partially synthetically produced in various
embodiments of the invention. An antibody may be derived from
natural sources (e.g., purified from a rodent, rabbit, chicken (or
egg) from an animal that has been immunized with an antigen or a
construct that encodes the antigen) partly or wholly synthetically
produced. An antibody may be a member of any immunoglobulin class,
including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
The antibody may be a fragment of an antibody such as an Fab',
F(ab').sub.2, scFv (single-chain variable) or other fragment that
retains an antigen binding site, or a recombinantly produced scFv
fragment, including recombinantly produced fragments. See, e.g.,
Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and
references therein. Preferred antibodies, antibody fragments,
and/or protein domains comprising an antigen binding site may be
generated and/or selected in vitro, e.g., using techniques such as
phage display (Winter, G. et al. 1994. Annu. Rev. Immunol.
12:433-455, 1994), ribosome display (Hanes, J., and Pluckthun, A.
Proc. Natl. Acad. Sci. USA. 94:4937-4942, 1997), etc. In various
embodiments of the invention the antibody is a "humanized" antibody
in which for example, a variable domain of rodent origin is fused
to a constant domain of human origin, thus retaining the
specificity of the rodent antibody. It is noted that the domain of
human origin need not originate directly from a human in the sense
that it is first synthesized in a human being. Instead, "human"
domains may be generated in rodents whose genome incorporates human
immunoglobulin genes. See, e.g., Vaughan, et al., Nature
Biotechnology, 16: 535-539, 1998. An antibody may be polyclonal or
monoclonal, though for purposes of the present invention monoclonal
antibodies are generally preferred.
[0032] "Approximately", as used herein, is generally taken to
include numbers that fall within a range of 10% in either direction
(greater than or less than) the number unless otherwise stated or
otherwise evident from the context (except where such number would
exceed 100% of a possible value). Where ranges are stated, the
endpoints are included within the range unless otherwise stated or
otherwise evident from the context.
[0033] "Biocompatible": A material is considered biocompatible with
respect to cells if it is substantially non-toxic to cells in
vitro, e.g., if its addition to cells in culture results in less
than or equal to 20% cell death. A material is considered
biocompatible with respect to a recipient, if it is substantially
non-toxic to the recipient's cells 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., an
immunological or inflammatory reaction, unacceptable scar tissue
formation, etc.
[0034] "Biodegradable" means capable of being broken down
physically and/or chemically within cells or 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 cells or within the body, etc., to form smaller chemical
species which can be metabolized and, optionally, reused, and/or
excreted or otherwise disposed of. Preferably a biodegradable
compound is biocompatible.
[0035] "Biomolecule", as used herein, a "biomolecule" refers to a
molecule such as a protein, peptide, proteoglycan, lipid,
carbohydrate, or nucleic acid having characteristics typical of
molecules found in living organisms. A biomolecule may be naturally
occurring or may be artificial (not found in nature and not
identical to a molecule found in nature). For example, a protein
having a sequence or modification resulting from the mental process
of man, and not occurring in nature, is considered an artificial
biomolecule. A protein (e.g., an oligonucleotide) having a sequence
or modification resulting from the mental process of man, and not
occurring in nature, is considered an artificial biomolecule.
[0036] "Chemotactic substance", as used herein, refers to a
substance having the ability to recruit cells to a site at which
the substance is present. Such cells may, for example, have the
potential to contribute to the formation or repair of a tissue
(e.g., by providing growth factors) or to contribute to an immune
response. Certain chemotactic substances may also function as
proliferation agents.
[0037] "Central nervous system": 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. Microglial cells
serve immune system functions.
[0038] "Complementary" means having the capability of forming ionic
or hydrogen bonding interactions between hydrophilic residues from
adjacent peptides in the scaffold. Each hydrophilic residue in a
peptide either hydrogen bonds or ionically pairs with a hydrophilic
residue on an adjacent peptide, or is exposed to solvent. Pairing
may also involve van der Waals forces.
[0039] "Effective amount", in reference to an active agent such as
a self-assembling peptide or biomolecule, refers to the amount
necessary to elicit a desired biological response. As will be
appreciated by those of ordinary skill in this art, the effective
amount of an agent may vary depending on such factors as the
desired biological endpoint, the agent to be delivered, the nature
of the site to which the agent is delivered, the nature of the
conditions for which the agent is administered, etc. For example,
the effective amount of a composition for treatment of an injury to
the nervous system may be an amount sufficient to promote healing
of an injury to a greater extent than would occur in the absence of
the composition, and/or to promote axon regrowth to a greater
extent than would occur in the absence of the composition. Axon
regrowth can include axon extension, axon regrowth across a lesion
or barrier, etc. An effective amount may be an amount sufficient to
promote functional recovery of one or more nervous system functions
to a greater extent than would occur in the absence of the
composition.
[0040] "Gene therapy vector" refers to a vector, as defined below,
that comprises a template for transcription of a therapeutic
nucleic acid molecule (e.g., an siRNA strand, shRNA strand,
antisense RNA strand, or ribozyme), or comprises a template for
transcription of a nucleic acid molecule that is translated to
produce a therapeutic polypeptide.
[0041] "Iso-osmotic solute" means a non-ionizing compound dissolved
in an aqueous solution such that the resulting solution (an
iso-osmotic solution) has an osmotic pressure or osmolality
compatible with cell viability over periods of time greater than 1
minute, preferably greater than 5 minutes, yet more preferably
greater than 10 minutes, yet more preferably at least 1 hour. In
general, a preferred iso-osmotic solution has an osmotic pressure
that approximates the osmotic pressure of the extracellular or
intracellular environment of cells (e.g., in tissue culture medium,
within a subject, etc.). For example, an iso-osmotic solution may
have an osmotic pressure that is within 290.+-.10 mosm/kg H.sub.2O.
Preferred iso-osmotic solutes include carbohydrates, such as
monosaccharides or disaccharides. Examples of preferred
carbohydrates include sucrose, glucose, galactose, fructose,
ribose, mannose, arabinose, and xylose. Still another preferred
iso-osmotic solute is glycerol, such as an aqueous solution of
glycerol that is between 5 to 20% (v/v) glycerol.
[0042] "Macroscopic" means having dimensions large enough to be
visible under magnification of 10-fold or less. In preferred
embodiments of the invention a macroscopic structure is visible to
the naked eye. A macroscopic structure may be transparent and may
be two-dimensional, or three-dimensional. If two-dimensional, in
certain embodiments of the invention it comprises more than a
single layer of molecules, e.g., 2, 3, or more layers of molecules.
Typically each dimension is at least 10 .mu.m, in size. In certain
embodiments at least two dimensions are at least 100 .mu.m, or at
least 1000 .mu.m in size. Frequently at least two dimensions are at
least 1-10 mm in size, 10-100 mm in size, or more. The relevant
dimensions may be, e.g., length, width, depth, breadth, height,
radius, diameter, circumference, in the case of structures that
have a regular two or three-dimensional shape such as a sphere,
cylinder, cube, etc., or an approximation of any of the foregoing
in the case of structures that do not have a regular two or
three-dimensional shape Other relevant dimensions may also be
used.
[0043] "Microfiber", as used herein, refers to a fiber having a
diameter of microscale dimensions. Typically a microscale fiber has
a diameter of 500 .mu.m or less, a diameter of less than 100 .mu.m,
a diameter of less than 50 .mu.m, a diameter of less than 20 .mu.m,
a diameter of between 10 and 20 .mu.m, or a diameter of between 5
and 10 .mu.m.
[0044] "Microscale", as used herein, generally refers to structures
having dimensions that may most conveniently be expressed in terms
of micrometers. For example, the term "microscale structure" may
refer to a structure having dimensions of approximately 500 .mu.m
or less, approximately 100 .mu.m or less, approximately 50 .mu.m or
less, approximately 20-50 .mu.m, approximately 10-20 .mu.m,
approximately 5-10 .mu.m, approximately 1-5 .mu.m, approximately 1
.mu.m, or between 0.5 and 1 .mu.m. One of ordinary skill in the art
will recognize that the length of such structures may run into the
millimeters, but that most dimensions are in the micrometer
range.
[0045] "Nanofiber" refers to a fiber having a diameter of nanoscale
dimensions. Typically a nanoscale fiber has a diameter of 500 nm or
less. According to certain embodiments of the invention a nanofiber
has a diameter of less than 100 nm. According to certain other
embodiments of the invention a nanofiber has a diameter of less
than 50 nm. According to certain other embodiments of the invention
a nanofiber has a diameter of less than 20 nm. According to certain
other embodiments of the invention a nanofiber has a diameter of
between 10 and 20 nm. According to certain other embodiments of the
invention a nanofiber has a diameter of between 5 and 10 nm.
According to certain other embodiments of the invention a nanofiber
has a diameter of less than 5 nm.
[0046] "Nanoscale", as used herein, generally refers to structures
having dimensions that may most conveniently be expressed in terms
of nanometers, or materials composed therefrom. For example, the
term "nanoscale structured material" or "nanoscale scaffold" may
refer to a material composed of structures (e.g., nanofibers)
having dimensions of approximately 500 nm or less, approximately
100 nm or less, approximately 50 nm or less, approximately 20-50
nm, approximately 10-20 nm, approximately 5-10 nm, approximately
1-5 nm, approximately 1 nm, or between 0.1 and 1 nm. The ranges
listed are assumed to include both endpoints. The relevant
dimensions may be, e.g., length, width, depth, breadth, height,
radius, diameter (e.g., pore diameter), circumference, in the case
of structures that do not have a regular two or three-dimensional
shape such as a sphere, cylinder, cube, etc., or an approximation
of any of the foregoing, e.g., in the case of structures that do
not have a regular two or three dimensional shape. Any other
relevant dimensions may also be used to determine whether a
material is a nanoscale structured material, depending, for example
on the shape of a structure formed therefrom. One of ordinary skill
in the art will recognize that one or more dimensions of a
nanoscale structure need not be in the nanometer range. For
example, the length of such structures may run into the micron
range or longer. However, generally most dimensions are in the
nanometer range.
[0047] "Nanoscale environment scaffold" or "nanoscale environment
structure" refers to a scaffold or structure comprising nanofibers.
According to certain embodiments of the invention at least 50% of
the fibers comprising the scaffold or structure are nanofibers.
According to certain embodiments of the invention at least 75% of
the fibers comprising the scaffold or structure are nanofibers.
According to certain embodiments of the invention at least 90% of
the fibers comprising the scaffold or structure are nanofibers.
According to certain embodiments of the invention at least 95% of
the fibers comprising the scaffold or structure are nanofibers.
According to certain embodiments of the invention at least 99% of
the fibers comprising the scaffold or structure are nanofibers. Of
course the scaffold or structure may also comprise non-fiber
constituents, e.g., water, ions, growth and/or
differentiation-inducing agents such as growth factors, therapeutic
agents, or other compounds, which may be in solution in the
scaffold or structure and/or bound to the scaffold or structure.
The term "scaffold" is not intended to impose any functional or
structural limitation on a composition of the invention but merely
serves to indicate that the composition may, for example, provide a
supporting framework for axon extension or growth, cell or tissue
growth, cell migration, extracellular matrix deposition, etc.
[0048] "Neural tissue", for purposes of this invention, refers to
one or more components of the central nervous system and/or
peripheral nervous system. Such components include brain tissue and
nerves. 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 extracellular matrix components, basal lamina (in
the PNS), endoneurium, perineurium, and epineurium in nerves, etc.
Additional normeural cells such as fibroblasts, endothelial cells,
macrophages, etc., are typically also present. See [86] for further
description of the structure of various neural tissues.
[0049] "Operably linked or operably associated" refers to a
relationship between two nucleic acid sequences wherein the
expression of one of the nucleic acid sequences is controlled by,
regulated by, modulated by, etc., the other nucleic acid sequences,
or a relationship between two polypeptides wherein the expression
of one of the polypeptides is controlled by, regulated by,
modulated by, etc., the other polypeptide. For example, the
transcription of a nucleic acid sequence is directed by an operably
linked promoter sequence; post-transcriptional processing of a
nucleic acid is directed by an operably linked processing sequence;
the translation of a nucleic acid sequence is directed by an
operably linked translational regulatory sequence; the transport,
stability, or localization of a nucleic acid or polypeptide is
directed by an operably linked transport or localization sequence;
and the post-translational processing of a polypeptide is directed
by an operably linked processing sequence. Preferably a nucleic
acid sequence that is operably linked to a second nucleic acid
sequence, or a polypeptide that is operatively linked to a second
polypeptide, is covalently linked, either directly or indirectly,
to such a sequence, although any effective three-dimensional
association is acceptable.
[0050] "Peptide", "polypeptide", or "protein", as used herein,
refers to a string of at least two amino acids linked together by
peptide bonds. A peptide generally represents a string of between
approximately 2 and 200 amino acids, more typically between
approximately 6 and 64 amino acids. Typically, the self-assembling
portion of a self-assembling peptide is about 8-24, frequently
about 12-20, or 16-20 amino acids. Peptide may refer to an
individual peptide or a collection of peptides. Inventive peptides
typically contain only natural amino acids, although non-natural
amino acids (i.e., compounds that do not occur in nature but that
can be incorporated into a polypeptide chain; see, for example, the
Web site having URL
www.cco.caltech.edu/.about.dadgrp/Unnatstruct.gif, which displays
structures of non-natural amino acids that have been successfully
incorporated into functional ion channels) and/or amino acid
analogs as are known in the art may alternatively be employed. In
particular, D amino acids may be used. Also, in various embodiments
of the invention one or more of the amino acids in an inventive
peptide may be altered or derivatized, for example, by the addition
of a chemical entity such as an acyl group, a carbohydrate group, a
carbohydrate chain, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation or
functionalization, etc. In certain embodiments of the invention a
peptide is branched, in which case it contains at least two amino
acid polymers, each of which consists of at least 3 amino acids
joined by peptide bonds, but the two amino acid polymers themselves
are not linked by a peptide bond.
[0051] "Peripheral nervous system (PNS)", for purposes of the
present invention, 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.
[0052] "Polynucleotide" or "oligonucleotide" refers to a polymer of
nucleotides. As used herein, an oligonucleotide is typically less
than 100 nucleotides in length. A polynucleotides or
oligonucleotide may also be referred to as a nucleic acid.
Naturally occurring nucleic acids include DNA and RNA. Typically, a
polynucleotide comprises at least three nucleotides. A nucleotide
comprises a nitrogenous base, a sugar molecule, and a phosphate
group. A nucleoside comprises a nitrogenous base linked to a sugar
molecule. In a polynucleotide or oligonucleotide, phosphate groups
covalently link adjacent nucleosides to form a polymer. The polymer
may include natural nucleosides (e.g., adenosine, thymidine,
guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,
deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and
2-thiocytidine), chemically modified bases, biologically modified
bases (e.g., methylated bases), intercalated bases, modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose). The phosphate groups in a polynucleotide or
oligonucleotide are typically considered to form the
internucleoside backbone of the polymer. In naturally occurring
nucleic acids (DNA or RNA), the backbone linkage is via a 3' to 5'
phosphodiester bond. However, polynucleotides and oligonucleotides
containing modified backbones or non-naturally occurring
internucleoside linkages can also be used in the present invention.
Such modified backbones include ones that have a phosphorus atom in
the backbone and others that do not have a phosphorus atom in the
backbone. Examples of modified linkages include, but are not
limited to, phosphorothioate and 5'-N-phosphoramidite linkages. See
U.S. Patent Application No. 20040092470 and references therein for
further discussion of various nucleotides, nucleosides, and
backbone structures that can be used in the polynucleotides or
oligonucleotides described herein, and methods for producing them.
Polynucleotides and oligonucleotides need not be uniformly modified
along the entire length of the molecule. For example, different
nucleotide modifications, different backbone structures, etc., may
exist at various positions in the polynucleotide or
oligonucleotide. Any of the polynucleotides described herein,
including siRNAs, shRNAs, ribozymes, antisense RNAs, may utilize
these modifications.
[0053] The polynucleotides may be of any size or sequence, and they
may be single- or double-stranded. A polynucleotide may be, for
example, a modified or unmodified circular plasmid, a linearized
plasmid, a cosmid, a viral genome, a modified viral genome, an
artificial chromosome, etc. In certain preferred embodiments, the
polynucleotide is greater than 100 base pairs long. In certain
other preferred embodiments, the polynucleotide is greater than
1000 base pairs long and may be greater than 10,000 base pairs
long. The polynucleotide is preferably purified and substantially
pure. Preferably, the polynucleotide is greater than 50% pure, more
preferably greater than 75% pure, and most preferably greater than
95% pure. The polynucleotide may be provided by any means known in
the art. In certain preferred embodiments, the polynucleotide has
been engineered using recombinant techniques (for a more detailed
description of these techniques, please see Ausubel et al. Current
Protocols in Molecular Biology (John Wiley & Sons, Inc., New
York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory
Press: 1989). The polynucleotide may also be obtained from natural
sources and purified from contaminating components found normally
in nature. The polynucleotide may be synthesized using enzymatic
techniques, either within cells or in vitro. The polynucleotide may
also be chemically synthesized in a laboratory. In a preferred
embodiment, the polynucleotide is synthesized using standard solid
phase chemistry. The polynucleotide may be modified by chemical or
biological means. In certain preferred embodiments, these
modifications lead to increased stability of the polynucleotide.
Modifications include methylation, phosphorylation, end-capping,
etc.
[0054] "Proliferation agent" and "mitogenic agent" are used herein
interchangeably to refer to the ability of a substance to enhance
the proliferation of cells.
[0055] "Regeneration", as used in reference to regeneration of
neural tissue in various embodiments of the invention, may include
any aspect of anatomical or functional restoration of the condition
of the neural tissue prior to an injury, which involves production
of new neural tissue (by which is meant either cells or portions of
cells). In certain embodiments of the invention production of new
neural tissue includes growth (e.g., increase in size along one or
more dimension, increase in volume, etc.) of existing cells, e.g.,
neurons. Regeneration may thus include growth of axons or other
neuron processes. Such processes may arise directly from the cell
body or may be extensions of processes that were severed or damaged
due to injury. The new tissue may replace tissue that was
previously present. In certain embodiments of the invention
production of new neural tissue includes division of existing cells
(cell proliferation).
[0056] "Repair", as used in reference to the repair of neural
tissue in various embodiments of the invention, may include any
aspect of anatomical or functional restoration of the condition of
the neural tissue prior to an injury. For example, it may include
restoration of physical continuity between portions of tissue that
were separated by an injury. Preferably such restoration of
physical continuity includes reapposition or reconnection of the
portions of tissue without appreciable separation by tissue of a
type that was not present prior to the injury, such as scar tissue.
Repair may thus include filling of a defect in neural tissue,
preferably by reapposition of portions of tissue separated by the
defect and/or by growth of new neural tissue, rather than by
development of scar tissue. Repair may, but need not, include
growth or development of new tissue. Thus regeneration may be
considered one aspect of repair, but repair can occur without
evidence of new tissue growth.
[0057] "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.
[0058] "Solution that is substantially free of ions" means a
solution to which no ions (or salts thereof) have been added or in
which the concentration of ions (or salts thereof) is less than
0.01 or 0.001 mM.
[0059] "Structurally compatible" means capable of maintaining a
sufficiently constant intrapeptide distance to allow structure
formation. In certain embodiments of the invention the variation in
the intrapeptide distance is less than 4, 3, 2, or 1 angstroms. It
is also contemplated that larger variations in the intrapeptide
distance may not prevent structure formation if sufficient
stabilizing forces are present. This distance may be calculated
based on molecular modeling or based on a simplified procedure that
has been previously reported (U.S. Pat. No. 5,670,483). In this
method, the intrapeptide distance is calculated by taking the sum
of the number of unbranched atoms on the side-chains of each amino
acid in a pair. For example, the intrapeptide distance for a
lysine-glutamic acid ionic pair is 5+4=9 atoms, and the distance
for a glutamine-glutamine hydrogen bonding pair is 4+4=8 atoms.
Using a conversion factor of 3 angstroms per atom, the variation in
the intrapeptide distance of peptides having lysine-glutamic acid
pairs and glutamine-glutamine pairs (e.g., 9 versus 8 atoms) is 3
angstroms.
[0060] "Subject", as used herein, refers to an individual to whom
an agent is to be delivered, e.g., for experimental, diagnostic,
and/or therapeutic purposes. Preferred subjects are mammals,
particularly domesticated mammals (e.g., dogs, cats, etc.),
primates, or humans.
[0061] "Therapeutic molecule, substance, compound, or agent" refers
to a molecule or combination of molecules of any type that, when
administered to a subject in need thereof, alleviates one or more
symptoms of a disease or undesired clinical condition, reduces the
severity of a disease or clinical condition, prevents or lessens
the likelihood of development of a disease or undesired clinical
condition, or facilitates repair or regeneration of tissue in a
manner other than simply providing general nutritional support to
the subject. It is to be understood that a therapeutic molecule is
generally to be administered in an effective amount, i.e., an
amount sufficient to achieve a clinically significant result. A
therapeutic molecule can be a small molecule, a biomolecule, etc.
See Goodman and Gilman's The Pharmacological Basis of Therapeutics,
10.sup.th Ed., and Katzung, Basic and Clinical Pharmacology, for
examples.
[0062] "Treating", as used herein, can generally include reversing,
alleviating, inhibiting the progression of, preventing or reducing
the likelihood of the disease, disorder, or condition to which such
term applies, or one or more symptoms or manifestations of such
disease, disorder or condition. "Preventing" refers to causing a
disease, disorder, condition, or symptom or manifestation of such,
or worsening of the severity of such, not to occur.
[0063] "Vector" is used herein to refer to a nucleic acid or a
virus or portion thereof (e.g., a viral capsid) capable of
mediating entry of, e.g., transferring, transporting, etc., a
nucleic acid molecule into a cell. Where the vector is a nucleic
acid, the nucleic acid molecule to be transferred is generally
linked to, e.g., inserted into, the vector nucleic acid molecule. A
nucleic acid vector may include sequences that direct autonomous
replication (e.g., an origin of replication), or may include
sequences sufficient to allow integration of part of all of the
nucleic acid into host cell DNA. Useful nucleic acid vectors
include, for example, DNA or RNA plasmids, cosmids, and naturally
occurring or modified viral genomes or portions thereof or nucleic
acids (DNA or RNA) that can be packaged into viral capsids. Plasmid
vectors typically include an origin of replication and one or more
selectable markers. Plasmids may include part or all of a viral
genome (e.g., a viral promoter, enhancer, processing or packaging
signals, etc.). Viruses or portions thereof (e.g., viral capsids)
that can be used to introduce nucleic acid molecules into cells are
referred to as viral vectors. Useful viral vectors include
adenoviruses, retroviruses, lentiviruses, vaccinia virus and other
poxviruses, herpex simplex virus, and others. Viral vectors may or
may not contain sufficient viral genetic information for production
of infectious virus when introduced into host cells, i.e., viral
vectors may be replication-defective, and such
replication-defective viral vectors may be preferable for
therapeutic use. Where sufficient information is lacking it may,
but need not be, supplied by a host cell or by another vector
introduced into the cell. The nucleic acid to be transferred may be
incorporated into a naturally occurring or modified viral genome or
a portion thereof or may be present within the virus or viral
capsid as a separate nucleic acid molecule. It will be appreciated
that certain plasmid vectors that include part or all of a viral
genome, typically including viral genetic information sufficient to
direct transcription of a nucleic acid that can be packaged into a
viral capsid and/or sufficient to give rise to a nucleic acid that
can be integrated into the host cell genome and/or to give rise to
infectious virus, are also sometimes referred to in the art as
viral vectors. Where sufficient information is lacking it may, but
need not be, supplied by a host cell or by another vector
introduced into the cell.
II. Overview
[0064] The development of new biological materials, particularly
biologically compatible materials that serve as permissive
substrates for cell growth, differentiation, and biological
function has broad implications for advancing medical technology
and for understanding basic biological characteristics of cells.
The present invention relates to materials and techniques for
enhancing repair and/or regeneration of neural tissue using
biocompatible materials that can serve as scaffolds or components
to support regrowth of neural tissue after injury. The materials
can also serve as delivery agents for biologically active molecules
that enhance the processes of regeneration and/or repair. The
materials create an environment that is permissive for regeneration
and repair of neural tissue, i.e., in the presence of the materials
neural tissue exhibits an enhanced capacity for regeneration and/or
repair. It is noted that materials that create an environment
permissive for regeneration may be used for the creation of neural
tissue structures that were not initially present in the subject,
rather than merely for restoration of previously existing
structures. For example, creation of an environment permissive for
regeneration will allow the creation of neural tissue bridges
around or through barriers such as areas of necrosis or scar
tissue. It will also allow the creation of new connections between
different portions of the brain (or elsewhere in the nervous
system), i.e., connections that do not necessarily resemble
structures that exist normally.
[0065] The inventors have previously described a class of
biomaterials that are made through self-assembly of ionic or polar
self-complementary peptides (See, e.g., references 32-24, 36, 38,
39 and U.S. Pat. Nos. 5,955,343 and 5,670,483). These peptides are
able to form hydrogels when contacted with water. In certain
embodiments of the invention these hydrogels contain approximately
99% or greater water content. The peptides self-assemble into
membranes or three-dimensional structures upon exposure to a
sufficient concentration of ions in solution, to form a stable
macroscopic porous matrix composed of orderly interwoven filaments
approximately 10-20 nm in diameter, with pore size on the order of
50-100 nm in linear dimension. An important characteristic of these
materials is that their properties and mechanical strength can be
controlled through manipulation of peptide parameters [33, 37-39].
For example, it has been shown that the stiffness of the gel
increases strongly with peptide concentration [39]. The sequences,
characteristics, and properties of the peptides and the structures
formed by them upon self-assembly are further discussed in the next
section.
[0066] The inventors have shown that these peptide structures are
able to support cell attachment, viability, and growth when cells
are cultured on the surface of the structure. In addition, the
structures are able to serve as substrates for neurite outgrowth
and synapse formation when neurons are grown on their surface [32].
In addition, the inventors have shown that it is possible to
encapsulate cells within the peptide hydrogels, thus placing the
cells in a three-dimensional arrangement within the peptide
scaffold, and that the cells maintain viability and function when
so encapsulated (see pending U.S. patent application Ser. Nos.
09/778,200, filed Feb. 6, 2001, Entitled "Peptide Scaffold
Encapsulation Of Tissue Cells And Uses Thereof" and 10/196,942
entitled "Liver Cellular Reprogramming in Peptide Hydrogel and Uses
Thereof").
[0067] The results described above indicated that the peptide
structures could support the growth of cell lines and of cells
isolated from the body in vitro. The work indicated that the
peptides provide a favorable environment for neuron attachment and
synapse formation in culture and also showed that they do not
elicit a detectable immune system response when implanted into a
mammalian subject. However, this work did not address the
possibility of employing the peptides to enhance repair or
regeneration of injured nervous tissue in vivo, i.e., in intact
mammalian subjects. In particular, the previous work did not
address the possibility of employing the peptides to enhance repair
or regeneration of injured neural tissue in the CNS, where
significant obstacles to such repair exist.
[0068] The nervous system possesses a number of distinctive
characteristics, indicating that strategies useful for repair of
other body tissues will not necessarily be effective for repair of
nervous system tissues, and vice versa. For example, nerves must
regenerate with the correct directionality to innervate the
appropriate target cells. Thus features common to wound healing,
such as development of scar tissue, often create barriers that
prevent functional repair. In addition, the presence of
fluid-filled cavities within the nervous system, which often arise
after injury, may prevent regenerating axons from reaching the
target location.
[0069] The response of neurons to injury is unique in a number of
respects. First of all, unlike many cells in the body, neurons are
unable to undergo mitosis. Their ability to regenerate a severed
portion or to sprout new processes (e.g., axons), varies depending
on the particular neural tissue involved. In the PNS, following a
complete nerve transection the distal portion of the nerve begins
to degenerate as a consequence of separation from the cell body and
activity of proteases. The proximal portion of the nerve swells,
but there is relatively little retrograde degradation. Phagocytic
cells such as macrophages and Schwann cells clear myelin and other
debris from the degenerating axons in addition to producing
molecules such as cytokines and neurotrophic factors shown to
enhance axon growth. Regeneration then begins from the proximal
portion as new axons sprout and extend. In humans, this typically
occurs at a rate of approximately 2-5 mm/day. Functional recovery
requires that the axons be able to find their distal target. Thus
large gaps and barriers within the tissue can compromise the extent
to which full recovery of function is achieved.
[0070] The CNS represents a unique environment in a number of
respects. First, the blood-brain or blood-spine barrier isolates
the CNS from the rest of the body. This may alter the wound healing
process, since it alters access by cells such as macrophages that
typically migrate to a site of injury, where they play a number of
roles, including removal of debris. Furthermore, the CNS is bathed
in cerebrospinal fluid (CSF), which has a unique chemical
composition and may react differently with implanted materials than
the extracellular fluids found elsewhere in the body.
[0071] In contrast to neurons in the PNS, CNS axons do not
typically undergo significant regeneration under native conditions
for a number of reasons. The physiological response to injury in
the CNS differs to that in the PNS in that, for example,
infiltration of the injury site by phagocytic cells such as
macrophages is slower, resulting in slower clearance of debris such
as myelin, which can inhibit axon growth. As discussed further
below, a number of molecules that inhibit axon growth are found at
sites of injury in the CNS, and astrocyte proliferation typically
results in formation of scar tissue, which can inhibit
regeneration. In addition, there appears to be little upregulation
of expression of endogenous growth-promoting molecules in the CNS.
In short, the CNS following injury does not present an environment
permissive for regeneration and repair of neural tissue.
[0072] The present invention encompasses the discovery that
introducing a biocompatible material having a nanoscale structure
at a site of injury within the nervous system results in creation
of an environment that is permissive for such regeneration and
repair. Unlike the great majority of natural or artificial
materials that have been used heretofore in an effort to facilitate
nerve regeneration, the materials of the present invention interact
with cells on a nanoscale rather than a microscale. The materials
are made of nanofibers rather than the microfibers typical of
various other materials whose use for this purpose has been
proposed. While not wishing to be bound by any theory, it is
believed that the small size of the fibers and/or the open weave
structure of the materials promote extension of cell processes and
allow diffusion of nutrients, waste products, etc., in a manner
that provides unique advantages for neural tissue regeneration. In
certain embodiments of the invention the nanofibers that comprise
the material are ordered during self assembly in a complementary
fashion due to weak interactive molecular forces. In certain
embodiments of the invention the nanofibers that comprise the
material are randomly ordered. In other words, while the fibers may
have an orderly internal structure, they may lack directionality or
alignment with respect to one another. For example, the fibers may
not be substantially parallel to one another. In addition, in
certain embodiments of the invention the fibers lack directionality
with respect to the injury or to locations proximal or distal to
the site of injury.
[0073] In general, the invention provides a method for enhancing
repair or regeneration of neural tissue at a site of injury in a
mammalian subject comprising: providing a nanoscale structured
material at or in the vicinity of the site of injury, wherein the
nanoscale structured material provides an environment that is
permissive for regeneration of neural tissue and allows axon growth
from one side of a site of injury or barrier to the other side of
site of injury or barrier. In other words, the injury or barrier
(which can be, for example, a tissue barrier such as necrotic
tissue, scar tissue, or a fluid-filled cavity) initially separates
a first location in which a regenerating axon is located, from a
second location. The nanoscale structured material can be provided
by introducing a precursor of the material, e.g., a composition
comprising components that assemble to form the material, at or in
the vicinity of a site of injury. An axon or portion thereof is
present at the first location. Presence of the material allows the
axon to extend such that a portion of the axon is present at the
second location. The first location can be on either side of the
injury or barrier. The two sides of the injury or barrier may be
identified by reference to the location of a cell body from which a
regenerating axon extends. For example, the side of the injury or
barrier on which the cell body of a regenerating axon is located
may be referred to as being proximal to the site of injury. The
side of the injury or barrier on which the cell body is not located
may be referred to as being distal to the site of injury. The
presence of the nanoscale structured material provides an
environment that allows axon growth from a location proximal to a
site of injury or barrier to a location distal to the site of
injury or barrier. Thus the invention provides a method for
enhancing repair or regeneration of neural tissue at a site of
injury in a mammalian subject comprising: providing a nanoscale
structured material at the site of injury, wherein the nanoscale
structured material provides an environment that is permissive for
regeneration of neural tissue and allows axon growth from a
location proximal to a site of injury or barrier to a location
distal to the site of injury or barrier. It will be appreciated
that in the case of certain injuries or barriers, particularly in
the CNS, cell bodies will be present on both sides of the injury or
barrier, in which case there can be bilateral growth. For example,
if an injury or barrier exists between two locations in the brain,
axons emerging from cell bodies at each location can regenerate
towards the other side of the lesion. The injury or barrier may,
for example, transect axons that extend between different regions
of the brain, between two nuclei (discrete collections of neurons),
etc.
[0074] The regeneration process involves extension of the axon and
also may involve a "knitting together" of the two sides of the
injury, so that an initial gap comes to be filled with living
tissue. It is to be understood that the phrase "at a site of
injury" includes locations in close proximity to such a site, such
that the material may readily come into physical contact with
tissue on one or both sides of the injury. The injury can be any
event that causes nerve damage, such that axons are separated from
their targets. For example, the injury may be due to surgery,
external trauma, stroke, or conditions such as tumors. The injury
may be caused by prior administration of an agent such as an
aminoglycoside antibiotic or neurotoxin, which causes damage to
neural tissue.
[0075] The target can either be the natural target of such axon,
i.e., the cell or cells on which such axon formed synapse(s) prior
to injury, or a new target. In the latter case, the invention
encompasses the introduction of nanoscale structured materials at a
site of injury to direct regenerating axons to innervate targets
with which they were not initially in communication prior to the
injury. For example, the methods and compositions of the invention
may be used in reconstructive brain surgery, in which axons are
routed so as to form new functional connections that replace or
compensate for neural tissue that has been permanently lost.
[0076] According to certain embodiments of the invention the
nanoscale structured material is provided by introducing a
precursor of the material into or in the vicinity of a site of
injury. By "precursor" is meant a composition comprising
component(s) that can assemble to form the nanoscale structured
material. In certain embodiments of the invention the components
can assemble in situ (i.e., within the body of a subject) The
nanoscale structured material may include, and/or its assembly may
involve, additional components present in situ, e.g., ions. In
certain embodiments the nanoscale strutured material is provided by
introducing a composition comprising self-assembling peptides into
a subject at or in the vicinity of a site of injury to the nervous
system. The peptides may be provided, for example, in solution or
in the form of a gel (i.e., already assembled). In general, the
amount of material introduced at or in the vicinity of the site of
injury will vary depending on various factors such as the size or
extent of the injury. For example, the volume introduced may range
from a few microliters to several milliliters or more, e.g., tens
of milliliters. The following section provides details regarding
certain self-assembling peptides suitable for use in the present
invention, mechanisms by which self-assembly occurs, and methods by
which the process of self-assembly and/or the features of the
assembled structure may be controlled. Subsequent sections describe
the methods and compositions of the invention in further
detail.
III. Self-Assembling Peptides
[0077] In certain preferred embodiments of the invention the
nanoscale structured material comprises self-assembling peptides.
These peptides comprise a family of complementary and structurally
compatible molecules. The peptides and their properties are
described in U.S. Pat. Nos. 5,955,343 and 5,670,483 and in
co-pending U.S. patent application Ser. Nos. 09/778,200, and
10/196,942. Prior to self-assembly the peptides may be dissolved in
a solution that is substantially free of monovalent ions or
contains only a low concentration of such ions, e.g., less than 10,
5, 1, or 0.1 mM. Self-assembly may be initiated by the addition of
an ionic solute to a peptide solution or by a change in pH [37,
38]. For example, NaCl at a concentration of between 5 mM and 5 M
induces the assembly of macroscopic structures within a few
minutes. Lower concentrations of NaCl may also induce assembly but
at a slower rate. Alternately, self-assembly may be initiated by
introducing the peptides (preferably dissolved in a solution that
is substantially free of monovalent ions) into a solution
comprising such ions, e.g., a physiological fluid such as CSF. The
peptides can thus self-assemble at a location in vivo. Preferred
ions include monovalent cations such as Li.sup.+, Na.sup.+,
K.sup.+, and Cs.sup.+. Preferably, the concentration of the ion is
at least 5, 10, 20, or 50 mM in order to induce self-assembly. One
of ordinary skill in the art will be able to select preferred
concentrations of ions based on the particular peptide and desired
speed of assembly.
[0078] In certain embodiments of the invention peptides forming the
macroscopic structure contain between 8 and 200 amino acids, 8 to
36 amino acids, or 8 to 16 amino acids, inclusive. In certain
embodiments of the invention the concentration of the peptides
prior to self-assembly is between 0.01% (0.1 mg/ml) and 99.99%
(999.9 mg/ml), inclusive. In certain embodiments of the invention
the concentration of the peptides prior to self-assembly is between
0.1% (1 mg/ml) and 10% (100 mg/ml), inclusive. In certain
embodiments of the invention the concentration of the peptides
prior to self-assembly is between 0.1% (1 mg/ml) and 5% (50 mg/ml),
inclusive, or between 0.5% (5 mg/ml) and 5% (50 mg/ml), inclusive.
In certain embodiments of the invention the concentration of the
peptides prior to self-assembly is approximately 5 mg/ml,
approximately 10 mg/ml, approximately 15 mg/ml, or approximately 20
mg/ml. In certain embodiments of the invention the peptides are
administered dry, e.g., in powder form, to a site of injury. The
peptides may then self-assemble following contact with body fluids
at the site of injury, e.g., at a fluid-filled cavity in the
CNS.
[0079] If desired, peptide scaffolds may be formed with a
predetermined shape or volume. To form a scaffold with a desired
geometry or dimension, an aqueous peptide solution may be placed in
a pre-shaped casting mold, and the peptides induced to
self-assemble into a scaffold by the addition of an ion, as
described herein. Alternately, the ion may be added to the peptide
solution shortly before placing the solution into the mold,
provided that care is taken to place the solution into the mold
before substantial assembly occurs. The resulting material
characteristics, time required for assembly, and geometry and
dimensions of the macroscopic peptide scaffold are governed by the
concentration and amount of peptide solution that is applied, the
concentration of ion used to induce assembly of the scaffold, and
the dimensions of the casting apparatus. Cells and agents such as
bioactive molecules (e.g., therapeutic compounds), may be
introduced into the peptide solution prior to self-assembly. The
self-assembly process then forms a structure that encapsulates the
cells or molecules. To achieve even distribution of the cells or
molecules within the structure it may be desirable to thoroughly
mix the solution prior to initiation of self-assembly. It may be
desirable to maintain the cells or agents in a solution that
contains substantially no ions or only low concentration of ions in
order to avoid initiation of self-assembly immediately upon
combining the cells or agents with the peptide solution. In this
case the cells are preferably maintained in an iso-osmotic solute
such as sucrose prior to combination with the peptide solution.
[0080] The side-chains of the peptides partition into two faces, a
polar face with charged ionic side chains and a nonpolar face with
alanines or other hydrophobic groups. These ionic side chains are
self-complementary to one another in that the positively charged
and negatively charged amino acid residues can form complementary
ionic pairs. These peptides are therefore called ionic,
self-complementary peptides, or Type I self-assembling peptides. If
the ionic residues alternate with one positively and one negatively
charged residue (-+-+-+-+), the peptides are described as "modulus
I;" if the ionic residues alternate with two positively and two
negatively charged residues (--++--++), the peptides are described
as "modulus II."
[0081] Many modulus I and II self-complementary peptides such as
EAKA 16-I, RADA16-I, RAEA16-I, and KADA16-I have been analyzed
previously (Table 1). These peptides are also referred to as
RAD16-I, RAE16-I, KAD16-I, etc. Modulus IV ionic self-complementary
peptides containing 16 amino acids; such as EAK16-IV, KAE16-IV,
DAR16-IV and RAD16-IV; have also been studied. If the charged
residues in these self-assembling peptides are substituted (i.e.,
the positive charged lysines are replaced by positively charged
arginines and the negatively charged glutamates are replaced by
negatively charged aspartates), there are essentially no
significant effects on the self-assembly process. However, if the
positively charged residues, lysine and arginine are replaced by
negatively charged residues, aspartate and glutamate, the peptides
can no longer undergo self-assembly to form macroscopic structures;
however, they can still form a beta-sheet structure in the presence
of salt. Other hydrophilic residues, such as asparagine and
glutamine, that form hydrogen bonds may be incorporated into the
peptides instead of or in addition to charged residues. If the
alanines in the peptides are changed to more hydrophobic residues,
such as leucine, isoleucine, phenylalanine or tyrosine, these
peptides have a greater tendency to self-assemble and form peptide
matrices with enhanced strength. Some peptides that have similar
compositions and lengths as these aforementioned peptides form
alpha-helices and random-coils rather than beta-sheets and do not
form macroscopic structures. Thus, in addition to
self-complementarity, other factors are likely to be important for
the formation of macroscopic structures, such as the peptide
length, the degree of intermolecular interaction, and the ability
to form staggered arrays.
[0082] It is noted that in certain embodiments of the invention a
group or radical such as an acyl group (RCO--, where R is an
organic group), e.g., an acetyl group (CH.sub.3CO--) is present at
the N terminus of the peptides in order to neutralize an extra
charge positive that may otherwise be present (e.g., a charge not
resulting from the side chain of the N-terminal amino acid).
Similarly, a group such as an amine group (NH.sub.2) may be used to
neutralize an extra negative charge that may otherwise be present
at the C terminus (e.g., a charge not resulting from the side chain
of C-terminal amino acid), thus converting the C terminus into an
amide (--CONH.sub.2). While not wishing to be bound by any theory,
the neutralization of charges on the terminal N and C molecules may
facilitate self-assembly. One of ordinary skill in the art will be
able to select other suitable groups.
[0083] Self-assembled nanoscale scaffolds can be formed with
varying degrees of stiffness or elasticity. The peptide scaffolds
typically have a low elastic modulus, in the range of 1-10 kPa as
measured in a standard cone-plate rheometer. Such low values permit
scaffold deformation as a result of cell contraction, and this
deformation may provide the means for cell-cell communication.
Scaffold stiffness can be controlled by a variety of means
including changes in peptide sequence, changes in peptide
concentration, and changes in peptide length [37, 38]. Other
methods for increasing stiffness can also be used, such as by
attaching a biotin molecule to the amino- or carboxy-terminus of
the peptides or between the amino- and carboxy-termini, which may
then be cross-linked.
TABLE-US-00001 TABLE 1 Representative Self-Assembling Peptides Name
Sequence (n-->c) Modulus RADA16-I n-RADARADARADARADA-c I (SEQ ID
NO: 1) RGDA16-I n-RADARGDARADARGDA-c I (SEQ ID NO: 2) RADA8-I
n-RADARADA-c I (SEQ ID NO: 3) RAD16-II n-RARADADARARADADA-c II (SEQ
ID NO: 4) RAD8-II n-RARADADA-c II (SEQ ID NO: 5) EAKA16-I
n-AEAKAEAKAEAKAEAK-c I (SEQ ID NO: 6) EAKA8-I n-AEAKAEAK-c I (SEQ
ID NO: 7) RAEA16-I n-RAEARAEARAEARAEA-c I (SEQ ID NO: 8) RAEA8-I
n-RAEARAEA-c I (SEQ ID NO: 9) KADA16-I n-KADAKADAKADAKADA-c I (SEQ
ID NO: 10) KADA8-I n-KADAKADA-c I (SEQ ID NO: 11) EAH16-II
n-AEAEAHAHAEAEAHAH-c II (SEQ ID NO: 12) EAH8-II n-AEAEAHAH-c II
(SEQ ID NO: 13) EFK16-II n-FEFEFKFKFEFEFKFK-c II (SEQ ID NO: 14)
EFK8-II n-FEFKFEFK-c I (SEQ ID NO: 15) ELK16-II
n-LELELKLKLELELKLK-c II (SEQ ID NO: 16) ELK8-II n-LELELKLK-c II
(SEQ ID NO: 17) EAK16-II n-AEAEAKAKAEAEAKAK-c II (SEQ ID NO: 18)
EAK12 n-AEAEAEAEAKAK-c IV/II (SEQ ID NO: 19) EAK8-II n-AEAEAKAK-c
II (SEQ ID NO: 20) KAE16-IV n-KAKAKAKAEAEAEAEA-c IV (SEQ ID NO: 21)
EAK16-IV n-AEAEAEAEAKAKAKAK-c IV (SEQ ID NO: 22 RAD16-IV
n-RARARARADADADADA-c IV (SEQ ID NO: 23 DAR16-IV
n-ADADADADARARARAR-c IV (SEQ ID NO: 24 DAR16-IV*
n-DADADADARARARARA-c IV (SEQ ID NO: 25 DAR32-IV
n-(ADADADADARARARAR)-c IV (SEQ ID NO: 26 EHK16 n-HEHEHKHKHEHEHKHK-c
N/A (SEQ ID NO: 27 EHK8-I n-HEHEHKHK-c N/A (SEQ ID NO: 28) VE20*
n-VEVEVEVEVEVEVEVEVEVE-c N/A (SEQ ID NO: 29) RF20*
n-RFRFRFRFRFRFRFRFRFRF-c N/A (SEQ ID NO: 30) N/A denotes not
applicable *These peptides form a .beta.-sheet when incubated in a
solution containing NaCl, however they have not been observed to
self-assemble to form macroscopic structures.
[0084] The list presented in Table 1 is representative rather than
exclusive. Other self-assembling peptides may be generated, e.g.,
by changing the amino acid sequence of any self-assembling peptide
by a single amino acid residue or by multiple amino acid residues.
To increase the mechanical strength of the structures, if desired,
cysteines may be incorporated into the peptides to allow the
formation of disulfide bonds, or residues with aromatic rings may
be incorporated and cross-linked by exposure to UV light. The in
vivo half-life of the structures may also be modulated by the
incorporation of protease cleavage sites into the structure,
allowing it to be enzymatically degraded. Combinations of any of
the above alterations may also be made to the same peptide
structure. Formation of cross-links by adding biotin to the
peptides and then cross-linking by addition of avidin may also be
used and may be a preferable approach.
[0085] The peptides may include L-amino acids, D-amino acids,
natural amino acids, non-natural amino acids, or a combination
thereof. If L-amino acids are present in the scaffold, degradation
produces amino acids that may be reused by the host tissue. The
fact that the basic monomeric subunit of the peptides in this
embodiment of the invention, i.e., L-amino acids, occurs naturally
within the body distinguishes this class of compounds from numerous
other biocompatible substances and may offer unique advantages. The
peptides may be chemically synthesized or purified from natural or
recombinant sources, and the amino- and carboxy-termini of the
peptides may be protected or not protected. The peptide scaffold
may be formed from one or more distinct molecular species of
peptides which are complementary and structurally compatible with
each other. Peptides containing mismatched pairs, such as the
repulsive pairing of two similarly charged residues from adjacent
peptides, may also form structures if the disruptive force is
dominated by stabilizing interactions between the peptides. Peptide
scaffolds may also be referred to herein as peptide hydrogels or
peptide hydrogel scaffolds.
[0086] Peptides capable of being cross-linked may be synthesized
using standard f-moc chemistry and purified using high pressure
liquid chromatography. The formation of a peptide scaffold may be
initiated by the addition of ions or salts thereof as described
herein. Hydrophobic residues with aromatic side chains may be
cross-linked by exposure to UV irradiation. The extent of the
cross-linking may be precisely controlled by the predetermined
length of exposure to UV light and the predetermined peptide
concentration. The extent of cross-linking may be determined by
light scattering, gel filtration, or scanning electron microscopy
using standard methods. Furthermore, the extent of cross-linking
may also be examined by HPLC or mass spectrometry analysis of the
structure after digestion with a protease, such as matrix
metalloproteases. Material strength may be determined before and
after cross-linking.
[0087] If desired, the peptide scaffolds formed from any of the
above peptides may be characterized using various biophysical and
optical techniques, such as circular dichroism (CD), dynamic light
scattering, Fourier transform infrared (FTIR), atomic force
microscopy (ATM), scanning electron microscopy (SEM), and
transmission electron microscopy (TEM) [32, 37-39]. For example,
biophysical methods may be used to determine the degree of
beta-sheet secondary structure in the peptide scaffold.
Additionally, filament and pore size, fiber diameter, length,
elasticity, and volume fraction may be determined using
quantitative image analysis of scanning and transmission electron
microscopy. The scaffolds may also be examined using several
standard mechanical testing techniques to measure the extent of
swelling, the effect of pH and ion concentration on scaffold
formation, the level of hydration under various conditions, and the
tensile strength. These methods allow one of ordinary skill in the
art to determine which of the various modifications and additions
to the peptides described below are suitable for use in the methods
of the invention.
IV. Model for Evaluation of Nerve Regeneration in the CNS
[0088] The experiments of Aguayo and co-workers [1, 2], using
peripheral nerve segments taken from an animal's leg and
transplanted into the CNS revealed that many CNS axons that will
not regenerate after being transected in the adult animal will
extend axons into the peripheral nerve (PN). A few years after the
discovery that the optic tract of newborn hamsters will regenerate
if transected during the first 3 days of postnatal life, in which
axons grew around the site of injury [3], So and Aguayo in Montreal
developed a new model for the study of CNS regeneration in adults.
A long segment of sciatic nerve was used as a bridge from the back
of the eye, where it was sutured after complete severance of the
optic nerve, to the midbrain tectum (superior colliculus, SC) [4].
Further studies using this approach have been conducted in several
laboratories. Many axons have been found to reconnect to the SC,
especially when retinal ganglion cell (RGC) survival is enhanced
with growth factor treatment [5-8]. Axon regeneration may be traced
using immunohistochemical techniques. Since the nerve bodies are
present in the retina, presence of tracer at sites distal to a site
of injury must represent axon regeneration from a location (the
retina) proximal to the site of injury. Some return of function has
also been found, with unlearned and learned responses to presence
or absence of light [7] [9] [10]. Electrophysiological recordings
in such hamsters have provided evidence of functional connections
[1, 11-13] although the amount of topographic organization has been
found to be very limited [14]. Morphology of single regenerated
axons has been described, revealing abnormally compact end arbors,
but these arbors have a full complement of synapses [15, 16].
[0089] The inventors have previously demonstrated some recovery of
visual orienting capability in hamsters in which the only
connection from the eyes is through one long peripheral nerve (PN)
(sciatic) bridge [17, 18]. This result depended on waiting a very
long time for the functional regeneration to occur (1.5 years or
more), but it demonstrated that functional recovery can occur and
can be effectively evaluated.
[0090] Other workers have tried using shorter PN bridges over the
site of a transected optic tract near the rostral border of the SC
[19]. In these experiments, only a small number of regenerating
axons were found to grow through the bridge. However, the inventors
have improved this paradigm using adult hamsters, with a
demonstration of fairly rapid and substantial recovery of visually
elicited orienting movements [18]. After the surgery in 3-wk old
hamsters, the anatomical and behavioral recovery was extensive, and
the animals appeared to be suffer little, if any, deficit. Even
with surgery at age 5.5 months, considerable recovery has been
found, with a very substantial quantity of regeneration of the
retinotectal projection [18]. These findings demonstrate that the
optic tract regeneration model is appropriate for evaluating
compositions and methods designed to facilitate neural tissue
regeneration in vivo. As described herein, the model has been used
to demonstrate anatomic and behavioral recovery in hamsters treated
with SAP.
V. Methods and Compositions of the Invention
[0091] A. Self-Assembling Peptides as Materials for Nerve
Regeneration
[0092] The inventors have identified the following criteria that
are important for development of an ideal material for neural
tissue regeneration: (1) building blocks are derived from molecules
normally found within the host, (2) basic units are amenable to
design and modification in order to achieve specific needs, (3)
controlled rate of biodegradation, (4) promotion of cell-substrate
interactions, (5) minimal or no cytotoxicity, (6) minimal or no
elicitation of immune responses and inflammation, (7) easy and
scaleable material production, purification and processing, (8)
readily transportable, (9) very high water content and
compatibility with aqueous solutions and physiological conditions,
(10) ability to integrate with the in-vivo environment, (11)
material is small enough to interact with the glycosolation of the
surrounding cells. Without wishing to be bound by any theory or to
limit the invention in any way, it is believed that the materials
described herein attain or come close to attaining these goals.
[0093] As described in further detail in the examples, when
introduced into a site of injury in the CNS in the animal model
described above, self-assembling peptides such as those described
in Section II above assembled into a scaffold that provided a
permissive environment in which nerve regeneration occurred. Young
animals were subjected to a surgically created injury in the brain
that severed axons in the optic tract. A solution containing a
self-assembling peptide (RADA16-I) was then injected into the site
of the lesion. Self-assembly of the peptides to form a peptide
scaffold was induced by ions that were present in the CSF. Axons
located proximally to the site of injury (in the retina) grew to
partially re-innervate a structure (the superior colliculus)
present at a location distal to the injury (Example 1). In another
set of experiments (Example 2), adult hamsters were subjected to a
surgically created injury in the brain that severed axons in the
brachium of the superior colliculus, resulting in blindness. FIG. 9
is a schematic diagram showing the site of the lesion. A solution
containing a self-assembling peptide (RADA16-I) was injected into
the site of the lesion at the time of surgery. Self-assembly of the
peptides to form a peptide scaffold was induced by ions that were
present in the CSF. Axons located proximally to the site of injury
(in the retina) grew to partially re-innervate a structure (the
superior colliculus) present at a location distal to the injury, as
shown in FIGS. 10 and 11. In addition, functional recovery
(restoration of response to a visual stimulus), was demonstrated as
described in Example 2 and shown in FIG. 12.
[0094] It is believed that the experiments described herein
represent the first demonstration that introduction of a
nanostructured material at a site of injury in the CNS can support
successful axon regrowth across the site of injury. Presence of the
SAP appears to permit the two faces of an injury to become
reapposed, allowing separated tissues to knit together. Presence of
the SAP appears to prevent the formation or deposition of
inhibitory scar tissue while not preventing, and perhaps directly
stimulating, the extension of nerve cell processes. Thus, the SAP
was shown to offer a new means of ameliorating tissue disruptions
caused by traumatic injury to the CNS, allowing regrowth of young
axons that are believed to have regenerative potential as well as
adult axons whose regeneration potential has heretofore been
believed to be extremely limited or nonexistent.
[0095] In general, any of the SAPs described above may be used in
the practice of the invention. However, it is possible that certain
of the SAPs will possess uniquely favorable characteristics for
nerve regeneration while others may prove less favorable. These
properties may be evaluated as described in the examples. The SAPs
may be introduced at a site of injury by injection, prior to
self-assembly, whereupon they self-assemble in vivo. They may also
be assembled in vitro and provided at the site of injury in any of
a variety of ways, e.g., by injection from a needle, by extrusion
from a syringe, by deposition of dry powder, or by direct
deposition of a preformed scaffold. The scaffold may, if desired,
be shaped into an elongated or tubular structure. In addition, the
SAPs may be incorporated into a composite structure. For example,
an outer sheath comprised of a different biocompatible material may
be filled with an SAP gel. The outer sheath may resemble, for
example, nerve guides or cuffs such as those used in the art to
facilitate nerve regeneration [see 86 for a review that describes a
number of such devices and lists references.]
[0096] B. Self-Assembling Peptide Scaffolds Incorporating
Additional Substances
[0097] In various embodiments of the invention one or more
additional substances is added to the peptide scaffold either prior
to or following self-assembly. The substance may serve any of a
number of purposes, including, but not limited to, those described
below. In certain embodiments of the invention neural tissue
contacts the substance as it extends or grows into the area
occupied by the peptide scaffold. In certain embodiments of the
invention the substance is released from the scaffold, e.g., by
diffusion, or by release from the scaffold as it degrades over
time. The peptide concentration and features such as cross-linking
may be selected to provide a desired rate of degradation and
release of the substance. The substance may contact neural tissue
at or near the site of injury and/or may enter the bloodstream and
travel to more distant locations. It is noted that in addition to
the various substances described below, which are selected
primarily based on their useful properties for enhancing neural
tissue regeneration, various other biologically active substances
can be incorporated into the scaffold. A biologically active
substance is any substance that can, either directly or indirectly,
produce a biological effect, preferably a substance that can confer
a therapeutic benefit. 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,
anti-inflammatory agents, immunosuppressive agents, chemotactic
agents, etc. In general, any biomolecule (e.g., protein, lipid,
nucleic acid, etc.), other organic molecule (e.g., a small
molecule) or inorganic molecule (e.g., a mineral), can be included
in the composition. The invention therefore provides a composition
comprising a nanoscale structured material or a precursor thereof
and an additional active substance. In certain preferred
embodiments of the invention the composition comprises
self-assembling peptides, wherein the peptides are amphiphilic
peptides that comprise substantially equal proportions of
hydrophobic and hydrophilic amino acids and are complementary and
structurally compatible. The invention provides compositions
comprising the precursor components of the nanoscale structured
material (e.g., self-assembling peptides or a solution thereof that
have not yet undergone assembly) and the active substance.
Preferably the peptides self-assemble to form a beta-sheet
macroscopic scaffold. Active substances can be provided at any
suitable concentration and in any suitable amount, e.g., in the
microgram or milligram range, or greater.
[0098] 1. Regeneration Promoting Factors
[0099] A number of substances that are known to increase neuron
survival and rate of neuronal growth have been found. Such
substances are referred to herein as neural regeneration-promoting
factors (RPF). RPFs may usefully be divided into extrinsic and
intrinsic factors. Extrinsic RPFs are generally factors that are
not by neurons themselves but which act on them to enhance
regeneration. Many of these factors were identified as endogenously
occurring molecules (e.g., peptides) that bind to the cell or
otherwise influence the cell from the extracellular environment,
e.g., by triggering a signaling cascade or blocking a program that
may send a cell down the apoptotic pathway. One example of a such a
factor of use in the present invention is ciliary neurotrophic
factor (CNTF), a factor used to preserve (i.e., maintain viability
of) neurons. With complete optic nerve section in rats, the
grafting of a PN segment to the optic nerve stump does not prevent
a massive loss of retinal ganglion cells--less than 10% are
reported to survive following this procedure [54-57]. Trying to
slow and reduce this cell death has been a major goal for
investigators using this or other paradigms for inducing CNS axon
re-growth. Positive results have been reported using various
extrinsic factors, primarily CNTF [58-60].
[0100] Additional RPFs of use in the present invention include a
family of neurotrophic factors referred to as neurotrophins. These
include nerve growth factor (NGF), brain derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5),
neurturin (NTN), persephin (PSP), and artemin (ART) [86, 99, 100].
Other factors that are known to influence nerve regeneration
include glial cell derived growth factor (GDNF), and acidic and
basic fibroblast growth factor (aFGF, bFGF). Particular factors
have been shown to enhance regeneration in various contexts within
the CNS and/or PNS [see 86 and references therein]. Additional
factors that may aid in nerve regeneration include adhesion
molecules such as L1, NCAM, and N-cadherin, molecules that
influence axon guidance and pathfinding (e.g., semaphorins, slits,
netrins, ephrins, etc.), chemotactic factors, synaptogenic factors
(e.g., agrin, laminin, and ARIA). These molecules may either be
provided exogenously, e.g., as a component of a SAP scaffold, or
their expression may be upregulated, as described below. RPFs (or
other active substances) can be provided at concentrations that
have been shown in the art to promote nerve regeneration or any
other desired effect. Growth factors are typically used at
concentrations ranging between about 1 fg/ml to 1 mg/ml. Frequently
growth factors are used at concentrations in the low nanomolar
range, e.g., 1-10 nM. In certain embodiments of the invention
growth factors are used at concentrations that are not typically
used in the prior art or that are not typically found in vivo under
normal conditions. For example, growth factors may be used at
concentrations that are 5 fold greater, 10 fold greater, 20 fold
greater, 100 fold greater, etc., than is typically required to
produce effects or than typically occurs in vivo. Titration
experiments can be performed to determine the optimal concentration
of a particular agent, such as a growth factor, depending upon the
particular effects desired. Factors may be added in purified form
or as components of a complex biological mixture such as serum.
Factors that promote cell proliferation may be referred to as
proliferation agents.
[0101] Another approach to increasing the regeneration capacity of
nerves is to alter the expression of a variety of
regeneration-associated genes, genes that encode components of the
neuronal cytoskeleton (e.g., actin, gelsolin, or other
actin-associated proteins), and/or genes that encode anti-apoptosis
factors. RPFs that are produced by neurons themselves may be
referred to as intrinsic factors. Regeneration-associated genes are
expressed transiently during development of the nervous system and
are typically not expressed in healthy adult nerves. However, upon
injury to peripheral nerves some of these genes are reexpressed,
and such reexpression is believed to be important for successful
regeneration. Its absence may be part of the reason why
regeneration does not occur in the injured CNS.
Regeneration-associated genes include GAP-43 and CAP-23 [see 86 and
references therein]. It has been reported that mutant mice that
overexpress two proteins found in axonal endings, GAP-43 and
CAP-23, show a considerable enhancement of regeneration of
transected dorsal column axons in a PN bridge [79].
[0102] The great decrease in regenerative capacity of the mammalian
optic nerve has been found to be caused by changes intrinsic to the
retinal cells [69]. It was found that down-regulation of Bcl-2 is a
major component of this change in retinal ganglion cells [45]. This
gene appears to play some as yet unidentified role in neurite
elongation, in addition to its better known role in preventing
apoptotic cell death, e.g., the neuronal death due to inadequate
uptake of neurotrophic factors. In addition, overexpression of
Bcl-2 protein in transgenic mice can greatly promote regenerative
capacity of retinofugal axons transected in the midbrain [45]. Such
experiments have demonstrated the great importance of intrinsic
factors in neurons for their ability to regenerate. Therefore, use
of methods to produce an increase in intrinsic factors like Bcl-2,
GAP-43, and CAP-23 may prevent much of the cell death that would
curb regenerative axon growth, and would also promote axon
elongation. Increasing production or release of other
anti-apoptotic substances or, conversely, decreasing expression of
pro-apoptotic substances (either produced by neurons or other cell
types), may also be useful.
[0103] Gene therapy techniques may be used to increase expression
of genes whose products enhance regeneration. Gene therapy
encompasses delivery of nucleic acids comprising templates for
synthesis of a molecule of interest (e.g., a therapeutic
polypeptide or nucleic acid) 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.
However, 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.
[0104] "Naked" nucleic acids, vectors (e.g., gene therapy vectors)
and vehicles that provide nucleic acids comprising templates for
synthesis of nucleic acids and proteins may be incorporated into
SAP scaffolds, from which they may be taken up by cells at or near
a site of injury. Preferably the nucleic acid includes a coding
sequence for a molecule to be expressed in a cell of interest and
also includes appropriate expression signals, e.g., promoters,
terminators, etc., to ensure proper expression, operably linked to
the coding sequence. The molecule to be expressed can be an RNA
that is then translated into a protein, or a therapeutic nucleic
acid such as a short interfering RNA, antisense RNA, ribozyme, etc.
In certain embodiments of the invention the expression signal(s)
are cell type specific, so that the gene will only be expressed in
cells of a particular cell type, e.g., neurons. A wide variety of
cell type specific proteins are known in the art. For example,
nestin is an intermediate filament protein expressed in
neuroepithelial neuronal precursor stem cells, and its expression
decreases with neuronal maturation [87]. Nestin is considered a
marker for immature neurons, and nestin-positive cells can
differentiate into either neurons or glia. NeuN is a
neuron-specific marker expressed in postmitotic cells [88]. Glial
fibrillarary acidic protein (GFAP) is a classic glial astrocyte
marker. Beta III tubulin is another neuron-specific protein.
Promoters from any of these cell type specific genes, or others
known in the art, may be used to selectively express a gene of
interest in the cell type in which the cell type specific gene is
normally expressed.
[0105] In general, either viral or non-viral vectors or vehicles
may be used. In certain embodiments of the invention the vector is
a viral vector that is able to infect neurons. For example, herpes
virus, adenovirus, adeno-associated virus, retroviruses, or
lentiviruses may be used. It may be preferable to avoid the use of
intact viruses in delivering templates to cells. Thus it may be
preferable to deliver nucleic acid (e.g., DNA) vectors or linear
nucleic acid 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. Such agents include a wide
variety of cationic polymers and modified cationic polymers,
lipids, etc. Cationic polymers are known to spontaneously bind to
and condense nucleic acids such as DNA into nanoparticles. For
example, naturally occurring proteins, peptides, or derivatives
thereof have been used. Synthetic cationic polymers such as
polyethylenimine (PEI), polylysine (PLL), polyarginine (PLA),
polyhistidine, etc., are also known to condense DNA and are useful
delivery vehicles. U.S. Pat. No. 6,013,240 and PCT application
WO9602655 provide further information on PEI Cationic polymers
modified by addition of groups such as acyl, succinyl, acetyl, or
imidazole groups, e.g., to reduce cytotoxicity, can be used.
Dendrimers can also be used. See [70, 80, and 89-91] for discussion
of and approaches for gene therapy. Cell-type specific targeting
ligands (e.g., the nontoxic neuronal-specific fragment C of tetanus
toxin), or an antibody that specifically binds to a molecule
expressed on a cell type of interest may be attached to a gene
therapy delivery agent to allow transfection of only certain cell
types. In general, the nucleic acid and any appropriate gene
therapy delivery agent (e.g., a cationic polymer) may be
incorporated into the scaffold in any of the ways discussed
below.
[0106] The invention therefore provides a composition comprising a
nanoscale structured material and a neural regeneration promoting
factor. In certain preferred embodiments of the invention the
composition comprises self-assembling peptides, wherein the
peptides are amphiphilic peptides that comprise substantially equal
proportions of hydrophobic and hydrophilic amino acids and are
complementary and structurally compatible. The invention provides
compositions comprising the precursor components of the nanoscale
structured material (e.g., self-assembling peptides or a solution
thereof that have not yet undergone self-assembly) and the neural
regeneration promoting factor. Preferably the peptides
self-assemble to form a beta-sheet macroscopic scaffold. Particular
extrinsic RPFs that may be incorporated into the scaffold include,
but are not limited to, CNTF, NGF, BDNF, NT-3, NT-4/5, GDNF, aFGF,
and bFGF. Particular intrinsic RPFs that may be incorporated into
the scaffold include, but are not limited to, nucleic acids that
provide templates for synthesis of a regeneration enhancing
protein. Particular regeneration enhancing proteins include, but
are not limited to, GAP-43, CAP-23, and Bcl-2. Additional RPFs that
may be incorporated into the scaffold include lithium (which has
been shown to have neuroprotective properties and also upregulates
genes that are present during early development as well as certain
genes that are believed to be critical for neuronal survival
[71-74]), laminin, agrin, acetylcholine receptor inducing activity
protein (ARIA), L1, NCAM, and N-cadherin, semaphorins, slits,
netrins, and ephrins.
[0107] 2. Substances that Counteract Inhibitory Molecules
[0108] One of the greatest challenges that faces efforts to enhance
regeneration and repair in the CNS is the development of scar
tissue, which typically results in the formation of a nonpermissive
environment that may inhibit axon growth and/or myelination [86].
Scarring involves a variety of cell types, including macrophages,
microglia, oligodendrocytes, and astrocytes. Various noncellular
components of a scar typically include myelin-associated molecules
(e.g., Nogo, myelin associated glycoprotein, oligodendrocyte-myelin
glycoprotein), chondroitin sulfate proteoglycans, collagens, etc.
Certain of these molecules have been shown to inhibit regeneration
of neural tissue. For example, it has been shown that
oligodendrocyte myelin inhibits nerve regeneration, and antibodies
that bind to the myelin protein NI-35 can block this inhibition [86
and references therein]. Antibodies that bind to growth inhibitory
molecules, e.g., molecules found in of scar tissue that forms after
injury to neural tissue, including, but not limited to, Nogo,
myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein
may be incorporated into the peptide scaffold. Such antibodies may
block interaction between the inhibitory molecule and a neural
tissue cell, e.g., a neuron or glial cell. A variety of enzymes
that degrade growth inhibitory molecules are known. For example,
chondroitinase ABC degrades chondroitin sulfate proteoglycan side
chains, which are upregulated following spinal cord injury and
inhibit axonal growth. Enzymes including, but not limited to,
chondroitinase ABC or any other enzyme that degrades chondroitin
sulfate proteoglycan side chains can be incorporated into the
peptide scaffold.
[0109] Another approach to decrease the amount of an inhibitory
molecule (e.g., an inhibitory molecule produced by a cell at or
near a site of injury) is to take advantage of the phenomenon of
RNA-mediated interference (RNAi) to reduce expression of a
transcript that encodes the inhibitory molecule [92-95]. Briefly,
it has been found that the presence of a short double-stranded RNA
molecule referred to as a short interfering RNA (siRNA), one strand
of which is substantially complementary to a transcript present in
a cell (the target transcript) within a cell results in inhibition
of expression of the target transcript. The mechanism typically
involves degradation of the transcript by intracellar 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, which can be processed
intracellularly into siRNA. SiRNA and shRNA has been demonstrated
to inhibit expression of target transcripts in mammalian cells both
in tissue culture and in vivo. An siRNA or shRNA whose presence
within a cell leads to inhibition of expression of a transcript by
RNA interference, whether by causing degradation of the transcript
or by causing translational repression, is said to be targeted to
the transcript. SiRNAs or shRNAs targeted to a transcript that
encodes an inhibitory molecule can be incorporated into the
scaffold. Transfection enhancing agents can also be included to
increase uptake of the siRNA or shRNA. Alternatively, 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 can be incorporated into the scaffold for delivery to cells
at or near the site of injury.
[0110] Any substance that acts to counteract the effect of a
molecule that is inhibitory for neural regeneration or repair,
whether by causing degradation, sequestering, reducing expression,
or blocking interaction of the molecule with a cell will be said to
counteract the inhibitory molecule. It is also noted that the SAP
itself may serve to sequester inhibitory molecules. The invention
therefore provides a composition comprising a nanoscale structured
material and a substance that counteracts a molecule that inhibits
regeneration or growth of neural tissue. In certain preferred
embodiments of the invention the composition comprises
self-assembling peptides, wherein the peptides are amphiphilic
peptides that comprise substantially equal proportions of
hydrophobic and hydrophilic amino acids and are complementary and
structurally compatible. The invention provides compositions
comprising the precursor components of the nanoscale structured
material (e.g., self-assembling peptides or a solution thereof that
have not yet undergone assembly) and the substance that counteracts
a molecule that inhibits regeneration or growth of neural tissue.
Preferably the peptides self-assemble to form a beta-sheet
macroscopic scaffold.
[0111] 3. Cells
[0112] As described above, repair and regeneration of neural tissue
can be enhanced by supplying regeneration promoting factors such as
neurotrophic factors, cell adhesion molecules, integrins, etc. One
way to provide such molecules (or others), is to deliver cells at
the site of injury. The cells may produce molecules that promote
regeneration or otherwise contribute to producing an environment
permissive for regeneration. Schwann cells, CNS glial cells,
macrophages, and olfactory ensheathing cells are among the cells
that may be useful in this regard [86 and references therein]. In
addition, various progenitor cells, e.g., neural progenitor cells,
glial progenitor cells, and/or stem cells, may also be useful. The
cells may or may not produce a regeneration promoting factor. Any
of these cell types may be incorporated into the scaffold. In
addition, any of these cell types (or others) can be genetically
modified, e.g., to increase the production of a regeneration
promoting factor, prior to incorporation into the scaffold. The
cell(s) may be autologous or non-autologous. They may be allogeneic
or non-allogeneic. They may be from the same species as the subject
into which they are introduced or from a different species. They
may be fetal or adult. In certain embodiments of the invention the
cells are neural cells. In certain embodiments of the invention the
cells are introduced to treat a disease such as Parkinson's disease
[98]. For example, the cells can be dopaminergic neurons.
[0113] The invention therefore provides a composition comprising a
nanoscale structured material and a cell. In general, the
composition will comprise a population of cells. In certain
preferred embodiments of the invention the composition comprises
self-assembling peptides, wherein the peptides are amphiphilic
peptides that comprise substantially equal proportions of
hydrophobic and hydrophilic amino acids and are complementary and
structurally compatible. The invention provides compositions
comprising the precursor components of the nanoscale structured
material (e.g., self-assembling peptides or a solution thereof that
have not yet undergone assembly) and the cell. Preferably the
peptides self-assemble to form a beta-sheet macroscopic
scaffold.
[0114] 4. Methods for Incorporation of Substances into SAP
Scaffold
[0115] In general, RPFs, blocking agents, cells, etc. may be
incorporated into the peptide scaffolds in a number of different
ways. For purposes of description any such molecule or cell is
referred to in this section as a "substance". Generally
incorporation may be via a bond such as a covalent bond, ionic
bond, etc., or may be by physical encapsulation. Formation of the
SAP scaffold may be divided into four stages, as follows:
[0116] Stage 1. Select the peptides and mix at the desired
concentration.
[0117] Stage 2. Introduce the material at the site of injury (e.g.,
by injection). A specific pattern of introduction may be
employed.
[0118] Stage 3. Self-assembly of peptide.
[0119] Stage 4. Post assembly deposition of RPFs.
[0120] Stage 4 is only necessary in the case of scaffolds that
incorporate additional substance(s), where the incorporation occurs
after introduction of the peptides at the site of injury. It will
be appreciated that stages 2 and 3 can take place in the opposite
order, i.e., the scaffold can be assembled in vitro prior to
introduction at a site of injury. In general, the stage at which
incorporation takes place may be selected based on the nature
and/or chemical makeup of the substance. Multiple substances
incorporated in each step or combination of steps. Incorporation at
the various stages is discussed below, roughly in reverse order of
degree to which incorporation may affect or involve the process of
assembly of the scaffold.
[0121] Incorporation at Stage 4: Perhaps the most expedient way of
incorporating substances into the scaffold is to deposit the SAP as
described above and then deposit the additional substance (e.g., as
a solution) at stage 4 on the scaffold after self-assembly has
occurred in vivo, e.g., a short period of time after introduction
of the peptide at the site of injury. This method offers certain
advantages, e.g., it will not perturb the peptide assembly.
[0122] Incorporation at Stage 3: The presence of ions (e.g., in the
form of a salt) is necessary for the self-assembly process of the
sapeptide. Certain RPFs are salts and may be able to initiate
self-assembly. One example would be an aqueous solution containing
LiCl (or another salt containing Li). LiCl can be added to the
peptide solution shortly before introducing it at the site of
injury. Li ions may then contribute to self-assembly in vivo.
Alternately, LiCl may be added to the peptide solution and
self-assembly allowed to occur in vitro. The assembled peptides may
then be introduced at the site of injury.
[0123] Incorporation at Stage 2: Co-deposition. The sapeptide and
the substance(s) may be co-loaded in a device used to introduce the
scaffold material at the site of injury, e.g., a syringe, and then
extruded.
[0124] Incorporation at Stage 1: Modification of the peptide and
use of other peptides:. Some of the RPFs include building blocks
for the neuron during its elongation stage, e.g., precursors or
substrates for synthesis of an endogenous molecule such as a
protein or peptide, nucleic acid, carbohydrate, lipid, etc. For
example, the substance may be a nucleoside such as inosine, which
can be used in the synthesis of nucleic acids. Such substances,
which are used in the normal metabolic and/or anabolic processes of
the cell, will be referred to collectively as nutrients. One
example is CDP-Choline. Since choline is used by every cell in the
body and there are choline-specific transporters, simple deposition
on the sapeptide may not be optimal since the molecule may simply
diffuse away. An approach to address this issue is to cause
incorporation of the substance into the structure of the sapeptide
or to tether the substance to the scaffold. The amount of choline
can be adjusted depending, for example, on the extent of damage to
neural tissue and the growth required for complete regeneration.
Also, it is noted that choline is one of the primary precursors for
the production of the neurotransmitter acetylcholine. In general,
in certain embodiments of the invention the substance is a
precursor of or substrate for synthesis of a neurotransmitter. It
is noted that the peptides themselves comprise amino acids, which
may serve as substrates for synthesis of proteins or peptides by
neural cells. In certain embodiments of the invention the substance
is a nutrient other than an amino acid provided by the peptides
themselves.
[0125] As the scaffold degrades over time neural tissue, e.g.,
axons and oligodendrocytes, will be provided with material to allow
and enhance regeneration. In general, peptides may be modified in
accordance with methods described in copending U.S. patent
application entitled "Self-Assembling Peptides Incorporating
Modifications", Ser. No. 10/877,068, filed Jun. 25, 2004, which is
incorporated herein by reference. Methods include covalent linkage
and modifying the sequence of the peptide, e.g., by including
particular motifs within the peptide. See 10/877,068 for further
details, including discussion of particular motifs that may enhance
neural regeneration or repair.
[0126] Simply mixing the sapeptide and the substance prior to
initiation of self-assembly will typically result in encapsulation
of the substance by the peptide. Combinations of the foregoing may
be used. For example, the overall process may include initial
addition of a stage 1 modification, e.g., CDP-choline, to the
sapeptide. Then a molecule LiCl can be used to cause the
self-assembly of the sapeptide prior to or following introduction
of the sapeptide at the site of injury (stage 3). Both the
sapeptide and an RPF can be delivered in a pattern that will modify
the direction of growth and/or enhance the growth rate, etc., of
the neurons. Introduction of another substance such as a growth
factor (at stage 4) could be used to promote axon sprouting. In
certain embodiments of the invention molecules to be incorporated
into the scaffold may be encapsulated in microparticles or
nanoparticles, e.g., polymeric microparticles or nanoparticles,
which are then themselves incorporated into the scaffold.
Preferably the polymers or other materials used for formation of
such drug delivery devices are biocompatible. Certain preferred
polymers are biodegradable. Suitable polymers include, but are not
limited to, poly(lactic-co-glycolic acid), polyanhydrides, ethylene
vinyl acetate, polyglycolic acid, chitosan, polyorthoesters,
polyethers, polylactic acid, and poly (beta amino esters).
Peptides, proteins such as collagen, and dendrimers (e.g., PAMAM
dendrimers) can also be used. Methods for making polymeric
microparticles and nanoparticles and/or for making polymer/nucleic
acid complexes, are described in U.S. patent application Ser. No.
10/446,444. It will be appreciated that a variety of different
substances other than the substances discussed above may be
incorporated
[0127] In general, a variety of devices may be used to introduce
the scaffold material at or in the vicinity of a site of injury.
The composition may be locally delivered at or near a site of
injury or degeneration by injection (e.g., using needle and
syringe), catheter, cannula, etc. The composition may be delivered
under imaging guidance, e.g., stereotactic guidance. A composition
can also be administered locally to its intended target tissue
during surgery, in which case it can, if liquid or gel, be
delivered using a syringe or poured from a suitable vessel.
Alternately, a material can be wetted with the composition and then
used to apply a composition to an area of tissue.
[0128] Delivery via a syringe is one convenient technique.
Multiport syringes may be used, in which case each port may contain
a different sapeptide/substance combination, or varying
concentrations of sapeptide/substance. In this manner directional
depostion of scaffold material can be achieved. Gradients of
substances can be introduced. Any suitable pattern can be employed,
depending upon the direction in which neural tissue regeneration is
desired and the particular stratification desired.
VI. Therapeutic Applications
[0129] In general, the methods and compositions of the invention
are useful in any situation involving injury to neural tissue. Such
injury may occur as a result of surgery, trauma, stroke, tumor,
neurodegenerative disease, or other diseases or conditions. The
injury may involve complete or incomplete transection of axons. The
injury may, but need not, involve death of neural cells. The
methods and compositions are useful to restore structural and/or
functional integrity to the neural tissue, i.e., to aid in
restoring the tissue to the functional or structural condition that
existed prior to the injury. Certain injuries may result in
physical barriers that can impede regeneration or repair of neural
tissue. Such barriers may include areas of necrosis, cavitation, or
scar tissue formation. In certain embodiments of the invention
introducing the materials described herein at a site of injury
allows axon growth from a location proximal to the site of injury
or barrier to a location distal to the site of injury or
barrier.
[0130] In addition, the methods and compositions of the invention
are useful to promote formation of neural tissue at locations or in
configurations other than those normally found in the subject. For
example, if a barrier such as a scar exists, the methods and
compositions of the invention may be employed to create new areas
of tissue that circumvent or eliminate the barrier. New neural
tissue that functionally compensates for or replaces neural tissue
that has been lost (e.g., due to stroke) can be created with the
aid of the methods and compositions described herein, offering the
possibility of reconstructive neurosurgery, e.g., reconstructive
brain surgery. The scaffolds described herein may form a bridge or
guide for development of new neural tissue.
[0131] Although the applications of the invention have primarily
been described in reference to regeneration of neural tissue in the
CNS, it is evident that they may also be employed to enhance
regeneration of nerves in the PNS, where the requirements for nerve
regeneration are less demanding.
[0132] The compositions and methods of the invention are also of
use for treating spinal dysfunction or damage due to any of a
variety of conditions. By "spinal dysfunction or damage" is meant
any condition resulting in a detrimental structural and/or
functional alteration in the normal motor, sensory, and/or support
functions of the spine, e.g., vertebral column (vertebrae and/or
discs) and/or spinal cord. The dysfunction may result in pain
either with or without an evident structural alteration or
abnormality.
[0133] In particular, the compositions and methods of the invention
are may be used in replacement, repair, and/or regeneration of
intervertebral disc tissue, e.g., nucleus pulposus tissue. The
intervertebral disc is a fibrocartilaginous disc serving as a
cushion between all of the vertebrae of the spinal column. The disc
consists of a nucleus (nucleus pulposus), composed primarily of
proteoglycans and Type II collagen with a capacity to absorb and
distribute load, and an outer, tougher annulus (annulus fibrosis)
with well-organized layer of Type I collagen that serves to
stabilize the motion segment. The structure and/or function of the
disc may be altered by various processes including normal
physiological aging, mechanical factors including trauma and
repetitive stress, segmental instability of the spine, and
inflammatory and biochemical factors. In the condition known as a
herniated disc, the nucleus pulposus is forced through a weakened
part of the disc. This results in back pain and leg pain (lumbar
herniation) or neck pain and arm pain (cervical herniation) due to
nerve root irritation.
[0134] Current approaches for treatment of damaged, e.g. herniated
discs include intervertebral disc excision (discectomy),
arthrodesis (fusion) of the spine using posterior, anterior, or
combined approaches. However, treatment of back pain and functional
preservation may be most effective if disc function is restored.
The compositions of the invention can be used to replace or augment
intervertebral disc tissue in conjunction with, or instead of, any
of the foregoing procedures. The intervertebral disc tissue to be
replaced or repaired may be tissue that is damaged due to trauma,
during surgery, etc., or may be tissue that has degenerated,
thinned, bulged, or herniated. Repair can include any aspect of
anatomical or functional restoration of the condition of the
intervertebral tissue to a normal condition, e.g., a conditions
prior to an injury or degenerative process. Disc repair can be
performed in conjunction with other aspects of spinal regeneration
including, but not limited to, use of the compositions of the
invention to repair and/or regenerate spinal neural tissue, i.e.,
spinal cord tissue and/or spinal nerve tissue.
[0135] The compositions, e.g., self-assembling peptides (either
pre-assembled or in solution), can be introduced into a damaged
disc space (e.g., within a region surrounded or partially
surrounded by annulus fibrosis), or can be used to create an
entirely new disc, which may be referred to as a replacement disc,
disc implant, etc. The replacement disc may consist essentially of
a composition of the invention, i.e., a nanoscale structured
material such as a composition comprising self-assembling peptides
as described above. The composition may optionally include one or
more additional agents, e.g., a biologically active substance such
as a therapeutic agent.
[0136] In certain embodiments of the invention the replacement disc
includes one or more supplementary materials. For example, a
composition of the invention may be used for nucleus pulpusos
replacement, while a second material (either synthetic or derived
from natural sources) can be used to functionally and/or
structurally replace all or part of the annulus fibrosis. For
example, fibrous tissue may be removed from elsewhere in a
patient's body and used to replace or repair a damaged annulus
fibrosis. Damaged or lost nucleus pulposus may be replaced with a
composition of the invention. Natural or synthetic polymeric
materials can also be used for this purpose. Metals, plastics,
e.g., polyethylene, or other biocompatible materials known in the
art can be used to fabricate a replacement disc. An intervertebral
disc replacement may comprise an inner portion and an outer
portion. The inner portion may comprise or consist essentially of
self-assembling peptides. The outer portion may also comprise or
consist of self-assembling peptides or may comprising or consist
essentially of one or more other materials. The other material(s)
may provide a greater strength or stiffness than the material(s)
that comprise the inner portion. For example, a replacement disc
may comprise an outer portion comprising a metal, plastic, or
polymer, and an inner portion comprising a nanoscale structured
material, e.g., a composition comprising self-assembling peptides.
A composition of the invention may provide a nucleus pulposus
implant, which may be used together with existing intervertebral
disc replacement devices. Alternately, different compositions of
the invention (e.g., peptide compositions of different stiffness)
can be used to replace nucleus pulposus and annulus fibrosis.
[0137] Any suitable diagnostic test or combinations thereof may be
used to identify a patient in need of replacement and/or repair of
intervertebral disc tissue. For example, the straight-leg raising
test may be used to diagnose presence of a herniated lumbar disc.
Myelograms and/or imaging studies such as MRI scans are also of
use.
[0138] The methods and compositions of the invention may be tested
using any of a variety of animal models for injury to, or
degeneration of, the nervous system or spine, e.g., intervertebral
disc. One such model, involving a knife wound to the optic tract in
hamsters, is described in detail herein. Other models that may be
used include, but are not limited to, a rodent model of anterior
ischemic optic neuropathy [96]; rodent models for thromboembolic
stroke [97], etc. See also references in 86.
EXAMPLES
Example 1
SAP Peptide Scaffolds Support Neural Tissue Regeneration In
Vivo
[0139] Materials and Methods
[0140] Animals and surgery. Syrian hamster pups at two days of age
(P2) were anesthetized with whole-body cooling. The scalp was
opened and the optic tract within the superior colliculus (SC) was
completely severed with a deep knife wound through a slot cut in
the cartilaginous skull, extending 1-2 mm below the surface from
the midline to a point beyond the lateral margin of the SC. At
surgery, 10 animals were treated by injection into the brain wound
of 10 microliters of 1% SAP RADA16-I (n-RADARADARADARADA-c; SEQ ID
NO: 1) prepared by dissolving peptide in sterile water. The
overlying scalp was then closed with tissue glue (cyanoacrylic).
The pups were returned to the cage, placed under a heat lamp, and
monitored until they recovered. After recovery the hamster pups
were returned to their mother. Control animals with the same lesion
included 3 with isotonic saline injection (10 microliters), and 27
earlier cases with knife cuts and no injection.
[0141] Axon tracing method. Four days before the animals were
sacrificed they were anesthetized with intraperitoneal injection of
Valium (50 mg/kg) and Nembutal (10 mg/kg). The animals with
survival times of 6, 30, and 60 days received intraocular
injections of 1 .mu.l of 1% Cholera-toxin subunit B (CT-B)
conjugated with fluorescein isothiocyanate (CTB-FITC) (List
Biological, Inc) into the vitreous humor of the left eye. This was
accomplished with the aid of glass micropipette (tip diameter
.about.10 .mu.m) attached to a General Valve Pico Spritzer as
previously described [52, 53]. The subjects were then returned to
their cages, under a heat lamp, and monitored until they
recovered.
[0142] Histology. The animals were sacrificed with an overdose of
anesthesia 4 days after the intraocular injection of CT-B and were
then perfused transcardially with PBS buffer (pH 7.4) followed by
2% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The brains
and eyes were removed and postfixed at 4.degree. C. until they
sank. The tissue was cut on a cryostat in 30 micron coronal
sections. These sections were mounted directly on gelatin-coated
slides. The mounted sections were first air dried, then washed with
PBS (pH 7.4) three times at 10 min intervals, and then preblocked
in PBS (pH 7.4) containing 2% Triton X-100, 2% normal rabbit serum,
and 2.5% bovine serum albumin for 30 min at room temperature. The
slides were then incubated with goat anti-cholaragenoid (List
Biological Lab, Inc.) (1:8000 dilution)+2% Triton X-100, 2% normal
rabbit serum, and 2.5% bovine serum albumin for 48 hours at room
temperature. Slides were then washed in PBS (pH 7.4) for three
rounds of 10 min each. Sections were incubated with fluorescent
donkey anti-IgG antibody Alexa-488 (secondary antibody from
Molecular Probes, Inc.) (1:200 dilution) for 1 her 30 min at room
temperature in a light protected chamber. Slides were then washed
in PBS (pH 7.4) 4 to 5 times at 5 min intervals. The slides were
then cover-slipped with Dabco. The slides were visualized through a
fluorescent microscope and pictures were taken with a Kodak DCS 620
digital camera.
[0143] Results
[0144] A tissue gap caused by deep transections of the optic tract
(OT) in the midbrain can completely block the re-innervation of the
superior colliculus (SC) by the retina, even when done at young
ages when the axons have regenerative potential. To assess the
ability of a self-assembling peptide (SAP) nanofiber scaffold to
facilitate the reconstruction of a tissue substrate that supports
regeneration across the tissue disruption, brain wounds were
inflicted in postnatal day-2 Syrian hamsters anesthetized by
whole-body cooling. The scalp was opened and the OT within the SC
was completely severed with a deep knife wound through a surgical
opening in the cartilaginous skull, extending 1-2 mm below the
surface from the midline to a point beyond the lateral margin of
SC.
[0145] FIG. 2 is a parasaggital view of an adult hamster brain
showing a schematic representation of the lesion (indicated with
arrows) that transected the optic tract in the middle of the
superior colliculus. FIG. 3 is a dorsal view reconstruction of the
hamster brain with cortex removed. Rostral is to the right and
caudal is to the left. The straight black line is the location of
the transection of the optic tract made at postnatal day 2 (P2).
Other areas of the brain shown include the superior colliculus
(SC), pretectal area (PT), lateral posterior nucleus (LP), medial
geniculate body (MGB), and inferior colliculus (IC).
[0146] Animals survived the procedure for 1, 3, 6, 30 or 60 days.
At surgery, 10 animals were treated by injection into the brain
wound of 10 ul of 1% SAP RADA16-I. Control animals with the same
lesion included 3 with isotonic saline injection (10 ul), and 27
earlier cases with knife cuts and no injection, surviving 6-9 days.
Additional animals were injected with SAP and the vital dye Congo
Red at 1% solution, in an effort to determine whether Congo Red
would stain the peptide structure (which it did not, under these
conditions). The Congo Red inhibits cell growth, thus these
injections served as an additional negative control. Initial
experiments had shown that addition of the SAP to CSF in vitro
resulted in self-assembly to form a peptide scaffold, confirming
that CSF contains sufficient a concentration of ions to support
self-assembly (data not shown).
[0147] Histological results revealed that only in the animals
treated with SAP alone, the tissue appears to have reconnected
across the lesion at all survival times (.chi..sup.2=34.8, df (1),
p<<0.001). FIGS. 4-7 show representative results obtained at
various times following infliction of the lesion. FIG. 4A shows a
schematic illustration of a parasagittal section of the dorsal
midbrain of a hamster, with the rectangle showing the approximate
location of images presented in panels 4A and 4B. FIG. 4B shows a
parasagittal section from the brain of a 1 month old hamster with a
saline-injected lesion inflicted at day P2. Arrows show the path
and extent of the knife cut. The longitudinal cavity shows the
failure of the tissue to form a bridge or grow together at the site
of the lesion. FIG. 4C is an image showing a part of a section
similar to that shown in FIG. 4B, from a 1 month hamster which had
a lesion inflicted at day P2 that was injected with 10 microliters
of 1% SAP RADA16-I. Retinal projections are shown in green. In
contrast to the saline-injected control, the proximal and distal
faces of the SAP-injected lesion are not separated by a large
cavity and appear to have grown together. In addition, axons have
extended from a location proximal to the lesion to a distal
location. FIG. 4D shows an enlarged view of area indicated with the
box in FIG. 4C. Regrown axons, traced with a standard cholera-toxin
labeling procedure using immunohistochemistry, are seen in white.
The bright region in the upper right is an area of dense
termination of axons that have crossed the lesion, seen with
fluorescence microcopy.
[0148] FIGS. 5-7 show results obtained with SAP injection at
additional time points following lesioning. FIG. 5 shows
parasaggital sections from animals sacrificed at 24 and 72 hours
postlesion and injection of SAP. 24 hour survival cases are shown
in FIG. 5A. 72 hour survival cases are shown in FIG. 5B. In each
panel, the image on the left shows lesions injected with SAP and
Congo Red, while the image on the right shows lesions injected with
SAP alone. FIG. 6 shows images of parasaggital sections from
animals one month postlesion and injection of SAP. FIG. 6A shows
images from two animals injected with SAP and Congo Red (similar to
saline controls not shown). FIG. 6B shows images from two animals
injected with SAP alone. FIG. 7A shows a dark-field photo
(composite) of a lesion site 2 months after infliction of the
lesion and injection of SAP. FIG. 7B is a corresponding
bright-field image. Note the minimal nature of the scar in the
bright field picture.
[0149] These results demonstrate that in the presence of the SAP
peptide, tissue bridges the gap created by the lesion, and partial
re-innervation of the caudal SC by the severed retinofugal axons
can occur. Thus, the SAP is shown to offer a new means of
ameliorating the tissue disruptions caused by injury to the CNS,
allowing regrowth of axons that have regenerative potential.
Example 2
Introduction of a SAP at a Site of InjuryPromotes Anatomic and
Functional Recovery in Adult Hamsters
[0150] We have previously demonstrated success using peripheral
nerve autografts to bridge sites of transection of the optic tract,
as assessed by behavioral recovery as well as by neuroanatomical
tracing of the retinal projections [18]. Syrian hamsters are a good
animal for such experiments because they become blind in tests of
visually elicited head turning after complete severance of the
brachium of the superior colliculus (BSC). Thus functional recovery
of severed axons can be assessed by evaluating visually elicited
head turning at various times after infliction of a lesion that
severs the brachium, such as those described in Example 1.
[0151] To determine the extent of return of vision the animals are
tested for visually elicited orienting responses [3, 51]. The
identity of the animal is unknown to the investigator during
testing periods. Before behavioral testing, the animals are
habituated to the testing environment. Videotaping of the response
is accomplished by the use of an overhead Sony digital video (DV)
recorder. Some of the trials are simultaneously taped from the side
with another Sony DV recorder. Later the video is analyzed
frame-by-frame on a 21-inch monitor. All of the cameras are time
synchronized. The first type of analysis is to determine if the
trial is valid or invalid. Once a trial is judged as valid it is
analyzed frame by frame, which enables us to plot the position of
the stimulus with respect to the initial head location. The angle
of the head at the beginning of the turning movement is set to
zero. The angular position of the stimulus is plotted with
reference to the initial head position. To plot the trajectory of
the turn we plot the changing position of the head over time and in
each frame we also plot the position of the stimulus. This insures
that if the stimulus is moved during the trial we can see the true
relative position of the stimulus with respect to the head at any
moment. The data obtained from these analyses are converted into
graphs of the elicited orienting movements. Leftward turns are
plotted as positive on the y axis and rightward turns are plotted
as negative values. Upon completion of testing the animals are
prepared for histological procedures.
[0152] FIG. 8 shows that an adult animal, (with a transected optic
tract after regeneration facilitated by surgically implanted PN
bridges) turns toward the stimulus in the affected left visual
field in small steps, prolonged here by movements of the stimulus
away from him. Each frame is taken from a single turning movement,
at times 0.00, 0.27, 0.53, and 0.80 sec from movement initiation.
The animal reached the stimulus just after the last frame. This is
about 0.20 sec slower than most turns by a normal animal.
[0153] The procedures described above were used to assess
functional recovery in adult hamsters that had been subjected to
deep transections of the brachium of the superior colliculus (BSC)
in the midbrain at 2 months of age. In untreated animals, such
transections can completely block the re-innervation of the
superior colliculus (SC) by the retina. The lesion was located as
shown in FIG. 9. At the time of surgery, animals were treated by
injection of 10 ul of 1% SAP RAD16-I into the brain wound. Animals
were allowed to recover from surgery and maintained under standard
laboratory conditions.
[0154] The animals were subjected to behavioral testing beginning
at day 44-46 after surgery. SAP treatment supported partial
re-innervation of the SC by the severed retinofugal axons
sufficient for significant behavioral recovery. Following SAP
treatment neural tissue reconnected across the site of the lesion
with axons growing through the site where the cut transected the
brachium of the superior colliculus. Behavioral testing started at
44-46 days following infliction of the lesion and injection of SAP.
Animals were tested by assessing their response when presented with
a stimulus (a seed) that was brought into the visual field from the
side. Normal animals turn toward the seed approximately 100% of the
time.
[0155] Initial data demonstrates a return of functional vision in
six animals, as shown in FIG. 12. The x axis in the figure shows
the dates tested. The y axis represents the number of times the
animal turned toward the stimulus during testing, expressed as a
percentage of the number of times the stimulus was presented on the
side that had received the lesion and SAP treatment. For example, a
value of 0.5 represents turning towards the stimulus on 50% of the
trials. Typically the stimulus was presented about 20 times on each
side to avoid development of a turning preference. Trend lines
representing the results of all trials on each day were plotted
using the logarithmic trendline function of Microsoft Excel to
calculate the least squares fit. An upward trend signifies
improving vision while a downward trend would signify worsening
vision. Note that the trend lines are up and to the right
signifying return of vision over time after complete transection of
the brachium of the and treatment with SAP. The dark blue line
represents the behavioral results for an adult animal, the
anatomical results for which are shown in FIG. 10. The aqua line is
believed to represent an animal that experienced a rapid return of
vision, which was complete prior to the start of behavioral
testing.
[0156] Following the conclusion of behavioral testing, animals were
subjected to intraocular injections of 1 .mu.l of 1% Cholera-toxin
subunit B (CT-B) conjugated with fluorescein isothiocyanate
(CTB-FITC) (List Biological, Inc) into the vitreous humor of the
right eye. Axon tracing and histology were performed as described
above to allow visualization of the lesion site and assessment of
axon regrowth. FIG. 10 is a parasagittal section of the dorsal
midbrain of a hamster. Rostral is left and caudual is right. The
section is from an 8 month old hamster and was taken 2 months
following infliction of the lesion. The bright yellow in the middle
of the picture that extends from the lower left to the upper right
is in the middle of the lesion site. Retinal projections are in
green. Note the axons extending through the center of the cut. The
area of densest crossing is in the upper right of the picture as
seen with fluorescence microscopy, however there are axons along
most of the site. In addition, the tissue has reconnected at the
lesion site in a similar manner to that observed in the younger
animals.
[0157] FIG. 11 is a parasagittal section of the dorsal midbrain of
a hamster. Rostral is left and caudual is right. The section is
from an 8 month old hamster and was taken 2 months following
injection of 10 .mu.l of 1% SAP RAD16-I into a lesion that
transected the brachium of the SC. The lesion site extends from top
of the picture to the bottom in the middle of the picture. White
arrows indicate the middle of the lesion. Retinal projections are
in green. Note the axons extending through the center of the area
where the cut was located. All of the tissue has repaired itself,
and axons have extended their processes to the superior colliculus.
The area of densest crossing is in the upper right of the picture
as seen with fluorescence microscopy. The re-grown axons were
traced with cholera-toxin fragment B labeling and using
immunohistochemistry for amplification of the tracer. This figure
was taken at lower magnification than FIG. 11 and thus shows
healing of the lesion over a larger scale. The site of the cut is
essentially indistinguishable from the surrounding unlesioned
tissue.
[0158] The foregoing description is to be understood as being
representative only and is not intended to be limiting. Alternative
systems and techniques for making and using the compositions and
devices of the invention and for practicing the inventive methods
will be apparent to one of skill in the art and are intended to be
included within the accompanying claims.
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Sequence CWU 1
1
31116DNAArtificial SequenceSelf-Assembling Peptides 1radaradara
darada 16216DNAArtificial SequenceSelf-Assembling Peptides
2radargdara dargda 1638DNAArtificial SequenceSelf-Assembling
Peptides 3radarada 8416DNAArtificial SequenceSelf-Assembling
Peptides 4raradadara radada 1658DNAArtificial
SequenceSelf-Assembling Peptides 5raradada 8616PRTArtificial
SequenceSelf-Assembling Peptides 6Ala Glu Ala Lys Ala Glu Ala Lys
Ala Glu Ala Lys Ala Glu Ala Lys1 5 10 1578PRTArtificial
SequenceSelf-Assembling Peptides 7Ala Glu Ala Lys Ala Glu Ala Lys1
5816PRTArtificial SequenceSelf-Assembling Peptides 8Arg Ala Glu Ala
Arg Ala Glu Ala Arg Ala Glu Ala Arg Ala Glu Ala1 5 10
1598PRTArtificial SequenceSelf-Assembling Peptides 9Arg Ala Glu Ala
Arg Ala Glu Ala1 51016DNAArtificial SequenceSelf-Assembling
Peptides 10kadakadaka dakada 16118DNAArtificial
SequenceSelf-Assembling Peptides 11kadakada 81217PRTArtificial
SequenceSelf-Assembling Peptides 12Ala Glu Ala Glu Ala Glu His Ala
His Ala Glu Ala Glu Ala His Ala1 5 10 15His138PRTArtificial
SequenceSelf-Assembling Peptides 13Ala Glu Ala Glu Ala His Ala His1
51416PRTArtificial SequenceSelf-Assembling Peptides 14Phe Glu Phe
Glu Phe Lys Phe Lys Phe Glu Phe Glu Phe Lys Phe Lys1 5 10
15158PRTArtificial SequenceSelf-Assembling Peptides 15Phe Glu Phe
Lys Phe Glu Phe Lys1 51616PRTArtificial SequenceSelf-Assembling
Peptides 16Leu Glu Leu Glu Leu Lys Leu Lys Leu Glu Leu Glu Leu Lys
Leu Lys1 5 10 15178PRTArtificial SequenceSelf-Assembling Peptides
17Leu Glu Leu Glu Leu Lys Leu Lys1 51816PRTArtificial
SequenceSelf-Assembling Peptides 18Ala Glu Ala Glu Ala Lys Ala Lys
Ala Glu Ala Glu Ala Lys Ala Lys1 5 10 151912PRTArtificial
SequenceSelf-Assembling Peptides 19Ala Glu Ala Glu Ala Glu Ala Glu
Ala Lys Ala Lys1 5 10208PRTArtificial SequenceSelf-Assembling
Peptides 20Ala Glu Ala Glu Ala Lys Ala Lys1 52116PRTArtificial
SequenceSelf-Assembling Peptides 21Lys Ala Lys Ala Lys Ala Lys Ala
Glu Ala Glu Ala Glu Ala Glu Ala1 5 10 152216PRTArtificial
SequenceSelf-Assembling Peptides 22Ala Glu Ala Glu Ala Glu Ala Glu
Ala Lys Ala Lys Ala Lys Ala Lys1 5 10 152316DNAArtificial
SequenceSelf-Assembling Peptides 23rarararada dadada
162416DNAArtificial SequenceSelf-Assembling Peptides 24adadadadar
ararar 162516DNAArtificial SequenceSelf-Assembling Peptides
25dadadadara rarara 162616DNAArtificial SequenceSelf-Assembling
Peptides 26adadadadar ararar 162716PRTArtificial
SequenceSelf-Assembling Peptides 27His Glu His Glu His Lys His Lys
His Glu His Glu His Lys His Lys1 5 10 15288PRTArtificial
SequenceSelf-Assembling Peptides 28His Glu His Glu His Lys His Lys1
52920PRTArtificial SequenceSelf-Assembling Peptides 29Val Glu Val
Glu Val Glu Val Glu Val Glu Val Glu Val Glu Val Glu1 5 10 15Val Glu
Val Glu 203020PRTArtificial SequenceSelf-Assembling Peptides 30Arg
Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe Arg Phe1 5 10
15Arg Phe Arg Phe 20314PRTArtificial SequenceSelf-Assembling
Peptides 31Arg Ala Asp Ala1
* * * * *
References