Compositions and Methods for Inhibiting the Activity of LAR Family Phosphatases

Abstract

To provide methods for inhibiting the activity, signaling and / or functions of leucocyte-common antigen related (LAR) family phosphatases induced by proteoglycans in cells of subjects.SOLUTION: Disclosed is a method to administer a therapeutic agent that inhibits one or more of catalytic activity, signal transduction and function of a LAR family phosphatase without inhibiting binding to or activation of the LAR family phosphatase by a proteoglycan. In some embodiments, the LAR family phosphatase is a receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide having about 65% homology to about 10-20 consecutive amino acids of the wedge domain of PTPσ. Also disclosed is a therapeutic agent comprising the therapeutic peptide for treating neuropathy.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority from U.S. Provisional Patent Application No. 61 / 621,623, filed on April 9, 2012, the subject matter of which is incorporated herein by reference in its entirety.

[0002] This application relates to compositions and methods for inhibiting or reducing the activity, signaling and / or function of phosphatases of the leukocyte common antigen-related (LAR) family, and to methods and compositions for treating diseases, disorders and / or conditions related to the activity, signaling and / or function of LAR family phosphatases.

Background Art

[0003] Spinal cord injury and other central nervous system (CNS) injuries can cause permanent physical disabilities or loss of movement (paralysis) and sensation below the site of the injury or disorder. Recovery after CNS injury is minimal, leading to substantial current interest in potential strategies to overcome this problem. A fundamental barrier to attempts to improve neuronal function after injury is that the mature CNS cannot regenerate.

[0004] Two well-known classes of regeneration inhibitors are myelin-associated inhibitors (MAG, Nogo and OMGP) and inhibitors in the scar tissue formed by glia at the site of injury (e.g., chondroitin sulfate proteoglycan (CSPG)). CSPG is involved in not only traumatic injuries but also a number of other CNS diseases including neurodegeneration. Examples of receptors for myelin-associated inhibitors include PirB and NgR.

[0005] Although CSPGs present a barrier to axonal regeneration, specific receptors for the inhibitory effects of CSPGs have not been identified to date. More specifically, CSPGs show dramatic upregulation after nerve injury, both in the extracellular matrix of scar tissue and in the perineuronal nets within more distant targets of transected axons. The inhibitory nature of CSPGs is reflected not only in the formation of dystrophic axonal constriction bulbs that cannot regenerate through the lesion, but also in the limited ability of spared fibers to undergo associated sprouting. It has been known for nearly 20 years that sulfated proteoglycans are a major contributing factor to the repulsive nature of the glial scar, but the exact mechanism of inhibition is poorly understood. Thus, an urgent need remains for a mechanism to regulate the function of CSPGs.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Embodiments described herein relate to methods of inhibiting or reducing the activity, signaling, and / or function of phosphatases of the leukocyte common antigen-related (LER) family in cells of a subject induced by proteoglycans. The methods include administering to the cells a therapeutic agent that inhibits one or more of the catalytic activity, signaling, and function of phosphatases of the LAR family without inhibiting the binding or activation of the phosphatases of the LAR family by proteoglycans.

Means for Solving the Problems

[0007] In some embodiments, the phosphatase of the LAR family is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent includes a therapeutic peptide having an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. For example, the therapeutic agent can include a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9-33.

[0008] In other embodiments, the phosphatase of the LAR family is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent can include a therapeutic peptide that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37. The therapeutic peptide can include, for example, conservative substitutions of at least 1, 2, 3, or 4 amino acids of residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37.

[0009] In some embodiments, the cell is a neuron, glial cell, glial progenitor cell, or neural progenitor cell.

[0010] In other embodiments, the therapeutic agent is linked to a therapeutic peptide and includes a transport moiety that facilitates uptake of the therapeutic peptide by the cell. For example, the transport moiety can be the Tat transport moiety of HIV.

[0011] In still other embodiments, the cell is in a subject to be treated, and the therapeutic agent is administered locally or systemically to the subject to be treated.

[0012] In still other embodiments, the therapeutic peptide is expressed in the cell.

[0013] Embodiments herein also relate to methods of treating diseases, disorders, and / or conditions associated with activation and signaling of phosphatases of the LAR family. The methods include administering to the cells of a subject a therapeutic agent that inhibits one or more of the catalytic activity, signaling, and function of the phosphatases of the LAR family without inhibiting binding to or activation of the phosphatases of the LAR family by proteoglycans.

[0014] In some embodiments, the diseases, disorders, and / or conditions include at least one of the diseases, disorders, and / or conditions of the nervous system.

[0015] In other embodiments, the diseases, disorders and / or conditions of the nervous system include at least one of neuropathy, neuropsychiatric disorder, nerve injury, neurotoxic disorder, neuropathic pain, and neurodegenerative disorder.

[0016] For example, neuropathy can include at least one of peripheral or cranial nerves, spinal cord or brain, traumatic or toxic injury to cranial nerves, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury. Neuropathy can also include at least one of Alzheimer's disease, dementia associated with Alzheimer's disease, Parkinson's disease, diffuse Lewy body disease, senile dementia, Huntington's disease, Tourette syndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, diabetic neuropathy, progressive supranuclear palsy, epilepsy, or Creutzfeldt-Jakob disease.

[0017] In some embodiments, nerve injury can be caused by, or associated with, epilepsy, cerebrovascular disease, autoimmune disease, sleep disorder, autonomic neuropathy, bladder disorder, abnormal metabolic state, muscular disorder, infectious and parasitic diseases, tumors, endocrine diseases, nutritional and metabolic diseases, immune diseases, diseases of the blood and blood-forming organs, mental diseases, nervous system diseases, sensory organ diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, urogenital system diseases, skin and subcutaneous tissue diseases, musculoskeletal and connective tissue diseases, congenital anomalies, or perinatal-originated conditions.

[0018] Still other embodiments described herein relate to therapeutic agents for promoting at least one of the proliferation, motility, viability, and plasticity of nerve cells. Therapeutic peptides are included having an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. For example, the therapeutic agent can include a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9-33.

[0019] In other embodiments, the therapeutic agent can include a therapeutic peptide that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 37. The therapeutic peptide can include, for example, conservative substitutions of at least 1, 2, 3, or 4 amino acids at residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37.

[0020] Still other embodiments described herein relate to pharmaceutical compositions. The pharmaceutical composition includes a therapeutic agent that includes a synthetic therapeutic peptide that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 37, linked to a therapeutic peptide, and a transport moiety that promotes cellular uptake of the therapeutic peptide.

[0021] In some embodiments, the therapeutic peptide includes, for example, conservative substitutions of at least one amino acid at residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37.

[0022] In other embodiments, the therapeutic peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 - 33 and 37.

[0023] In still other embodiments, the therapeutic agent is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 - 66 and 70.

Brief Description of the Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] The embodiments described in this specification are not limited to specific methodologies, protocols, reagents, etc., and can be changed as such. The terms described in this specification are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims. Except in the examples of operation or as otherwise indicated, all numbers representing the amounts of components or reaction conditions used in this specification should be understood to be modified by the term "about" in all cases.

[0026] All specified patents and other publications are hereby expressly incorporated by reference into this specification for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All references to dates or representations regarding the content of these documents are based on the information available to the applicant and do not constitute an admission as to the accuracy of the dates or content of these documents.

[0027] Unless defined otherwise, scientific and technical terms used herein should have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, the singular forms shall include the plural, and the plural forms shall include the singular. Generally, the nomenclature utilized in connection with the cell and tissue culture, molecular biology, and chemistry of proteins and oligonucleotides or polynucleotides and hybridization described herein, as well as the techniques thereof, are those well known and commonly employed in the art.

[0028] As used herein, "one or more of a, b, and c" means a, b, c, ab, ac, bc, or abc. The use of "or" herein is an inclusive or.

[0029] As used herein, the term "administering" to a patient includes delivering, dispensing, or applying an active compound in a pharmaceutical formulation to a subject by a route suitable for delivering the active compound to a desired location in the subject (e.g., to contact a desired cell such as a desired neuron thereby), including administration into the cerebrospinal fluid or across the blood-brain barrier, delivery by any of parenteral or oral routes, intramuscular injection, subcutaneous or transdermal injection, intravenous injection, buccal administration, transdermal delivery, and administration by rectal, colonic, vaginal, or respiratory routes. The agent can be administered, for example, to an unconscious, anesthetized, or paralyzed subject via intravenous injection, or to a pregnant subject via intravenous injection to stimulate fetal axonal growth. Specific routes of administration can include topical administration (e.g., eye drops, creams applied under the eyelids or erosive formulations, injection into the outer layer of the eye such as via intraocular injection into an aqueous or vitreous humor, subconjunctival injection or sub-tenon injection, parenteral administration or administration via an oral route).

[0030] As used herein, the term "antibody" includes synthetic antibodies including human and animal mAbs, and polyclonal antibodies, recombinant antibodies (antisera), humanized antibodies, chimeric antibodies including anti-idiotype antibodies and their derivatives. A part or fragment of an antibody refers to a region of the antibody that retains at least part of the ability (specificity and affinity of binding) to bind to a specified epitope. The term "epitope" or "antigenic determinant" refers to the site on an antigen to which the paratope of an antibody binds. Epitopes formed by adjacent amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually contain at least 3, at least 5 or 8-10, or about 13-15 amino acids in a unique spatial arrangement. Methods for determining the spatial arrangement of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, 66, EPITOPE MAPPING PROTOCOLS IN METS.IN MOLECULAR BIO. (Morris, ed., 1996); Burke et al., 170, J.Inf.Dis. 1110-19 (1994); Tigges et al., 156, J.Immunol. 3901-10).

[0031] As used herein, the term "axon growth" or "axon elongation" (also referred to herein as "neurite outgrowth") includes the process by which an axon or dendrite extends from a neuron. Outgrowth occurs at a new neurite or by expansion of a previously existing cell process. Axon elongation can include linear expansion of axonal processes up to 5 cell diameters or more. Growth processes of neurons, including neuritogenesis, can be evidenced by expression of GAP-43 detected by methods such as immunostaining. "Stimulating axon growth" means promoting axon elongation.

[0032] As used herein, "neurons of the central nervous system (CNS)" includes neurons of the brain, cranial nerves and spinal cord.

[0033] As used herein, the term "axonal retraction" refers to the retraction of an axon resulting from trauma to the axon.

[0034] As used herein, a "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence that defines a domain that is foreign and not substantially homologous to the domain of the first polypeptide (e.g., a portion of a polypeptide). A chimeric protein may present a foreign domain, which is found in an organism (although it is a different protein), and the first protein is also expressed, or it may be a "interspecies", "intergenic", etc. fusion of protein structures expressed by different types of organisms.

[0035] As used herein, the terms "contacting a neuron" or "treating a neuron" refer to any mode of delivery or "administration" of an agent to a cell or whole organism in which the agent is capable of exhibiting its pharmaceutical effect on the neuron. "Contacting a neuron" includes methods of bringing the agent of the present invention into the vicinity of the neuron, both in vivo and in vitro. Suitable modes of administration can be determined by those skilled in the art, and such modes of administration can vary between agents. For example, when stimulating axonal growth of neurons in vitro, the agent can be administered, for example, by transfection, lipofection, electroporation, infection with a viral vector, or by addition to the growth medium.

[0036] As used herein, an effective amount of an agent or therapeutic peptide is an amount sufficient to achieve a desired therapeutic or pharmaceutical effect, e.g., an amount capable of activating neuron growth. As defined herein, an effective amount of an agent can vary according to factors such as, for example, the condition, age, and weight of the subject, and the ability of the agent to elicit a desired response in the subject. A dosing regimen can be adjusted to provide an optimal therapeutic response. An effective amount is also one in which the therapeutically beneficial effects outweigh the toxic or detrimental effects of the active compound. As used herein, the term "therapeutically effective amount" refers to an amount effective at the administered dosage and for the period of time necessary to achieve the desired therapeutic outcome. The therapeutic outcome can be, for example, a reduction in symptoms, an extension of survival, improved motility, etc. The therapeutic outcome need not be "cure."

[0037] As used herein, the term "expression" refers to the process by which a nucleic acid is translated into a peptide or transcribed into an RNA that can be translated into a peptide, polypeptide, or protein. If the nucleic acid is derived from genomic DNA, expression includes mRNA splicing when an appropriate eukaryotic host cell or organism is selected. For a heterologous nucleic acid to be expressed in a host cell, it must first be delivered to the cell and then, once inside the cell, ultimately be present in the nucleus.

[0038] As used herein, the term "gene therapy" includes the introduction of heterologous DNA into mammalian, particularly human, cells that are in a disorder or condition for which treatment or diagnosis is sought. The DNA is introduced into target cells selected in such a way that the heterologous DNA is expressed and thereby the encoded therapeutic product is produced. Alternatively, the heterologous DNA intervenes in some way in the expression of the DNA encoding the therapeutic product; it may encode a product such as a peptide or RNA that intervenes in some way directly or indirectly in the expression of the therapeutic product. Gene therapy may be used to deliver a nucleic acid encoding a gene product to replace a defective gene or to complement a gene product produced by the mammalian or cell into which it is introduced. The nucleic acid introduced may encode a therapeutic compound that is not normally produced in the mammalian host or is not produced in therapeutically effective amounts or for a therapeutically useful time, for example, a growth factor or an inhibitor thereof, a tumor necrosis factor or an inhibitor thereof, such as a receptor therefor. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the affected host to enhance or otherwise alter the product or its expression.

[0039] As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid that includes an open reading frame encoding a polypeptide that includes both exon and (optionally) intron sequences.

[0040] As used herein, the term "non-homologous nucleic acid sequence" generally refers to DNA encoding RNA and proteins that are not normally produced in vivo by the cells in which they are expressed, or DNA that intervenes or encodes a mediator that modifies the expression of endogenous DNA by affecting transcription, translation, or other regulatory biological processes. Non-homologous nucleic acid sequences are also referred to as foreign DNA. DNA that is recognized or considered by one of ordinary skill in the art to be non-homologous or foreign to the cell in which it is expressed is encompassed herein by non-homologous DNA. Examples of non-homologous DNA include, for example, DNA encoding a trackable marker protein such as a protein conferring drug resistance, DNA encoding a therapeutically effective substance such as an anti-cancer agent, an enzyme, and a hormone, and DNA encoding other types of proteins such as an antibody. Antibodies encoded by non-homologous DNA can be secreted or expressed on the surface of the cell into which the non-homologous DNA has been introduced.

[0041] As used herein, the terms "homology" and "identity" are used synonymously throughout and refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for purposes of comparison. If the positions in the aligned sequences are occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0042] As used herein, the term "neuropathy" includes diseases, disorders or conditions that directly or indirectly affect the normal function or biological structure of the nervous system of a subject. The term "stroke" is recognized in the art and includes the sudden attenuation or loss of consciousness, sensation and voluntary movement caused by the rupture or occlusion (e.g., due to a thrombus) of an artery in the brain. "Traumatic brain injury" is recognized in the art and includes a state in which a traumatic blow to the head causes injury to the brain or the connecting spinal cord, with or without penetration of the skull. Usually, the initial trauma can cause expanding hematomas, subarachnoid hemorrhage, cerebral edema, increased intracranial pressure, and cerebral hypoxia, which in turn lead to serious secondary events due to low cerebral blood flow.

[0043] As used herein, the term "neuronal migration" refers to the ability of neuronal cells or migrating neuronal processes to migrate, such as axonal migration or dendritic migration.

[0044] As used herein, the phrases "parenteral administration" and "administered parenterally" mean, when used herein, a mode of administration by ordinary injection other than enteral administration and topical administration, and includes, without limitation, intravenous, intramuscular, intraarterial, intraventricular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, sub-synovial, intraspinal, and intrasternal injections and infusions.

[0045] As used herein, the phrases "systemic administration" and "administered systemically" mean, when used herein, the administration of a compound, drug or other substance other than direct administration to a target tissue (e.g., the central nervous system), so that it enters the whole body of an animal and is thus subject to metabolism and other similar processes, e.g., subcutaneous administration.

[0046] As used herein, the terms "patient" or "subject" or "animal" or "host" refer to any mammal. The subject can be a human, but can also be a mammal in need of veterinary treatment, such as a pet (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, chicken, pig, horse, etc.), and a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

[0047] As used herein, "neurons of the peripheral nervous system (PNS)" includes neurons that are present or extend outside of the CNS. The PNS is intended to include neurons generally understood to be classified in the peripheral nervous system, including sensory neurons and motor neurons.

[0048] As used herein, the terms "polynucleotide sequence" and "nucleotide sequence" are also used interchangeably herein.

[0049] As used herein, the terms "peptide" and "polypeptide" are used interchangeably herein and refer to a compound consisting of about 2 to about 90 amino acid residues, including the linking of the amino group of one amino acid to the carboxyl group of another amino acid by a peptide bond. Peptides can be derived or extracted from native proteins, for example, by enzymatic cleavage or chemical cleavage, and can be prepared by conventional peptide synthesis methods (e.g., solid-phase synthesis) or molecular biological techniques (see Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989)). "Peptide" can include suitable L- and / or D-amino acids, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., P-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and rare amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl, and / or other functional groups on the peptide can be free (e.g., unmodified) or can be protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, and means for adding or removing protecting groups are known in the art. See, for example, Green & Wuts, PROTECTING GROUPS IN ORGANIC SYNTHESIS (John Wiley & Sons, 1991). The functional groups of the peptide can also be derivatized (e.g., alkylated) using methods known in the art.

[0050] Peptides can be synthesized and assembled into libraries containing a large number of distinct molecular species, from 2 to 3. Such libraries can be prepared using well-known methods of combinatorial chemistry, as described herein, or screened using other suitable methods for measuring whether the library contains peptides that can antagonize the CSPG / PTPσ interaction. Antagonists of such peptides can then be isolated by suitable means.

[0051] As used herein, the term "peptidomimetic" refers to a protein-like molecule designed to mimic a peptide. Peptidomimetics are typically generated from modifications of existing peptides or by designing similar systems that mimic peptides, such as peptoids and β-peptides. Regardless of the approach, a modified chemical structure is designed to advantageously tune molecular properties such as stability or bioactivity. These modifications involve alterations to non-naturally occurring peptides (e.g., altered backbone and incorporation of non-natural amino acids).

[0052] As used herein, the term "progenitor cell" is a cell that is generated during the differentiation of a stem cell and has some, but not all, of the characteristics of its ultimately differentiated progeny. For example, a defined progenitor cell, such as a "neural progenitor cell," is lineage-related but not related to a specific or ultimately differentiated cell type.

[0053] As used herein, the term "stem cell" means a cell that is capable of undergoing self-renewal (i.e., progeny having the same differentiation potential) and that can give rise to progeny cells that are further restricted in their differentiation potential. Within the context of the present invention, stem cells include, for example, more differentiated cells that have been dedifferentiated by, for example, nuclear transfer, fusion with more primitive stem cells, introduction of specific transcription factors, or culture under specific conditions. See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying et al., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1199-1203 (2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al., Nature, 448:313-317 (2007); and Takahashi et al., Cell, 131:861-872 (2007).

[0054] As used herein, the term "retraction" refers to the withdrawal of axons from the site of injury, for example, from the site where a glial scar forms. Here, the tip of the regenerating axon stops growing and becomes dystrophic. These dystrophic tips can then further withdraw from the glial scar and the site of injury.

[0055] A polynucleotide sequence (DNA, RNA) is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of the polynucleotide sequence. The term "operably linked" includes having an appropriate start signal (e.g., ATG) before the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequence and to allow production of the desired polypeptide encoded by the polynucleotide sequence.

[0056] As used herein, the term "recombinant" as used herein means that a protein is derived from a prokaryotic expression system or a eukaryotic expression system.

[0057] As used herein, the term "tissue-specific promoter" means a nucleic acid sequence that serves as a promoter, i.e., regulates the expression of a selected nucleic acid sequence operably linked thereto and affects the expression of the selected nucleic acid sequence in specific cells of a tissue such as epithelial cells. The term also encompasses so-called "leaky" promoters that primarily regulate the expression of a selected nucleic acid in one tissue but also cause expression in other tissues. The term "transfection" is used to refer to the uptake of exogenous DNA by a cell. A cell is transfected when exogenous DNA has been introduced inside the cell membrane. A number of transfection methods are generally known in the art. See, e.g., Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu et al., Gene, 13:197 (1981). Using such techniques, one or more exogenous DNA moieties, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into a suitable host cell. The term encompasses chemical, electrical, and virus-mediated transfection procedures.

[0058] As used throughout this specification, the term "transcription regulatory sequence" is a general term used to refer to nucleic acid sequences such as initiation signals, enhancers, and promoters that induce or control the transcription of a sequence encoding a protein to which they are operably linked. In some instances, the transcription of a recombinant gene is under the control of a promoter sequence (or other transcription regulatory sequence), which controls the expression of the recombinant gene in the cell type in which expression is intended. It will also be understood that the recombinant gene may be under the control of transcription regulatory sequences that control the transcription of the naturally occurring form of the protein, which may be the same as or different from these sequences.

[0059] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Preferred vectors are those capable of autonomous replication and / or expression of the nucleic acids to which they are bound. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector".

[0060] As used herein, the term "wild-type" refers to a naturally occurring polynucleotide encoding a protein, or a part thereof, or a protein sequence, or a part thereof, such as it normally exists in vivo. As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) and, as appropriate, ribonucleic acid (RNA). The term is to be understood to include RNA or DNA equivalents, analogs, single-stranded (sense or antisense) and double-stranded polynucleotides made from nucleotide analogs and applicable to the desired embodiments.

[0061] Agents, compounds, compositions, antibodies, etc. used in the methods described herein are contemplated to be purified and / or isolated prior to use. A purified substance is typically "substantially pure" and means that a nucleic acid, polypeptide, or fragment thereof, or other molecule, is separated from the components that naturally accompany it. Typically, a polypeptide is substantially pure if it contains less than 60%, 70%, 80%, 90%, 95% or even 99% by weight of the proteins and other organic molecules with which it is naturally associated. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in cells that do not normally express the protein, or by chemical synthesis. An "isolated substance" has been removed from its natural location and environment. In the case of an isolated or purified domain or protein fragment, the domain or fragment substantially does not contain the amino acid sequences that flank the protein in the naturally occurring sequence. The term "isolated DNA" means that the DNA substantially does not contain the genes that flank a given DNA in the genome in which it naturally occurs. Thus, the term "isolated DNA" encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.

[0062] As used herein, the terms "portion", "fragment", "variant", "derivative" and "analogue", when referring to a polypeptide of the invention, include polypeptides that retain at least some of the biological activity (e.g., inhibition of an interaction such as binding) as referenced herein. Polypeptides as described herein can include, without limitation, portions, fragments, variants, or derivative molecules as long as the polypeptide still serves its function. The polypeptides of the invention or portions thereof can include proteolytic fragments, deletion fragments, and in particular, fragments that more readily reach the site of action, especially when delivered to an animal.

[0063] This application relates to compositions and methods for inhibiting and / or reducing the activity, signal transduction and / or function of phosphatases of the leukocyte common antigen-related (LAR) family, such as LAR and receptor protein tyrosine phosphatase sigma (PTPσ), and methods for treating diseases, disorders and / or conditions associated with the activation and signal transduction of phosphatases of the LAR family, such as LAR and PTPσ.

[0064] The phosphatases of the LAR family consist of three members: LAR itself, receptor protein tyrosine phosphatase sigma (PTPσ) and receptor protein tyrosine phosphatase delta (PTPδ). PTPσ and PTPf (LAR itself) are involved as receptors for chondroitin sulfate proteoglycan (CSPG), a major component of the glial scar and perineuronal net. The sugar side chains of CSPG can bind to LAR and PTPσ expressed by cells such as neurons, and can inhibit the proliferation, plasticity, regeneration and sprouting failure in neurons.

[0065] PTPσ knockout neurons have been found to show a reduction in inhibition in assays mediated by various CSPGs, such as increased regeneration after nerve injury, such as after spinal cord injury and optic nerve crush. The results in LAR knockout have reached no conclusion with both increases and decreases in the regenerative phenotype being found. Since CSPG is a major inhibitor of regeneration and plasticity in the damaged mature nervous system, functional inhibitors of these LAR family phosphatases can be used as therapeutic agents to promote the proliferation, plasticity, regeneration and sprouting of neurons.

[0066] Accordingly, some embodiments described herein relate to methods for promoting the proliferation, motility, survival, and / or plasticity of cells (i.e., neurons, neural progenitor cells, neural stem cells, or endothelial cells) that express phosphatases of the LAR family and that are activated and / or can potentially be activated by proteoglycans such as CSPG. The methods can include administering to the cells a therapeutically effective amount of a therapeutic agent effective to inhibit the catalytic activity, signaling, and / or function of phosphatases of the LAR family. Inhibition of the activity, signaling, and / or function of phosphatases of the LAR family can be used to promote the cell proliferation, motility, survival, and plasticity of these cells.

[0067] In certain embodiments, cells that express phosphatases of the LAR family include neurons and glial cells. Other examples of cells include endothelial cells. Still other examples of cells that express phosphatases of the LAR family and that can be activated by proteoglycans can be readily screened using known assays.

[0068] The activity, signal transduction, and / or function of the LAR family of phosphatases can be suppressed, inhibited, and / or blocked in several ways, including direct inhibition of the activity of the intracellular domain of the LAR family of phosphatases (e.g., by using small molecules, peptidomimetics, or dominant inhibitory polypeptides); activation of genes and / or proteins that inhibit one or more of the activity, signal transduction, and / or function of the intracellular domain of the LAR family of phosphatases (e.g., by enhancing the expression or activity of the genes and / or proteins); inhibition of genes and / or proteins that are mediators downstream of the LAR family of phosphatases (e.g., by blocking the expression and / or activity of the mediator genes and / or proteins); induction of genes and / or proteins that negatively regulate one or more of the activity, signal transduction, and / or function of the LAR family of phosphatases (e.g., by using recombinant gene expression vectors, recombinant viral vectors, or recombinant polypeptides); or, for example, gene replacement with hypomorphic mutants of the LAR family of phosphatases (e.g., by homologous recombination, overexpression using recombinant gene expression or viral vectors, or mutagenesis).

[0069] Therapeutic agents that inhibit or reduce one or more of the activity, signal transduction, and / or function of the LAR family of phosphatases include agents that decrease and / or suppress the activity, signal transduction, and / or function of the LAR family of phosphatases without inhibiting the binding or activation of the LAR family of phosphatases by proteoglycans such as CSPG. Such agents can be delivered intracellularly and, once delivered intracellularly, can promote the intrinsic proliferative capacity of cells such as neurons, activate neuronal proliferative pathways (e.g., the CNS), and produce neurobeneficial effects.

[0070] Neurobeneficial effects can include responses or results that are beneficial to the health or function of neurons, a part of the nervous system, or the nervous system in general. Examples of such effects include improvements in the ability of neurons or a part of a nerve to withstand, regenerate, maintain desired functions, proliferate, or survive damage. Neurobeneficial effects can include causing or achieving such responses or improvements in the function or resilience among the components of the nervous system. Examples of causing a neurobeneficial effect include, after damage to neurons, stimulating axonal outgrowth; making neurons resistant to apoptosis; making nerves resistant to toxic compounds such as, for example, β-amyloid, ammonia, or other neurotoxins; reversing age-related neuronal atrophy or loss of function; reversing age-related loss of cholinergic innervation; reversing and / or reducing axonal retraction, and / or promoting nerve sprouting.

[0071] One possible mechanism for the regulation, modulation, and / or inhibition of LAR family phosphatases involves dimerization of the intracellular portion of LAR family phosphatases. In contrast to receptor tyrosine kinases that are active as dimers and inactive as monomers, several protein tyrosine phosphatases (PTPσ) are inactive in their dimerized state and active as monomers. These include PTPα, PTP1B, and CD45. Each of these molecules can be crystallized in both their active monomeric form and their inactive dimeric form. In addition, LAR and CD45 exhibit homotypic binding under certain oxidation conditions, while PTPσ can dimerize in response to ligand binding. This suggests that ligands for LAR family phosphatases can dictate the activation state of LAR family phosphatases such as LAR and PTPσ. Thus, mimicking dimerization by intracellularly targeted therapies can directly inactivate LAR family phosphatases without altering the intracellular matrix or other ligands.

[0072] We have found that peptidomimetics of the intracellular portion of the LAR family of phosphatases can inhibit and / or reduce LAR activity induced by CSPG activation. Intracellular inhibition of LAR family activity, signaling and / or function in response to CSPG activation promotes neuronal outgrowth, including restoration of growth cone motility, neurite outgrowth, sprouting and promotion of neuronal survival and plasticity, and has also been found to inhibit neuronal axonal retraction.

[0073] In one embodiment, therapeutic agents that inhibit or reduce one or more of the activity, signaling and / or function of the phosphatases of the LAR family include therapeutic peptides or small molecules that bind to and / or complex with the intracellular domain of at least one phosphatase of the LAR family to inhibit the activity, signaling and / or function of the LAR family of phosphatases. Thus, cell growth, motility, viability and plasticity of these cells can be promoted using therapeutic peptides or small molecules that bind to and / or complex with the intracellular domain of at least one phosphatase of the LAR family in neurons.

[0074] In some embodiments, the therapeutic agent can be a peptidomimetic of the wedge-shaped domain (i.e., the wedge domain) of the intracellular catalytic domain of the phosphatases of the LAR family. Structural and sequence analysis reveals that all members of the LAR family contain a 24-amino acid wedge-shaped helix / loop / helix motif conserved in the first intracellular catalytic domain that can potentially mediate homotypic / heterotypic receptor interactions. Table 1 lists the amino acid sequences of the intracellular portions of members of the LAR family of phosphatases containing the wedge domain. These 24-amino acid wedge domains of the intracellular portions of the phosphatases of the LAR family are identified by underlining. While the specific structure of the wedge domain is conserved in most of the wedge domains of the LAR family, the exact amino acids that make up the wedge domain vary between individual proteins and subfamilies.

Table 1

[0075] The wedge domains of members of a particular LAR family were found to be involved in homotypic interactions or binding with members of that particular LAR family. For example, in pull-down assays, the wedge domain of LAR was able to specifically interact with full-length LAR, but not with other family members such as PTPσ. In addition, in vitro binding assays showed that the wedge domain peptides of PTPmu and LAR (wedge domain + HIV-TAT) specifically aggregated homotypically rather than binding randomly to each other. Particularly interestingly, the fact that the wedge domain of LAR was unable to bind to sigma indicates specificity even among members of similar families.

[0076] Thus, using peptide mimics of these wedge domains of phosphatases of the LAR family when expressed in cells (e.g., neurons) or conjugated to intracellular transport moieties, it is possible to abolish LAR family signaling in neurons activated by CSPG and promote cell proliferation, motility, and viability. The binding of these therapeutic peptides to the intact wedge domains of specific PTPs potentially (i) interferes with the ability of that PTP to interact with target proteins such as phosphatase targets; (ii) interferes with the activity that promotes intermolecular interactions between the PTP and another domain contained within the PTP, such as the catalytically inactive second phosphatase domain D2; prevents access of the protein to the active phosphatase site; (iii) competes with the normal interactors of the wedge domain; and / or sterically inhibits phosphatase activity.

[0077] In some embodiments, the peptidomimetic (i.e., therapeutic peptide) can comprise, consist essentially of, and / or consist of about 10 to about 20 amino acids and have an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to about 10 to about 20 consecutive amino acid portions of the wedge domain amino acid sequence of a phosphatase of the LAR family.

[0078] In other embodiments, the peptidomimetic can comprise, consist essentially of, and / or consist of about 10 to about 20 amino acids and have an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or 100% identical to about 10 to about 20 consecutive amino acid portions of the wedge domain of PTPσ. We have found that a peptidomimetic (e.g., therapeutic peptide) corresponding to or substantially identical to the wedge domain of PTPσ with a cytoplasmic carrier can remove CSPG-mediated inhibition and allow neurons to move on CSPG substrates instead of typical inhibition. This effect is volume-dependent and dependent on responsive cells expressing PTPσ. Astrocytes that do not express PTPσ at the protein level do not respond to peptide inactivation, while satellite glia that express PTPσ respond to the peptide. Furthermore, this peptide can be systemically administered to promote plasticity and functional recovery after severe spinal cord injury.

[0079] As shown in Table 2, the sequence of the wedge domain of PTPσ is highly conserved among higher mammals, with only one amino acid change (from threonine to methionine at position 6) between mouse and rat preventing 100% identity.

Table 2

[0080] As shown in Table 2, the first α - helix of the wedge - shaped domain of PTPσ includes amino acids 1 - 10, the turn region includes amino acids 11 - 14, and the second α - helix includes amino acids 15 - 24. For example, the first α - helix of the wedge - shaped domain of human PTPσ has the amino acid sequence of DMAEHTERLK (SEQ ID NO: 67), the turn has the amino acid sequence of ANDS (SEQ ID NO: 68), and the second α - helix has LKLSQEYESI (SEQ ID NO: 69).

[0081] The wedge - shaped domain also shares sequence homology with other members of the LAR family, LAR and PTPδ. These amino acids seem to be necessary for the overall structure of the wedge - shaped domain. The conserved amino acids include alanine at position 13, which marks the end of the first α - helix and the start of the turn, making it plausible that it is necessary for the general size and structure of the wedge shape.

[0082] Since the secondary and tertiary structures of the wedge - shaped domain remain consistent in most receptor PTPs, several conservative substitutions can be made to therapeutic peptides targeting PTPσ to obtain similar results. Examples of conservative substitutions include, for example, substitution between non - polar (hydrophobic) residues such as isoleucine, valine, leucine, or methionine, substitution of one polar (hydrophilic) residue for another, for example, between arginine and lysine, between glutamine and asparagine, between glycine and serine, substitution between basic residues such as lysine, arginine, or histidine, and / or substitution between acidic residues such as aspartic acid or glutamic acid.

[0083] These conservative substitutions can occur in domains that are not unique in the helix or turn, particularly positions 1 - 3 and 7 - 10 in the first α - helix; positions 12 and 13 in the turn; and positions 15, 16, 18 - 24 in the second α - helix. These amino acids may be necessary for the overall structure of the wedge - shaped domain but not for the specificity of the wedge - shaped binding to PTPσ.

[0084] Unique amino acids for PTPσ, particularly those differentially expressed between PTPσ and LAR, have been found to be required for the specificity of the wedge domain binding. These include the EH domain at positions 4 and 5 of the first α - helix and the subsequent threonine or methionine at position 6 (substitutions in rat and mouse). In the turn, there is a unique serine at position 14 in all higher mammals. Finally, in the second α - helix, there is a unique leucine at position 17. The possible roles of these unique amino acids are discussed below.

[0085] The serine residue at position 14 in the turn is of particular interest for the position of the wedge domain. This amino acid, located in the turn between the α - helices, extends somewhat from the general secondary and tertiary structure of PTPσ, allowing it to be utilized for binding interactions. In addition, serine is known to facilitate several homo - and hetero - binding events, such as hydrogen bonds between adjacent serines, due to its hydroxyl group and the polarity it contains. Serine is also known to undergo various modifications such as phosphorylation, increasing the likelihood of its necessity for specificity. Focusing on the turn of the wedge domain, it is possible that a small peptide containing the conserved serine could provide even greater stability with a similar function. Such a peptide could be synthesized as a loop with either cysteine terminus to create disulfide bonds.

[0086] Unique amino acids in the first α - helix include glutamic acid at position 4, histidine at position 5, and threonine or methionine at position 6. Histidine is involved in the consensus wedge domain, which is not found in LAR, PTPδ, PTPmu, or CD45. Since all three of these amino acids are charged or polar, it is thought that this sequence or one of its components is required for the wedge specificity of PTPσ.

[0087] Furthermore, the second α - helix contains a unique leucine at position 17. Leucine is said to be related to an adhesive molecule that is crucial for the three - dimensional structure of the leucine zipper. In these molecules that are structurally similar to the wedge - shaped domain, leucines of opposing α - helices, which are located at approximately 7 - residue intervals, interact with the hydrophobic regions of the opposing α - helices. Since the first α - helix also has a leucine at position 9, this unique leucine is thought to be necessary for the integrity of the entire wedge - shaped tertiary structure of PTPσ.

[0088] Accordingly, in other embodiments, the therapeutic peptide can comprise, consist essentially of, or consist of about 14 to about 20 amino acids, and includes the amino acid sequence EHX1ERLKANDSLKL (SEQ ID NO: 37), wherein X1 is T or M in the sequence. The therapeutic peptide comprising SEQ ID NO: 37 can include at least 1, at least 2, at least 3, at least 4, or at least 5 conservative substitutions such that the therapeutic peptide has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 37.

[0089] In some embodiments, the conservative substitutions are those of amino acid residues 4E, 5R, 6L, 7K, 9N, 10D, 12L, or 13K of SEQ ID NO: 37. By way of example, amino acid residue 4E can be substituted with D or Q, amino acid residue 5R can be substituted with H, L or K, amino acid residue 6L can be substituted with I, V or M, amino acid residue 7K can be substituted with R or H, amino acid residue 9N can be substituted with E or D, amino acid residue 10D can be substituted with E or N, amino acid residue 12L can be substituted with I, V or M, and / or amino acid residue 13K can be substituted with R or H.

[0090] The therapeutic peptides described herein can be the subject of various other modifications, substitutions, insertions, and deletions such that such changes provide certain advantages in their use. In this regard, a therapeutic peptide that binds to and / or forms a complex with the wedge-shaped domain of a phosphatase of the LAR family can have one or more changes made to it and still be capable of inhibiting or reducing one or more of the activities, signaling, and / or functions of a phosphatase of the LAR family, and can be corresponding or substantially homologous rather than identical to the cited polypeptide sequence that retains such ability.

[0091] The therapeutic peptide can be any of various forms of polypeptide derivatives including amides, conjugates with proteins, cyclic polypeptides, polymeric polypeptides, analogs, fragments, chemically modified polypeptides, and similar derivatives.

[0092] It will be fully understood that conservative substitutions can also include using chemically derivatized residues in place of non-derivatized residues provided that such peptides exhibit the requisite binding activity.

[0093] "Chemical derivative" refers to a polypeptide to a subject having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzyl group, t-butoxycarbonyl group, chloroacetyl group, or formyl group. A free carboxyl group can be derivatized to form a salt, methyl ester and ethyl ester, or other types of esters or hydrazides. A free hydroxyl group can be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine can be derivatized to form N-im-benzene histidine. Also included as chemical derivatives are polypeptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. 4-Hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; ornithine may be substituted for lysine. The polypeptides described herein also include any polypeptide having one or more additions and / or deletions or residues to the sequence of the polypeptide shown herein, as long as the required activity is maintained.

[0094] In still other embodiments, the therapeutic agent can be a mimetic or competitive inhibitor of a downstream protein activated by a phosphatase of the LAR family. Some downstream proteins and pathways have been shown to act downstream of the LAR family outside of phosphatase activity. Among these, Caskin (Ckn) and LAR interacting protein (replin-α) have roles in both synapse formation and axon guidance.

[0095] In a yeast two-hybrid interaction system, mCkn1 directly binds to mLAR and mPTPRδ, and mCkn2 directly binds to mLAR and mPTPσ. The interaction of Ckn with phosphatases of the LAR family was mapped to a region containing two sterile α motif (SAM) domains that constitute the phenotype. The first SAM domain is conserved among Ckn family members.

[0096] Thus, in some embodiments, the therapeutic agent can be a peptide mimetic or competitive inhibitor of Ckn that inhibits the phosphatase / Ckn binding of the LAR family and alleviates downstream LAR family signaling. The peptide can have an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to a sequence of about 10 to about 30 contiguous amino acids of a portion of mCkn1 and mCkn2. Examples of peptides having an amino acid sequence that is substantially identical to a sequence of about 10 to about 30 contiguous amino acids of a portion of mCkn1 and mCkn2 are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0097] In other embodiments, the therapeutic agent can be a peptide mimetic or competitive inhibitor of REPRIN-α that inhibits the phosphatase / REPRIN-α binding of the LAR family and alleviates downstream LAR family signaling. Family members of REPRIN play a decisive role in synapse development and maintenance. REPRIN-α can act in downstream signaling of the LAR phosphatase. Yeast two-hybrid interaction screening implicates the first SAM domain of REPRIN-α family members as the binding region for family members of the LAR phosphatase. The peptide can have an amino acid sequence that is substantially identical to a sequence of about 10 to about 30 contiguous amino acids of a portion of REPRIN-α. An example of a peptide having an amino acid sequence that is substantially identical to a sequence of about 10 to about 30 contiguous amino acids of a portion of REPRIN-α is SEQ ID NO: 41.

[0098] Similar to the above-described therapeutic peptides that bind to or form a complex with the wedge-shaped domain, therapeutic polypeptides that are mimetics or competitive inhibitors of mCkn1, mCkn2, or Repin-α can be the subject of various changes, substitutions, insertions, and deletions such that such changes provide certain advantages in their use. For example, the therapeutic polypeptide can be any of various forms of polypeptide derivatives including amides, conjugates with proteins, cyclic polypeptides, polymeric polypeptides, analogs, fragments, chemically modified polypeptides, and similar derivatives.

[0099] One or more peptides of the therapeutic peptides described herein can be modified by natural processes such as, for example, post-translational processing and / or chemical modification methods known in the art. Modifications can be present in the peptide backbone, amino acid side chains, and at the amino or carboxy terminus of the peptide. It will be fully understood that the same type of modification can be present at the same or different degrees at several sites of a given peptide. Modifications include, for example, without limitation, acetylation, acylation, addition of an acetamidomethyl (Acm) group, ADP-ribosylation, amidation, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, formation of a disulfide bond, demethylation, formation of a covalent cross-link, formation of cystine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and addition of an amino acid to a protein mediated by transfer RNA such as ubiquitination (see, for example, Protein-structure and molecular properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New-York, 1993 for reference).

[0100] The peptides and / or proteins described herein may include, for example, biologically active mutants, variants, fragments, chimeras, and analogs; include amino acid sequences having truncations of one or more amino acids, where the truncations can originate from the amino terminus (N-terminus), carboxy terminus (C-terminus), or within the protein. The analogs of the present invention involve insertions or substitutions of one or more amino acids. Variants, mutants, fragments, chimeras, and analogs can function as inhibitors of phosphatases of the LAR family (without being limited to the examples of the present invention).

[0101] The therapeutic polypeptides described herein can be prepared by methods known to those of skill in the art. The peptides and / or proteins can be prepared using recombinant DNA. For example, one preparation can include culturing a host (bacterial or eukaryotic cell) under conditions that provide for the expression of the peptide and / or protein in the cell.

[0102] Purification of the polypeptide can be performed by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity, or other purification methods commonly used for protein purification. The purification steps can be carried out under non-denaturing conditions. On the other hand, if a denaturing step is required, the protein can be refolded using techniques known in the art.

[0103] In some embodiments, the therapeutic peptides described herein can include additional residues that can be added at either end of the polypeptide for the purpose of providing a linker by which the polypeptide can be conveniently linked and / or attached to other polypeptides, proteins, detectable moieties, labels, solid matrices, or carriers.

[0104] The linker of amino acid residues is usually at least one residue and can be up to 40 or more residues, more often 1 to 10 residues. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic acid, aspartic acid, etc. In addition, the polypeptide of interest can vary by sequences that are modified by terminal -NH2 acylation, for example, by acetylation or by terminal carboxyl amidation, for example, by amidation of thioglycolic acid by terminal modification with ammonia, methylamine, etc. Terminal modification, as is well known, is useful for reducing susceptibility to protease digestion and thus serves to extend the half - life of the polypeptide in solutions where proteases may be present, especially in biological fluids. In this regard, cyclization of the polypeptide is also a useful terminal modification and is particularly preferred in view of the stable structure formed by cyclization and the biological activities observed in such cyclic peptides as described herein.

[0105] In some embodiments, the linker can be a flexible peptide linker that links a therapeutic peptide to other polypeptides, molecules such as proteins and / or detectable moieties, labels, solid matrices or carriers. The flexible peptide linker can be up to about 20 amino acids in length. For example, the peptide linker can contain up to about 12 amino acid residues, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. Optionally, the peptide linker contains two or more of the following amino acids: glycine, serine, alanine and threonine.

[0106] In some embodiments, the therapeutic agent comprising the therapeutic peptide described herein can be provided in the form of a conjugate protein or drug delivery construct that includes at least a transport subdomain or moiety (i.e., transport moiety) linked to the therapeutic peptide. The transport moiety can facilitate the uptake of the therapeutic polypeptide into mammalian (i.e., human or animal) tissues or cells (e.g., nerve cells). The transport moiety can covalently bind to the therapeutic polypeptide. Covalent bonds can include peptide bonds and labile bonds (e.g., bonds that are readily cleavable or are susceptible to chemical change in the internal target cell environment). Further, the transport moiety can crosslink (e.g., chemically crosslink, UV crosslink) to the therapeutic polypeptide. The transport moiety can also be linked to the therapeutic polypeptide together with the linking polypeptide described herein.

[0107] The transport moiety can repeat more than once with the therapeutic polypeptide. Repeating the transport moiety can affect (e.g., increase) the uptake of the peptide and / or protein by the desired cells. The transport moiety can be located in either or both of the amino-terminal or carboxy-terminal regions of the therapeutic peptide.

[0108] In one embodiment, the transport moiety can include at least one transport peptide that, once linked to the transport moiety, enables the therapeutic polypeptide to penetrate cells by a receptor-independent mechanism. In one example, the transport peptide is a synthetic peptide containing a Tat-mediated delivery sequence and at least one of SEQ ID NOs: 9-33 and 37-41. These peptides can each have the amino acid sequences of SEQ ID NOs: 42-66 and 70-74.

[0109] Other examples of known transport moieties, subdomains, etc. are, for example, Canadian Patent Document No. 2,301,157 (a conjugate containing the homeodomain of antennapedia), which is hereby incorporated by reference in its entirety, and U.S. Patent Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604 (TatHIV protein; amino acids of the herpes simplex virus-1 DNA binding protein VP22; a histidine tag in the range of 4 to 30 histidine repeats, or a conjugate containing a mutagenized derivative or analog thereof capable of promoting the uptake of an active cargo moiety by a receptor-independent method).

[0110] A 16-amino acid region of the third α-helix of the antennapedia homeodomain has been shown to enable a protein (made as a fusion protein) to cross the cell membrane (PCT International Publication No. WO99 / 1809 and Canadian Patent Application No. 2,301,157). Similarly, the HIV Tat protein has also been shown to be able to cross the cell membrane.

[0111] In addition, the transport moiety can include a polypeptide having a region rich in basic amino acids covalently linked to an active agent moiety (e.g., a fragment inhibitor peptide containing an intracellular domain). As used herein, the term "region rich in basic amino acids" relates to a region of a protein having a high content of basic amino acids such as arginine, histidine, asparagine, glutamine, and lysine. A "region rich in basic amino acids" can have, for example, 15% or more basic amino acids. In some cases, a "region rich in basic amino acids" can have less than 15% basic amino acids but still function as a transporter region. In other examples, the basic amino acid region will have 30% or more basic amino acids.

[0112] The transport portion may further include a proline-rich region. As used herein, the term "proline-rich region" refers to a region of a polypeptide that has 5% or more (up to 100%) proline in its sequence. In some cases, the proline-rich region may have between 5% and 15% proline. Further, the proline-rich region refers to a region of a polypeptide that contains more proline than is generally observed in naturally occurring proteins. The proline-rich region of the present application can function as a transporter region.

[0113] In one embodiment, the therapeutic peptides described herein can non-covalently bind to a delivery agent. Examples of polypeptide delivery agents that non-covalently bind are the Chariot protein delivery system (see U.S. Patent No. 6,841,535, which is hereby incorporated by reference in its entirety, J Biol. Chem. 274(35):24941-24946; and Nature Biotec. 19:1173-1176).

[0114] In other embodiments, the therapeutic peptide can be expressed in cells to be treated using gene therapy, thereby inhibiting LAR family signal transduction. Gene therapy can use a vector containing a nucleotide encoding the therapeutic peptide. A "vector" (sometimes also referred to as a "vehicle" for gene delivery or gene transfer) refers to a macromolecular or molecular complex that contains a polynucleotide to be delivered to a cell. The polynucleotide to be delivered can include the coding sequence in gene therapy. Vectors include, for example, viral vectors (e.g., adenovirus (Ad), adeno-associated virus (AAV), and retrovirus), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate the delivery of polynucleotides to target cells.

[0115] Vectors can also contain other components or functional groups that further regulate gene delivery and / or gene expression, or provide beneficial properties to target cells. Such other components include components that affect binding or targeting to cells (including components that mediate cell type- or tissue-specific binding); components that affect uptake of the vector nucleic acid by cells; components that affect the intracellular localization of the polynucleotide after uptake (e.g., agents that mediate nuclear localization); and components that affect the expression of the polynucleotide. Such components can also include markers such as detectable and / or selectable markers that can be used to take up, select for, and detect cells that have expressed the nucleic acid delivered by the vector. Such components can be provided as natural features of the vector (e.g., use of specific viral vectors having components or functionality that mediate binding and uptake), or the vector can be modified to provide such functional groups.

[0116] Selectable markers can be positive, negative, or bifunctional. Positive selectable markers allow for the selection of cells carrying the marker, while negative selectable markers allow for the selective removal of cells carrying the marker. A variety of such marker genes are described, including bifunctional (i.e., positive / negative) markers (see, e.g., WO92 / 08796 to Lupton, S., published May 29, 1992; and WO94 / 28143 to Lupton, S., published December 8, 1994). Such marker genes can provide additional criteria for control that can be advantageous in the context of gene therapy. A wide variety of such vectors are known in the art and are generally available.

[0117] Vectors for use in this specification include viral vectors, lipid-based vectors, and other non-viral vectors capable of delivering nucleotides encoding the therapeutic peptides described herein to target cells. The vector can be a targeted vector, particularly a targeted vector that preferentially binds to neurons. Vectors for use in this application include those that exhibit low toxicity to target cells and induce cell-specific production of therapeutically useful amounts of therapeutic peptides.

[0118] Examples of viral vectors are those derived from adenovirus (Ad) or adeno-associated virus (AAV). Both human and non-human viral vectors can be used, and recombinant viral vectors can be replication-deficient in humans. When the vector is an adenovirus, the vector can include a polynucleotide having a promoter operably linked to a gene encoding a therapeutic peptide and is replication-deficient in humans.

[0119] Other viral vectors that can be used in this specification include vectors based on herpes simplex virus (HSV). HSV vectors lacking one or more immediate-early genes (IE) are generally non-toxic, survive in a latent-like state, and are advantageous as they provide efficient transduction of target cells. Recombinant HSV vectors can incorporate up to about 30 kb of non-homologous nucleic acid.

[0120] Retroviruses such as murine leukemia virus (MLV) and lentivirus can also be used in this application. See, for example, Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors can contain up to 5 kb of non-homologous (therapeutic) DNA in place of the viral genes. The non-homologous DNA can include a tissue-specific promoter and a nucleic acid encoding a therapeutic peptide. In methods of delivery to neurons, it can also encode a ligand for a tissue-specific receptor.

[0121] Additional retroviral vectors that can be used include replication-defective lentiviral vectors based on the human immunodeficiency virus (HIV). See, for example, Vigna and Naldini, J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157, 1998. Lentiviral vectors are advantageous in that they are capable of infecting both actively dividing cells and non-dividing cells.

[0122] The lentiviruses for use in the present application can be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include tissue-specific promoters operably linked to nucleic acids encoding therapeutic peptides as well as nucleic acid sequences required for vector propagation. The former can include viral LTRs, primer binding sites, polypurine tracts, att sites and capsid-forming sites.

[0123] In some embodiments, lentiviral vectors can be employed. Lentiviruses have been found to be capable of transducing different types of CNS neurons (Azzouz et al., (2002) J Neurosci. 22: 10302-12) and can be used in some embodiments because of their large cloning capacity.

[0124] Lentiviral vectors can be packaged in the capsid of any lentivirus. Replacing one particle protein with another particle protein from a different virus is called "pseudotyping". The vector capsid can contain a viral envelope derived from another virus, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV). The use of the VSV G protein results in high vector titers and high stability of the vector virus particles.

[0125] Alpha virus-based vectors such as those made from Semliki Forest virus (SFV) and Sindbis virus (SIN) can also be used in the present application. The use of alpha viruses is described in Lundstrom, K., Intervirology, 43:247-257, 2000 and Perri et al., Journal of Virology, 74:9802-9807, 2000.

[0126] Recombinant replication-defective alpha virus vectors are advantageous because they can express high levels of heterologous (therapeutic) genes and can infect a wide range of target cells. Alpha virus replicons can be targeted to specific cell types by displaying a functional heterologous ligand or binding domain on the virion surface that enables selective binding to target cells expressing cognate binding partners. Since alpha virus replicons can establish latency, they can establish long-term heterologous nucleic acid expression in target cells. Replicons can also exhibit transient heterologous nucleic acid expression in target cells.

[0127] In many viral vectors compatible with the methods of the present application, it is possible to introduce more than one promoter into the vector to enable more than one heterologous gene to be expressed by the vector. Additionally, the vector can include a sequence encoding a signal peptide or other moiety that facilitates expression of a therapeutic peptide from the target cell.

[0128] To combine the advantageous properties of two viral vector systems, hybrid viral vectors can be used to deliver nucleic acids encoding therapeutic peptides to target neurons, cells or tissues. Standard techniques for constructing hybrid vectors are well known to those skilled in the art. Such techniques can be found, for example, in Sambrook et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y or in numerous laboratory manuals discussing recombinant DNA techniques. Double-stranded AAV genomes in the capsids of adenoviruses containing a combination of AAV and adenovirus ITRs can be used to transduce cells. In another variation, AAV vectors can be placed into "deficient in power", "helper-dependent" or "high-capacity" adenovirus vectors. Adenovirus / AAV hybrid vectors are discussed in Lieber et al., J. Virol. 73:9314-9324, 1999. Retrovirus / adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186, 2000. The retroviral genome contained within the adenovirus can integrate into the genome of the target cell and achieve stable gene expression.

[0129] Other nucleotide sequence elements that facilitate the expression of therapeutic peptides and the cloning of vectors are further contemplated. For example, the presence of an enhancer upstream of the promoter or a terminator downstream of the coding region can, for example, enhance expression.

[0130] According to another embodiment, a tissue-specific promoter can be fused to the nucleotide encoding the therapeutic peptide described herein. By fusing such a tissue-specific promoter in an adenoviral construct, the expression of the transgene is limited to a specific tissue. Using the recombinant adenoviral system of the present application, the degree of effectiveness and specificity of gene expression provided by the tissue-specific promoter can be determined. For example, neuron-specific promoters and vectors such as the promoter of platelet-derived growth factor β-chain (PDGF-β) are well known in the art.

[0131] In addition to virus vector-based methods, nucleic acids encoding therapeutic peptides can be introduced into target cells using non-viral methods. An overview of non-viral methods of gene delivery is provided in Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. An example of a non-viral gene delivery method according to the present application employs plasmid DNA to introduce nucleic acids encoding therapeutic peptides into target cells. Gene delivery methods based on plasmids are generally known in the art.

[0132] Synthetic gene delivery molecules can be designed to form multimolecular aggregates with plasmid DNA. These aggregates can be designed to bind to target cells. Cationic amphiphilic substances containing lipopolyamines and cationic lipids can be used to provide nucleic acid delivery independent of receptors on target cells.

[0133] In addition, preformed cationic liposomes or cationic lipids can be mixed with plasmid DNA to generate a complex that can be transfected into cells. Methods involved in the formation of cationic lipids are reviewed in Felgner et al., Ann. N.Y. Acad. Sci. 772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev. 20:221-266, 1996. For gene delivery, DNA can also be conjugated to amphiphilic cationic peptides (Fominaya et al., J. Gene Med. 2:455-464, 2000).

[0134] According to the present application, methods comprising virus-based and non-virus-based components can be used. For example, Epstein-Barr virus (EBV)-based plasmids for therapeutic gene delivery are described in Cui et al., Gene Therapy, 8:1508-1513, 2001. Further, DNA / ligand / polycationic adducts conjugated to adenovirus are described in Curiel et al., Nat. Immunol. 13:141-164, 1994.

[0135] Furthermore, nucleic acids encoding a therapeutic peptide can be introduced into target cells by transfecting the target cells using the electroporation method. The electroporation method is well-known and can be used to facilitate transfection of cells with plasmid DNA.

[0136] Optionally, a vector encoding the expression of a therapeutic peptide can be delivered in vivo to target cells in the form of an injectable formulation containing a pharmaceutically acceptable carrier such as, for example, physiological saline. According to the present application, other pharmaceutical carriers, formulations and preparations can also be used.

[0137] When the target cells include neurons to be treated such as quiescent neurons or dormant neurons, the vector can be delivered by direct injection in an amount sufficient to express the therapeutic peptide to an extent that enables highly effective treatment. By injecting the vector directly into or near the periphery of the neurons, transduction of the vector can be targeted more effectively than expected, and loss of the recombinant vector can be minimized as much as possible. This type of injection enables local transduction of a desired number of cells, particularly at the site of CNS injury, thereby maximizing the therapeutic efficacy of gene transfer and minimizing the possibility of an inflammatory response to viral proteins. Other methods of administering the vector to the target cells can be used, which will depend on the particular vector employed.

[0138] The therapeutic peptide can be expressed in the target cells for a desired length of time, including transient expression and stable long-term expression. In one aspect of the present application, the nucleic acid encoding the therapeutic peptide will be expressed for a length of time defined by a therapeutically effective amount effective to induce the activity and proliferation of the transfected cells. In another aspect of the present application, the nucleic acid encoding the therapeutic peptide will be expressed for a length of time defined by a therapeutically effective amount effective to restore the lost function in the targeted neurons after CNS injury.

[0139] A therapeutically effective amount is an amount capable of producing a medically desirable result in the treated animal or human. As is well known in the medical art, the dosage for any one animal or human will depend on a number of factors including the size of the subject, body surface area, age, the particular composition being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The specific dosage of a protein and nucleic acid can be readily determined by one of ordinary skill in the art using the experimental methods described below.

[0140] The therapeutic agents described herein can be further modified (e.g., chemically modified). Such modifications can be devised to facilitate manipulation and purification of the molecule, enhance solubility of the molecule, facilitate administration, target a desired location, and increase or decrease the half-life. A number of such modifications are known in the art and can be applied by a skilled practitioner.

[0141] In some embodiments, the therapeutic agents and pharmaceutical compositions comprising the therapeutic agents described herein can be delivered to neurons of the CNS and / or PNS. Such neurons can be damaged or diseased. Alternatively, such neurons can be healthy, non-damaged neurons. Such neurons can be located at the site of injury or at sites associated with the injury. The neurons targeted for therapeutic administration, delivery / contact of the agents and compositions described herein will be those neurons for which it is believed that neuronal outgrowth would be beneficial to the subject. Such determination is within the capabilities of a skilled practitioner through minimal routine experimentation.

[0142] The therapeutic agents and pharmaceutical compositions for treatment described herein can also be delivered to non-neuronal cells of the CNS and / or PNS, such as, for example, non-neuronal cells that provide support to neurons. Such cells include, without limitation, glial cells (e.g., astrocytes, oligodendrocytes, ependymal cells, radial glia in the CNS; and Schwann cells, satellite glia, enteric glia in the PNS).

[0143] In the therapeutic methods disclosed herein, a therapeutically effective amount of a therapeutic agent is administered to a subject. In one embodiment, the formulation comprising the therapeutic agent is administered to the subject within a period of about 100 hours from the time of injury to the nervous system, e.g., within 24, 12, or 6 hours from the time of injury, after the injury has occurred.

[0144] In one embodiment, the administration is specific to one or more specific locations within the subject's nervous system. The preferred mode of administration can vary depending on the specific agent and specific target selected.

[0145] When the therapeutic agents are to be delivered to a subject, they can be administered, for example, orally (e.g., in capsules, suspensions or tablets), systemically, or by a suitable route including parenteral administration. Parenteral administration includes, for example, intramuscular, intravenous, intra-articular, intra-arterial, subarachnoid, subcutaneous or intraperitoneal administration. The agents can also be administered orally, transdermally, topically, by inhalation (e.g., intratracheally, intranasally, oral inhalation, or nasal drops), or rectally. The administration can be local or systemic, if appropriate for the indication.

[0146] Both local and systemic administrations are contemplated herein. Desirable features of local administration include achieving an effective local concentration of the therapeutic agent and avoiding adverse side effects resulting from systemic administration of the therapeutic agent. In one embodiment, the therapeutic agent can be administered by introduction into the cerebrospinal fluid of the subject. In certain embodiments, the therapeutic agent can be introduced into the ventricles, lumbar region or cisterna magna. In another embodiment, the therapeutic agent can be introduced into a nerve or at the site of spinal cord injury, at the site of pain or neurodegeneration, or intravitreally in contact with retinal cells.

[0147] A pharmaceutically acceptable formulation can be suspended in an aqueous solvent and introduced via a conventional subcutaneous injection needle or using an infusion pump.

[0148] In another embodiment, a therapeutic agent can be administered under the arachnoid membrane of a subject. As used herein, the term "subarachnoid administration" is intended to include delivering a therapeutic agent directly to the cerebrospinal fluid of a subject by a technique that includes injection into the lateral ventricle via a burr hole or a cisternal puncture or a lumbar puncture (described in Lazorthes et al., 1991, and Ommaya, 1984, the contents of which are incorporated herein by reference). The term "lumbar region" is intended to include the region between the third and fourth lumbar vertebrae (the lumbar spine). The term "cistern" is intended to include the region at the back of the skull where the skull ends and the spinal cord begins. The term "ventricle" is intended to include the cavities that continue into the central canal of the spinal cord. Administration of the therapeutic agent to any of the above-described sites can be achieved by direct injection of the therapeutic agent or by use of an infusion pump. An implantable or external pump or catheter can be used.

[0149] For injection, the therapeutic agent can be formulated in a liquid solution, usually a physiologically compatible buffer such as, for example, Hank's solution or Ringer's solution. In addition, the therapeutic agent can be formulated in solid form and redissolved or suspended immediately prior to use. The lyophilized form is also included. The injection can be, for example, in the form of a bolus injection of the therapeutic agent or in the form of a continuous drip (e.g., using an infusion pump).

[0150] In one embodiment, the therapeutic agent can be administered by intracerebroventricular injection into the brain of a subject within generally 100 hours (e.g., including within 6, 12, 24, or 100 hours from the time of injury) when an injury (resulting in a condition characterized by abnormal elongation of axons of neurons in the central nervous system) occurs. The injection can be performed through a burr hole made in the subject's skull. In another embodiment, the therapeutic agent can be administered through a shunt surgically inserted into the ventricle of the subject within generally 100 hours (e.g., including within 6, 12, or 24 hours from the time of injury) when the injury occurs. For example, even if injections into the third and fourth cerebellar ventricles can also be performed, the injection can be further performed on the larger lateral ventricle. In yet another embodiment, the therapeutic agent can be administered by injection into the cisterna magna or lumbar region of the subject within 100 hours (e.g., including within 6, 12, or 24 hours from the time of injury) when the injury occurs.

[0151] Additional means of administration to intracranial tissue include application to the olfactory epithelium with subsequent transmission to the olfactory bulb and migration to more proximal parts of the brain. Such administration is possible with a nebulized or atomized formulation.

[0152] In another embodiment, the therapeutic agent can be administered to the subject at the site of injury within generally 100 hours (e.g., including within 6, 12, or 24 hours from the time of injury) when the injury occurs.

[0153] In further embodiments, the ophthalmic compositions of the therapeutic agents described herein are used to prevent or reduce damage to retinal and optic nerve head tissues and to improve functional recovery after injury to eye tissues, as well as to prevent or reduce damage to these tissues. Ophthalmic conditions that can be treated include, but are not limited to, retinopathies (including diabetic retinopathy and retrolental fibroplasia), macular degeneration, ophthalmic ischemia, and glaucoma. Other conditions treated by the methods of the invention include injuries associated with damage to ophthalmic tissues, such as ischemic perfusion injury, photochemical injury, and injuries associated with ophthalmic surgery, particularly injuries to the retina or optic nerve head due to exposure to light or surgical instruments. The ophthalmic compositions can also be used as adjuncts to ophthalmic surgery, for example, by intravitreal injection or subconjunctival injection following ophthalmic surgery. The therapeutic agents can be used for the acute treatment of transient conditions or, particularly in the case of degenerative diseases, chronically. The ophthalmic compositions can be used prophylactically, particularly prior to ophthalmic surgery or non-invasive ophthalmic procedures or other types of surgery.

[0154] In some embodiments, the therapeutic agent can be administered to the subject for a long period of time to produce optimal axonal elongation or sprouting and / or to inhibit axonal retraction. For example, sustained contact with the active compound can be achieved by repeated administration of the active compound over a period of time, such as one week, several weeks, or one month or more. A pharmaceutically acceptable formulation used to administer the therapeutic agent can also be formulated to provide sustained delivery to the subject. For example, the formulation can deliver the active compound for at least 1, 2, 3, or 4 weeks following an initial administration to the subject. For example, a subject being treated according to the invention is treated with the active compound for at least 30 days (by either repeated administration or use of a sustained delivery system or both).

[0155] Sustained delivery of the therapeutic agent can be demonstrated, for example, by the continuous therapeutic effect of the therapeutic agent over an extended period of time (e.g., sustained delivery of the agent can be demonstrated by continuous elongation of the axons of CNS neurons in the subject). Alternatively, sustained delivery of the therapeutic agent can be demonstrated by detecting the presence of the therapeutic agent in vivo over an extended period of time.

[0156] Approaches to sustained delivery include the use of polymeric capsules, minipumps for delivering formulations, biodegradable implants or transplanted introduced gene autologous cells (see U.S. Patent No. 6,214,622). Implantable infusion pump systems (INFUSAID pumps (Twanda, Pennsylvania)); see Zierski et al., 1988; Kanoff, 1994) and osmotic pumps (sold by Alza Corporation) are commercially available and are known in the art. Another mode of administration is via an implantable and externally programmable infusion pump. Infusion pump systems and reservoir systems are also described in U.S. Patent Nos. 5,368,562 and 4,731,058.

[0157] Vectors encoding therapeutic peptides are not as frequently administered as other types of therapeutic agents. For example, the effective amount of such vectors ranges from about 0.01 mg / kg to about 5 or 10 g / kg and is administered daily, weekly, biweekly, monthly or less frequently.

[0158] The ability to deliver or express a therapeutic peptide enables the regulation of cell activity in a number of different cell types. The therapeutic peptide can be expressed, for example, in heart cells via a heart-specific promoter that regulates heart contraction (or excitability), in the spinal cord via the HB9 promoter that regulates the activity of motor neurons after spinal cord injury, and, for example, in neurons in brain regions affected by a degenerative disease such as Parkinson's disease to control excitability in selected neuronal brain regions.

[0159] In some embodiments, neurons derived from the central or peripheral nervous system can be contacted with a therapeutic agent ex vivo to promote axonal outgrowth in vitro. Thus, neurons can be isolated from a subject, grown in vitro using techniques well known in the art, and then treated to regulate axonal outgrowth. Briefly, a neuronal culture can be obtained by allowing neurons to migrate from fragments of neural tissue attached to a suitable substrate (e.g., a culture dish), or by mechanically or enzymatically dissociating the tissue to yield a suspension of neurons. For example, the enzymes trypsin, collagenase, elastase, hyaluronidase, deoxyribonuclease, pronase, or various combinations thereof can be used. Methods for isolating neuronal tissue and dissociating the tissue to obtain isolated cells are described in Freshney, CULTURE OF ANIMAL CELLS, A MANUAL OF BASIC TECHNIQUE, (3rd ed., 1994). Such cells can then be contacted with a therapeutic agent in the amounts and for the times as described above. Once regulation of axonal outgrowth is achieved in the neurons, these cells can be re-administered to a subject, for example, by transplantation.

[0160] The ability of an agent to promote nerve regeneration in a subject can be evaluated using any of a variety of known procedures and assays. For example, after injury, the ability of an agent to re-establish nerve connectivity and / or function can be determined histologically (by making sections of neuronal tissue and looking at neuronal branching or by demonstrating cytoplasmic transport of a dye). An agent can also be evaluated by monitoring its ability to fully or partially restore electroretinograms after injury to the retina or optic nerve, or to fully or partially restore the pupillary light reflex in an injured eye.

[0161] Other tests that can be used include standard tests of nerve function in human subjects or animal models of spinal cord injury (e.g., standard reflex tests, urological tests, uroflowmetry tests, deep and superficial pain sensation tests, proprioceptive placing of the hindlimbs, walking tests, and evoked potential tests). In addition, nerve impulse conduction can be measured in a subject, for example, by measuring the conduction action potential as an indicator of the production of a nerve beneficial effect.

[0162] Animal models that can be used herein include a partial transection rat model that examines how well a compound can enhance the survival and sprouting of the remaining intact fragments of the almost completely transected spinal cord. Thus, after administration of a candidate agent, these animals can be evaluated for the recovery of specific functions, for example, how well a rat can manipulate a food pellet with its forelimb (the relevant spinal cord is transected by 97%).

[0163] Another animal model that can be used in the assay includes a rat model of stroke. Using the administration of an agent to these animals, it can be evaluated whether a given compound, administration route, or dose provides a nerve regeneration effect such as, for example, an increase in the level of function, or an increase in the rate of function reacquisition, or an increase in the degree of function retention in the test animal.

[0164] The ability of an agent to produce a nerve beneficial effect in a subject can be evaluated using standard neurological evaluations used to assess the progression in human patients after stroke. Such standard neurological evaluations are routine in medical technology and are described, for example, in "Guide to Clinical Neurobiology" Edited by Mohr and Gautier (Churchill Livingstone Inc. 1995).

[0165] In some embodiments, a therapeutic agent can be used to treat diseases, disorders or conditions associated with elements of the nervous system, including central components, somatic components, autonomic components, sympathetic and parasympathetic components, neural sensory tissue in the eye, ear, nose, mouth or other organs, as well as glial tissue associated with neurons and their cellular and structural components. Neuropathy can be caused, for example, by damage to neurons such as mechanical damage or damage by toxic compounds, by abnormal growth or development of neurons, or by dysregulation such as, for example, downregulation of neuronal activity. In one embodiment, the therapeutic agent can be applied to damaged nerves, the site of nerve injury or the site of nerve injury repair. In some embodiments, the therapeutic agent is applied to the site of primary nerve repair. Damage to a nerve can be equivalent to nerve severance (neurotmesis), where the nerve is partially or completely severed, or a small area is damaged and surgically removed.

[0166] Neuropathy can detrimentally affect the functions of the nervous system, such as, for example, sensory function (the ability to sense changes inside and outside the body); integrative function (the ability to interpret changes) and motor function (the ability to respond to interpretation by initiating actions such as muscle contraction or secretion of granules).

[0167] Examples of neuropathies include peripheral or cranial nerves, spinal cord or brain, traumatic or toxic injury to cranial nerves, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury. Other neuropathies include, for example, Alzheimer's disease, dementia associated with Alzheimer's disease (such as Pick's disease), Parkinson's disease, and other diffuse Lewy body diseases, senile dementia, Huntington's disease, Gilles de la Tourette syndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy (Charcot - Marie - Tooth disease), diabetic neuropathy, progressive supranuclear palsy, epilepsy and Creutzfeldt - Jakob disease. Autonomic dysfunction includes hypertension and sleep disorders.

[0168] Also treated by the therapeutic agents described herein are, for example, neurological and psychiatric disorders such as depression, schizophrenia, schizoaffective disorder, Korsakoff's psychosis, mania, anxiety or phobic disorders, learning or memory disorders (e.g., memory loss and age-related memory loss), attention deficit hyperactivity disorder, mood swings disorder, major depressive disorder, mania, obsessive-compulsive disorder, neuroactive substance use disorder, anxiety disorder, pathological phobia, panic disorder, bipolar mood disorder, psychogenic pain syndrome, and eating disorders. Other examples of neurological disorders include damage to the nervous system by infectious diseases (e.g., meningitis, fever of various etiologies, HIV, syphilis, or post-polio syndrome), and damage to the nervous system by electricity (including contact with electricity or lighting, complications of electro-conductive psychiatric therapies). Neurological disorders associated with ophthalmic symptoms include damage to the retina and optic nerve, glaucoma, and age-related macular degeneration.

[0169] The developing brain is a target for neurotoxicity in the development of the central nervous system at many stages of infancy and childhood, as well as during pregnancy, and the methods of the invention can be utilized to prevent or treat neurological deficits in embryos or fetuses in utero, premature infants, or young children who require such treatment, including those with congenital neurological deficits. Further neurological disorders include, for example, those listed in HARRISON’S PRINCIPLES OF INTERNAL MEDICINE (Braunwald et al., McGraw-Hill, 2001) and in the AMERICAN PSYCHIATRIC ASSOCIATION’S DIAGNOSTIC AND STATISTICAL MANUAL OF MENTAL DISORDERS DSM-IV (American Psychiatric Press, 2000).

[0170] The therapeutic agents described in this specification can be used in a method for treating medical conditions associated with nerve damage. The medical conditions can refer to any movement disorder, epilepsy, cerebrovascular disease, autoimmune disease, sleep disorder, autonomic neuropathy, bladder disorder, abnormal metabolic state, muscle disorder, infectious and parasitic diseases and tumors, endocrine disorder, nutritional and metabolic diseases, immune diseases, diseases of the blood and blood-forming organs, mental disorders, nervous system diseases, sensory organ diseases, cardiovascular system diseases, respiratory system diseases, digestive system diseases, urogenital system diseases, skin and subcutaneous tissue diseases, musculoskeletal and connective tissue diseases, congenital anomalies, certain perinatal-originated conditions, and symptoms, signs, and undefined conditions.

[0171] Treatable cerebrovascular disorders can be caused by conditions including, but not limited to, aneurysms, strokes, arrhythmias, myocardial infarctions, ischemia-reperfusion injuries, and cerebral hemorrhages.

[0172] Treatable autoimmune diseases include, but are not limited to, multiple sclerosis.

[0173] Sleep disorders treatable by the present application can be caused by conditions including, but not limited to, sleep apnea and parasomnias.

[0174] Autonomic neuropathies treatable by the present application can be caused by conditions including, but not limited to, gastrointestinal motility disorders, vomiting, nausea, diarrhea, chronic hiccups, gastroesophageal reflux disease, and excessive gastric acid secretion, gastrointestinal disorders, autonomic insufficiency, excessive epiphoresis, excessive rhinorrhea, and cardiovascular diseases including, but not limited to, cardiac arrhythmias and irregularities, hypertension, and carotid sinus disease.

[0175] Bladder disorders treatable by the present application can be caused by conditions including, but not limited to, spinal cord injury and spastic or flaccid bladder.

[0176] The abnormal metabolic conditions treatable by the present application may be caused by conditions including, but not limited to, hyperthyroidism or hypothyroidism. The muscle disorders treatable by the present application can include, but are not limited to, muscular dystrophy and spasms of the upper airway and face.

[0177] Using a therapeutic agent to treat neuropathic pain caused by conditions including, but not limited to, migraine with aura, migraine without aura, menstrual migraine, transformed migraine, atypical migraine, complicated migraine, hemiplegic migraine, conversion migraine, chronic daily headache, episodic tension-type headache, analgesic rebound headache, episodic cluster headache, chronic cluster headache, cluster transformation, chronic paroxysmal hemicrania, persistent unilateral headache, post-traumatic headache, post-traumatic neck pain, post-herpetic neuralgia including the head and face, pain resulting from spinal fractures associated with osteoporosis, pain of arthritis in the spinal cord, pain associated with cerebrovascular disorders and stroke, headache due to vascular disorders, reflex sympathetic dystrophy, anterior neck pain (obtainable from various causes including, but not limited to, muscular, intervertebral disc-originated or degenerative causes including arthritis, posture-related, metastasis), glossalgia, carotidynia, chondrotomodynia, otalgia due to middle ear lesions, stomachache, sciatica, maxillary neuralgia, laryngeal headache, myalgia of the neck muscles, trigeminal neuralgia (sometimes also called tic douloureux), headache after lumbar puncture, headache due to low cerebrospinal fluid pressure, temporomandibular joint disorder, atypical facial pain, ciliary neuralgia, paratrigeminal neuralgia (sometimes also called Raeder's syndrome), pyramidal neuralgia, Eagle syndrome, idiopathic intracranial hypertension, orofacial pain, myofascial pain syndrome including the head, neck and shoulders, cluster headache, neck headache, paratrigeminal paralysis, SPG neuralgia (sometimes also called lower half migraine, lower facial pain syndrome, Sluder neuralgia, and Sluder's syndrome), carotidynia, vidian neuralgia, burning pain and / or combinations of the above.

[0178] As used herein, the term "headache" can refer to migraine, tension headache, cluster headache, trigeminal neuralgia, secondary headache, tension-type headache, chronic and transient headache, drug-induced headache / rebound headache, chronic paroxysmal hemicrania, persistent one-sided headache, post-traumatic headache, post-herpetic headache, vascular headache, headache associated with reflex sympathetic dystrophy, cervicogenic headache, internal carotid artery aneurysm headache, sciatic neuralgia headache, trigeminal neuralgia headache, occipital headache, maxillary headache, hypnic headache, paratrigeminal headache, pyramidal headache, shoulder headache, vidian headache, low CSF pressure headache, TMJ headache, burning pain headache, pleural headache, all primary headaches (e.g., primary stabbing headache, primary cough headache, primary exertional headache, primary headache associated with sexual activity, headache during sleep, and new daily persistent headache), trigeminal-autonomic headache (e.g., short-lasting unilateral neuralgiform headache attacks, SUNCT, all suspected TACs and SUNA), chronic daily headache, occipital neuralgia, atypical facial pain, neuropathic trigeminal neuralgia, and other types of headache.

[0179] In still other embodiments, a therapeutic agent can be used to promote the viability, plasticity and / or proliferation of stem cells or progenitor cells. The stem cells can include stem cells expressing a LAR phosphatase receptor including neural stem cells or progenitor cells. The therapeutic agent can be administered to the stem cells or progenitor cells in vitro, ex vivo or in vivo. When administered to the stem cells or progenitor cells in vitro or ex vivo, the stem cells or progenitor cells can be transplanted into a subject for therapeutic application.

[0180] For neural stem cells / progenitor cells, a method of transplanting neural stem cells / progenitor cells into a desired region commonly used in the field of regenerative medicine can be employed in combination with the administration of a therapeutic agent to the cells or region. More specifically, for example, a method of transplanting neural stem cells / progenitor cells into the region can be exemplified by suspending neural stem cells / progenitor cells in phosphate buffered saline together with a therapeutic agent and adding / injecting the resulting cell suspension into the region.

[0181] In other embodiments, the therapeutic agents described herein can be applied to nerve grafts. Grafts can include tissues intended for implantation into humans or animals. For example, various types of grafts such as autografts, isografts, allografts, and xenografts are contemplated within the scope of the subject invention. The size of the graft (e.g., length and diameter) is not critical. For example, the length of a nerve graft can be from about 1 centimeter to about 10 centimeters or more than about 10 centimeters. The diameter of the nerve graft can be tailored to the diameter of the damaged nerve or portion of nerve as needed. The nerve graft can be a structurally complete nerve fragment that bridges a gap along the length of the recipient nerve or to replace a distal end, i.e., for end-to-end transplantation. Alternatively, the nerve graft can be a partial nerve fragment or an eccentrically shaped fragment (e.g., a nerve flap) that is intended to reconstruct a lacerated nerve that has some structural disruption but retains its physical continuity.

[0182] When a therapeutic agent is applied to a nerve graft, the entire graft is treated. The therapeutic agent can be applied to the entire nerve graft in a batch. Batch treatment can be applied to fresh or pre-frozen nerve grafts. The therapeutic agent can also be applied to the nerve graft before, during, or after transplantation. For example, the therapeutic agent can be applied to any part of the graft such as one or both ends that are joined to the base of the damaged nerve. If applying the therapeutic agent to a damaged nerve, for example, at or adjacent to the site of injury, the therapeutic agent can be applied to the area of the damaged nerve that promotes repair of the damaged nerve.

[0183] The therapeutic agent can be placed in a culture medium for application to the nerve graft. The culture medium can be an undefined medium, a defined medium, or a defined medium supplemented with, for example, serum. The embodiments described herein also include a preservation solution for preserving the nerve graft prior to transplantation. The preservation solution contains a culture medium and at least one therapeutic agent. The preservation solution can also contain other biologically active agents such as the growth factors described below.

[0184] In other embodiments, spinal cord injuries such as a unilateral transection of C2 elicit an increase in inhibitory proteoglycans at the level of the phrenic motor nucleus in the extracellular matrix and perineuronal net on the same side as, but distal to, the spinal cord lesion. As discussed in U.S. Patent Application 10 / 754,102, which is incorporated herein by reference, treatment with chondroitinase ABC (chABC) degrades these matrix molecules that are strongly inhibitory.

[0185] It is contemplated herein that enzymatically modifying (via chondroitinase (chABC)) the inhibitory extracellular matrix in motor neurons surrounding PNN in combination with administration of a therapeutic agent can maximize the sprouting ability and functional impact of the remaining nerve fibers. It is further contemplated that enhancing and / or effecting much greater total fiber sprouting, in combination with increasing the physiological output of the neurons themselves, acts synergistically to improve spinal cord injury. Accordingly, in another embodiment, a subject can be administered chondroitinase ABC in addition to the therapeutic agents described herein to provide a more enhanced recovery than each of the treatments used alone. In some embodiments, a bolus injection of ChABC near the CNS lesion can improve motor function in the subject.

[0186] The methods described herein can further include administering or contacting the cells with an agent that blocks a regeneration inhibitor, such as a compound that inhibits the blockade derived from myelin of neurogenesis. Known inhibitors of neuron outgrowth (e.g., of regeneration at the site of CNS injury) are myelin-derived inhibitors (e.g., Nogo-A, MAG, OMgp, EphrinB3, Sema4D, and Sema5A), astrocyte-derived inhibitors (e.g., CSPG, KSPG, EphrinB2, and Slit), and fibroblast-derived inhibitors (e.g., Sema3A). The second agent can be an antagonist to any of these inhibitors. In one embodiment, the cells are further contacted with one or more such agents. In one embodiment, the agent inhibits myelin inhibitors of nerve regeneration (e.g., myelin-associated glycoprotein (MAG), Nogo, oligodendrocyte myelin glycoprotein (OMgp)). Inhibitors of MAG are disclosed in U.S. Patent No. 5,932,542. Inhibitors of Nogo are disclosed in U.S. Patent Application Publication No. 2009 / 215691. Inhibitors of OMgp are disclosed in U.S. Patent Application Publication No. 2008 / 188411. The cells can be contacted with the agent that inhibits the interaction of CSPG with PTPσ before, after, and / or simultaneously with this agent.

[0187] In some embodiments, cells can also be contacted with an agent that activates the proliferation pathway of neurons (e.g., the CNS). Some agents include, but are not limited to, neurotrophic factors such as inosine, mannose, glucose, or glucose-6-phosphate as described by Li et al., 23, J. Neurosci. 7830 (2003); Chen et al., 99, PNAS, 1931 (2002); and Benowitz et al., 273, J. Biol. Chem. 29626 (1998). Oncomodulin as described by TGFβ and Yin et al., 23, J. Neurosci. 2284 (2003) is also an agent. In addition, polypeptide growth factors such as BDNF, NGF, NT-3, CNTF, LIF, and GDNF can also be used. In one embodiment, a method comprising an agent that stimulates neuron extension further comprises contacting a neuron (e.g., the CNS) with a cAMP regulator that increases the concentration of intracellular cAMP (e.g., cAMP) and / or a polyamine (Cai et al., 35 Neuron 711 (2002)). For example, the ability of ganglion cells of mature rats to respond to mannose requires an increase in cAMP (Li et. al., 2003).

[0188] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the claims. Example 1

[0189] This example shows that long-term exposure to CSPG causes growth cones to collapse, stabilize, and adhere excessively. In this example, the spot assay used to measure CSPG induced the stabilization of mature sensory neurons. Method

[0190] Mature female rat dorsal root ganglion (DRG) neurons were obtained and grown on a gradient of the chondroitin sulfate proteoglycan aggrecan as previously described. Delta T imaging dishes with glass bottoms were coated with poly-L-lysine (PLL) and incubated overnight at room temperature. Spots were created by dissolving 2 mg / ml of aggrecan in calcium- and magnesium-free Hank's balanced salt solution (HBSS). Eight 2-μl dots were placed on half of each dish and allowed sufficient time to dry on the glass. Finally, the dishes were coated with 10 μg / ml of laminin for 3 hours at 37°C. After incubation, 6000 mature dissociated DRG neurons were added to each dish in Neurobasal A medium supplemented with Glutamax, Penn / strep, and B27. The cells were grown for 4 - 6 days.

[0191] Immediately prior to the start of imaging, the Delta T dish was sealed with a glass coverslip. 100x microscopy imaging was performed while warming the object and sample stage to keep the cells at 37°C. Images were obtained every 30 seconds and compiled together to create an imaging movie. The dynamics of growth cones and filopodia were tracked and manually quantified using Metamorph. Result

[0192] Figures 1 and 2 (A - D) show that mature sensory neurons exposed to the CSPG aggrecan gradient stabilize in specific regions at the edge of the spots and excessive adhesion to the substrate results in the formation of growth cones, elongation of filopodia, and lack of motility. Of the 24 growth cones examined, 22 (92 percent) became immobile and lost motility within 4 - 6 days in vitro. Example 2

[0193] This example shows that the expression of LAR is higher in growth cones that are more stable than motile cones. In this example, the technique used in microscope cinematography was slightly changed to perform a spot assay on a glass coverslip. After PLL treatment, the coverslip was dried and coated with a small amount of nitrocellulose to enhance the adhesion interactions required for spot formation. After drying the nitrocellulose, four spots were made on each coverslip (one per quadruplicate) using 700 μg / ml of aggrecan and 5 μg / ml of laminin dissolved in HBSS. After drying, the coverslips were coated with 5 μg / ml of laminin for 3 hours at 37°C. 2,000 dissociated postnatal dorsal root ganglion neurons were added to each coverslip in Neurobasal A medium supplemented with Glutamax, Penn / strep, and B27. In addition, the peptide was added at the concentration required at the time of plating on the plate. After growing the cells for 5 days, they were fixed with 4% paraformaldehyde.

[0194] The slides were fixed and stained with goat anti-PTPσ (1:100, R&D Systems) and mouse anti-βIII tubulin (1:500, Invitrogen). Axons and growth cones were imaged at 100× magnification. The density of PTPσ in the growth cone and axon compartments was analyzed using ImageJ (N = 40) for each trophic and non-trophic neuron.

[0195] Figure 3 shows that the density of PTPσ was significantly enriched in trophic and stabilized growth cones compared to motile growth cones on laminin. Example 3

[0196] As schematically illustrated in FIG. 4, the leukocyte common antigen-related (LAR) family of phosphatases consists of three members: LAR itself, receptor protein tyrosine phosphatase sigma (RPTPσ), and receptor protein tyrosine phosphatase delta (RPTPδ). Recent studies have shown a binding interaction between the sugar side chains of chondroitin sulfate proteoglycan (CSPG), a molecule that is highly inhibitory to nerve growth, plasticity, and regeneration, and LAR or RPTPσ. Furthermore, crystallography and sequence analysis have shown that all three family members contain exactly the same binding domain and binding pocket, providing evidence that RPTPδ may also be a functional receptor for CSPG. Structural and sequence analyses have revealed that all members of the LAR family contain a wedge-shaped helix / loop / helix motif in the first intracellular catalytic domain that mediates homophilic / heterophilic receptor interactions. Using a peptide mimetic of this wedge-shaped domain tagged with the TAT sequence localized in the cytoplasm, LAR activity was successfully abolished in the paradigm of neurotrophic factor signaling. We used NIH's BLAST to identify orthologous sequences of RPTPσ and RPTPδ and designed peptides of the wedge-shaped domain for each target. The peptides were a newly created intracellular LAR blocking peptide (ILP), an intracellular sigma blocking peptide (ISP), and an intracellular delta blocking peptide (IDP). Interestingly, the fact that this domain is highly conserved in higher vertebrates indicates that it is a functionally important region.

[0197] Wedge shape of rat and mouse PTPσ: DMAEHMERLKANDSLKLSQEYESI (SEQ ID NOs: 20 and 21)

[0198] Wedge shape of human PTPσ: DMAETMERLKANDSLKLSQEYESI (SEQ ID NO: 33)

[0199] The peptide is conjugated as a tag HIV-TAT to create a functional blocking peptide. HIV-TAT

[0200] NH2 GRKKRRQRRRC DMAEHMERLKANDSLKLSQEYESI-NH2 PTP sigma mouse / rat (SEQ ID NO: 53)

[0201] NH2 GRKKRRQRRRC DMAETMERLKANDSLKLSQEYESI-NH2 PTP sigma human (SEQ ID NO: 54)

[0202] NH2 GRKKRRQRRRC DLADNIERLKANDGLKFSQEYESI-NH S LAR (SEQ ID NO: 55)

[0203] NH2 GRKKRRQRRRCE LADHIERLKANDNLKFSQEYESI-NH2 PTP delta (SEQ ID NO: 56)

[0204] NH2 GRKKRRQRRRC IREDDSLMLYALAQEKKESNMHES-NH2 mixed sigma (SEQ ID NO: 57)

[0205] These peptides were ordered from Genscript, dissolved in water, and stored long-term at -80°C. The peptides were added to the medium when plating neurons.

[0206] After 5 days in vitro, the cells were fixed and stained with mouse anti-B3 tubulin (green). The number of processes that completely bridged the gradient was counted and normalized to the number of neuronal cell bodies at each individual spot. Result

[0207] Figures 5-7 show that peptides of the wedge domain of PTPσ (ISP) enable neurons to extend processes that cross the CSPG gradient. The process was dose-dependent, and optimal crossing was 2.5 μm. In addition, the LAR peptide (ILP) also enabled neurons to cross the CSPG (Figure 6). Neither the solvent control nor scrambled ISP / TAT allowed extension of neurons across the gradient. Time-lapse imaging showed that treatment with ISP prevented axonal destabilization and excessive adhesion, allowing the growth cone to extend filopodia and maintain motility. 65% of the growth cones analyzed remained motile in vitro at 4-6 days, in contrast to 8% under control conditions. Example 4

[0208] This example describes a method for generating spinal cord injury (SCI) in adult female Sprague-Dawley rats using an Infinite Horizon device to produce a moderate / severe impact for use in the SCI assays of Examples 5-8. Briefly, rats were deeply anesthetized with a cocktail of ketamine / xylazine. Immediately under anesthesia, the back was shaved and disinfected with iodine and ethanol. Entering from the back, the 7th to 10th lumbar vertebrae were exposed by skin incision, and laminectomy was performed at the 8th and 9th segments to expose the intact spinal cord. Prior to the impact shock, the spinal column and spinal cord were fixed in a stereotaxic frame. Finally, the rats received a 250 kd Infinite Horizon impact centered without dwell time. After suturing the muscle and closing the skin, the animals were placed on a 37°C hot pad and given free access to food and water upon awakening from surgery. Pain was monitored, and painful animals were given a low dose of Marcaine at the injection site. Physiological saline and gentamicin (antibiotic) were given for 5 days postoperatively to prevent bladder infection. This experiment was performed 3 times from start to finish. N = 15, ISP; N = 11, solvent; N = 6, ILP

[0209] Animals were randomly divided into three groups: solvent control, ISP, or ILP. Lyophilized ISP or ILP was first dissolved in sterile water at a concentration of 2.5 mM. To perform individual treatments for each animal, the peptide was further diluted to a concentration of 5 μM in a solvent solution of 5% DMSO in sterile saline. 25 ml of each treatment (ILP, ISP, or solvent) was dispensed into 50 individual Eppendorf tubes, each containing 500 μl. The drugs were stored at -20°C and thawed immediately before use. Starting on the first day after injury and continuing for 7 weeks, each animal received daily subcutaneous administration of either the solvent, 5 μM ISP, or 5 μM ILP on the back over the lesion (49 treatments, 11 μg / rat / day). Example 5

[0210] This example shows the hindlimb movement and spontaneous movement pattern according to the Basso-Beattie-Bresnahan scale for spontaneous movement after SCI in solvent-treated SCI animals and LAR peptide-treated SCI animals. Method

[0211] While the animals were allowed to freely wander on the table for 3 minutes, the movement of their hindlimbs and the spontaneous movement pattern were scored by a blinded observer according to the Basso-Beattie-Bresnahan scale (Basso et al, 1995) for spontaneous movement after SCI. Behavioral tests were performed on days 1, 4, 7 after injury, and then weekly for an additional 10 weeks. Statistical analysis was performed by two-way analysis of variance for repeated measures.

[0212] Figures 8 and 9 show that following the initial period of spinal cord impact, all treatment groups had recovered basic hindlimb movement by 2 weeks post-injury, with an average BBB score of 9 (supporting weight with the hindlimbs without stepping). Over the next 10-week period, both the vehicle-treated animals and the LAR peptide-treated animals recovered only slightly beyond this point, on average regaining the ability to walk with occasional weight-bearing. On average, the ISP-treated animals continued to recover, reaching a score of 12 at 6 weeks (occasional consistent stepping with hindlimb and forelimb coordination) and exceeding 13 by 7 weeks (between frequent and consistent coordination). Individually, animals reached a score of 19, which was nearly complete spontaneous movement, with a highly raised tail, consistent toe clearance during stepping, and correct foot placement. Additional animals reached nearly normal scores of 18.5 and 18. Five out of seven animals regained at least frequent coordinated stepping. Example 6

[0213] This example shows the results of the grid walking test in vehicle-treated SCI animals and LAR peptide-treated SCI animals. Method

[0214] Twelve weeks after spinal cord injury, animals were allowed to freely roam on a wire grid (100 cm × 75 cm, 1 cm gaps between wires). A camera overhead tracked and calculated the total distance moved (Ethovision), while the number of slips was manually counted by blinded observers. Data are presented as the total number of left and right slips per meter moved. The grid walking test was performed only once to prevent animals from training and artificially improving (the rehabilitation phenomenon). 2 Result

[0215] ​The sensorimotor coordination and balance recovery were measured using the grid walking test. Figures 10 and 11 show that the solvent-treated animals made an average of 6 missteps per meter traveled on the grid. The ILP treatment led to a very slight and non-significant improvement in missteps in the grid walking test. The ISP treatment made significantly fewer missteps on average than the solvent-treated and ILP-treated animals. In addition, the fact that several animals made less than 3 missteps suggests a nearly complete recovery of this behavior. Example 7

[0216] This example shows the recovery of micturition function in solvent-treated SCI animals and ISP peptide-treated SCI animals. Method (metabolic cage)

[0217] The animals were placed in metabolic cages overnight in the dark cycle (16 hours). Urine was separated and collected into a syringe connected to a force transducer. The increase in force corresponding to each individual micturition was plotted on Spike 2. The graph was extracted into Excel and manually checked to measure the total number of micturitions and the average volume of each micturition. Urodynamics

[0218] In the final experiment, the animals were anesthetized with urethane at 14 weeks post-injury. This anesthesia prevents excessive movement while protecting the bladder reflex. A catheter was inserted into the bladder via the urethra to allow slow perfusion with physiological saline. In addition, two electrodes were inserted into the external urethral sphincter via the vagina to measure muscle activity. Muscle activity and muscle pressure (measured via the catheter) were plotted on Spike 2. Result

[0219] Recovery of urination behavior was measured using a metabolic cage. During the dark cycle at 4, 8, and 12 weeks post-injury, animals were placed in a metabolic cage where urination was measured by a force transducer. Figure 12 shows that although no significant recovery was seen on average at 4 or 8 weeks, ISP led to a significant increase in the number of urinations at 12 weeks post-injury. Untreated animals urinated an average of 2 times per hour, while solvent-treated and ILP-treated animals had a significantly decreased number, once every 2 hours, with a significantly increased volume / urination. ISP treatment significantly increased the number of urinations post-injury by up to 2-fold on average, and multiple animals reached the normal (untreated) urination number level.

[0220] To examine whether the animals had complete control over the contractility of the bladder muscle and sphincter, at 14 weeks post-injury, the animals underwent terminal urodynamic analysis. Under urodynamics and slow perfusion of saline into the bladder, untreated animals contracted their bladder muscle, resulting in a sharp increase in pressure in the bladder (upper trace). The decrease in pressure corresponded to the relaxation of the external urethral sphincter, assisting the animals in urination. Both of these behaviors were completely lost after spinal cord injury, at which time a gentle increase in pressure in the bladder resulted in the maximum value finally reached and the saline leaked out. The external urethral sphincter relaxed occasionally but was not correlated with the contraction of the bladder detrusor muscle that caused inappropriate urination. Figures 13(A - B) show that after ISP treatment, many animals recovered coordinated contraction of the bladder in harmony with patterned relaxation of the external urethral sphincter (marked by red arrows).

[0221] Recovery beyond that of solvent-treated animals was defined as being better than 2 times the standard deviation above the solvent mean values (urination and BBB). Animals were placed in each group. 13 out of 15 animals showed significant improvement in behavior, and 4 were within the significant functional range for all three behaviors. Example 8

[0222] This example shows the expression of 5HT in solvent-treated SCI animals and ISP peptide-treated SCI animals. Method

[0223] Animals were perfused transcardially with 4% paraformaldehyde and the spinal column was excised. One day after additional exposure to PFA, the spinal column was removed and cryoprotected with 30% sucrose for 3 - 7 days. The corresponding segments of L1 - L3 were embedded and 20 - μm transverse sections were mounted on slides.

[0224] Slides were blocked with 5% goat serum and probed with a primary antibody against 5HT (1:500, Immunostar). Following washing and incubation with the appropriate secondary antibody, the slides were covered with coverslips and sealed. Images were taken with a fluorescence microscope at 2× magnification under the same exposure, gain, gamma, and offset, and comparisons between slides were made.

[0225] For axonal density analysis, the contours of the gray and white matter were traced and the mean pixel intensity was calculated using ImageJ. Since minimal staining was observed in the dorsal columns of the spinal cord, the pixel intensity in this region was subtracted as the internal background for each individual section. Once sections were randomly selected, analysis was performed every 200 μm over a total distance of 2 cm (10 segments). The remaining 8 out of the highest and lowest pixel intensities were averaged. Result

[0226] 5HT is an important neurotransmitter in the spinal cord whose role is to control the acquisition and excitability of the motor network. When combined with other treatments, 5HT agonists can significantly enhance motor behavior after spinal cord injury. We stained for 5HT expression at the lumbar spinal cord and multiple segments below the level of the injury. The lumbar spinal cord contains the pacemaker for hindlimb spontaneous movement and bladder control and contains motor neurons for the muscles of the foot and bladder. Figure 14 shows that in untreated animals, 5HT expression or axonal density was very high with a uniform staining pattern in the left and right gray and white matter. At high magnification, fibers penetrating the white matter can be seen. Fourteen weeks after spinal cord injury, 5HT expression was significantly lower in vehicle control animals, and only 2 or 3 small patches remained in the gray and white matter. ISP treatment resulted in a dramatic increase in 5HT staining throughout the gray and white matter. The staining was remarkably robust and varied greatly from section to section in discrete, non-uniform patches throughout the gray and white matter. This pattern suggests sprouting and / or regeneration of 5HT to spared motor output centers. Since 2 ISP non-responders did not show a dramatic increase in 5HT expression, the increase in 5HT expression correlated well with behavioral recovery. Quantification again showed a significant increase in 5HT expression throughout the gray and white matter (Figure 15). Example 9

[0227] We identified several proteins and pathways that act downstream of the LAR family outside of phosphate activity (Figure 4). Among these, Caskin (Ckn) and LAR interacting protein α (Replin-α) have important roles in both synapse formation and axon guidance. In a yeast two-hybrid interaction system, mCkn1 directly binds mLAR and mPTPRδ, and mCkn2 directly binds mLAR and mPTPσ. The interaction of Ckn with phosphatases of the LAR family was mapped to a region containing two sterile α motif (SAM) domains that constitute the phenotype. We created a homology map for Ckn in Drosophila, mouse, rat, and human using BLAST to align the acceptor sequences of the proteins shown in Table 3.

[0228] Table 3 shows that the first SAM domain is conserved among Ckn family members. We designed a 20 - amino - acid peptide, hypothesizing that we would act as a small - molecule competitor for LAR family phosphatase / Ckn binding and move downstream of LAR family signaling. Working in the Drosophila system, the C - terminal region of dCkn was identified as necessary for downstream signaling events. We designed 20 - amino - acid peptides for both mCkn1 and mCkn2, which are their targets but may act as small - molecule competitors for downstream of the target.

Table 3

[0229] Repelin family members play important roles in synapse development and maintenance (we hypothesize that Repelin - α may act in signaling downstream of the LAR phosphatase). Yeast two - hybrid interaction screening relates the first SAM domain of Repelin - α family members as a binding region for LAR phosphatase family members. We designed a 20 - amino - acid peptide corresponding to the same region within the first SAM domain of all four members of the Repelin - α family (Table 4). It is interesting that the four orthologs of Repelin - α have retained this same region through evolution, suggesting this region as functionally important. We hypothesize that we can disrupt the LAR / Repelin - α1 - 4 interaction using this small - molecule competitor and disrupt signaling downstream of LAR phosphatase family members.

Table 4

[0230] The present invention has been particularly shown and described with reference to its preferred embodiments, but it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims. All patents, publications, and references cited in the foregoing specification are hereby incorporated by reference in their entirety.

[0231] This application also encompasses the following inventions. (1) A method of inhibiting and / or reducing the activity, signal transduction, and / or function of a phosphatase of the leukocyte common antigen-related (LAR) family induced by proteoglycan in a target cell, comprising administering to the cell a therapeutic agent that inhibits one or more of the catalytic activity, signal transduction, and function of the LAR family phosphatase without inhibiting the binding of the proteoglycan to the LAR family phosphatase or its activation. (2) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 65% homologous to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. (3) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 75% homologous to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. (4) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 85% homologous to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. (5) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 95% identical to about 10 to about 20 consecutive amino acids of the wedge domain of PTPσ. (6) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 33. (7) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide consisting of SEQ ID NO: 37. (8) The method of (1), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide that is at least about 65% identical to SEQ ID NO: 37. (9) The method of (8), wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid at residue 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37. (10) The method according to any one of (2) to (8), wherein the cell is a neuron, a glial cell, a glial progenitor cell, or a neural progenitor cell. (11) The method according to any one of (2) to (8), wherein the therapeutic agent comprises a transport moiety linked to the therapeutic peptide and facilitating the uptake of the therapeutic peptide by the cell. (12) The method of (11), wherein the transport moiety is the Tat transport moiety of HIV. (13) The method of (11), wherein the cell is in a subject to be treated, and the therapeutic agent is administered systemically to the subject to be treated. (14) The method of (11), wherein the cell is in a subject to be treated, and the therapeutic agent is administered locally to the cell. (15) A method according to any one of (2) to (8) in which the therapeutic agent is expressed in cells. (16) A method of treating a disease, disorder and / or condition associated with the activation and signal transduction of LAR family phosphatases, comprising administering to a cell of a subject expressing LAR family phosphatase a therapeutic agent that inhibits one or more of the catalytic activity, signal transduction and / or function of LAR family phosphatase without inhibiting the binding of LAR family phosphatase to proteoglycan or its activation. (17) The method of (16), wherein the disease, disorder and / or condition comprises at least one of a disease, disorder and / or condition of the nervous system. (18) The method of (17), wherein the disease, disorder and / or condition of the nervous system comprises at least one of neuropathy, neuropsychiatric disorder, nerve injury, neurotoxic disorder, neuropathic pain, and neurodegenerative disorder. (19) The method of (18), wherein the neuropathy comprises at least one of peripheral nerve or cranial nerve, spinal cord or brain, traumatic or toxic injury to cranial nerve, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury. (20) The method of (18), wherein the neuropathy comprises at least one of Alzheimer's disease, dementia associated with Alzheimer's disease, Parkinson's disease, diffuse Lewy body disease, senile dementia, Huntington's disease, Tourette syndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, diabetic neuropathy, progressive supranuclear palsy, epilepsy, or Jakob-Creutzfeldt disease. (21) A method according to (18), wherein nerve injury is caused by, or associated with, at least one of epilepsy, cerebrovascular disease, autoimmune disease, sleep disorder, autonomic neuropathy, bladder disorder, abnormal metabolic state, muscular disorder, infectious and parasitic diseases, tumors, endocrine diseases, nutritional and metabolic diseases, immune diseases, diseases of the blood and blood-forming organs, mental diseases, nervous system diseases, sensory organ diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, urogenital system diseases, skin and subcutaneous tissue diseases, musculoskeletal and connective tissue diseases, congenital anomalies, or perinatal origin conditions. (22) A method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 65% identical to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (23) A method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 75% identical to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (24) A method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 85% identical to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (25) A method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), the therapeutic agent comprises a therapeutic peptide, and the therapeutic peptide has an amino acid sequence that is at least about 95% identical to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (26) The method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 33. (27) The method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide consisting of SEQ ID NO: 37. (28) The method according to (18), wherein the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide that is at least about 65% homologous to SEQ ID NO: 37. (29) The method according to (28), wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid at residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37. (30) The method according to any one of (22) to (29), wherein the cell is a nerve cell, a glial cell, a glial progenitor cell, or a neural progenitor cell. (31) The method according to any one of (22) to (29), wherein the therapeutic agent is linked to a therapeutic peptide and comprises a transport moiety that facilitates the uptake of the therapeutic peptide by the cell. (32) The method according to (31), wherein the transport moiety is the Tat transport moiety of HIV. (33) The method according to (31), wherein the therapeutic agent is administered systemically to the subject to be treated. (34) The method according to (31), wherein the therapeutic agent is administered locally to the cell. (35) The method according to any one of (22) to (19), wherein the therapeutic agent is expressed in the cell. (36) A method for treating nerve injury in a subject, A method comprising administering to damaged nerve cells of a subject a therapeutic agent that inhibits one or more of the catalytic activity, signal transduction, and / or function of LAR family phosphatases without inhibiting the binding or activation of LAR family phosphatases by proteoglycans. (37) A method according to (36), wherein the nerve injury comprises at least one of a peripheral nerve or cranial nerve, spinal cord or brain, traumatic or toxic injury to a cranial nerve, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury. (38) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide having an amino acid sequence that is at least about 65% homologous to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (39) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide having an amino acid sequence that is at least about 75% homologous to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (40) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide having an amino acid sequence that is at least about 85% homologous to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (41) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide having an amino acid sequence that is at least about 95% homologous to about 10 to about 20 contiguous amino acids of the wedge domain of PTPσ. (42) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 33. (43) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide consisting of SEQ ID NO: 37. (44) (36) A method, wherein the therapeutic agent comprises a therapeutic peptide that is at least about 65% homologous to SEQ ID NO: 37. (45) A method according to (44) wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid at residues 4, 5, 6, 7, 9, 10, 12 or 13 of SEQ ID NO: 37. (46) A method according to any one of (38) to (45) wherein the therapeutic agent is linked to the therapeutic peptide and comprises a transport moiety that facilitates cellular uptake of the therapeutic peptide. (47) A method according to (36) wherein the transport moiety is the Tat transport moiety of HIV. (48) A method according to (36) wherein the therapeutic agent is administered systemically to a subject to be treated. (49) A method according to (36) wherein the therapeutic agent is administered locally to cells. (50) A method according to any one of (38) to (45) wherein the therapeutic agent is expressed in cells. (51) A therapeutic agent that promotes at least one of neuronal proliferation, motility, viability and plasticity, the therapeutic agent comprising a synthetic peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33. (52) A therapeutic agent according to (51) further comprising a transport moiety that is linked to the therapeutic peptide and facilitates cellular uptake of the therapeutic peptide. (53) A therapeutic agent according to (52) wherein the transport moiety is the Tat transport moiety of HIV. (54) A therapeutic agent that promotes at least one of neuronal proliferation, motility, viability and plasticity, the therapeutic agent comprising a synthetic therapeutic peptide that is at least about 65% homologous to SEQ ID NO: 37. (55) A therapeutic agent according to (54) wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid at residues 4, 5, 6, 7, 9, 10, 12 or 13 of SEQ ID NO: 37. (56) A therapeutic agent according to (54) wherein the therapeutic peptide comprises SEQ ID NO: 37. (57) A therapeutic agent according to (54) to (56), further comprising a transport moiety linked to the therapeutic peptide and facilitating the uptake of the therapeutic peptide by cells. (58) A therapeutic agent according to (57), wherein the transport moiety is the Tat transport moiety of HIV. (59) A pharmaceutical composition comprising: a therapeutic agent comprising a synthetic therapeutic peptide that is at least about 65% homologous to SEQ ID NO: 37 and a transport moiety linked to the therapeutic peptide and facilitating the uptake of the therapeutic peptide by cells. (60) A pharmaceutical composition according to (59), wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid at residue 4, 5, 6, 7, 9, 10, 12 or 13 of SEQ ID NO: 37. (61) A pharmaceutical composition according to (59), wherein the therapeutic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33 and 37. (62) A pharmaceutical composition according to (59), wherein the transport moiety is the Tat transport moiety of HIV. (63) A pharmaceutical composition according to (59), wherein the therapeutic agent is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 66 and 70.

Claims

1. A peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33, and a transport moiety linked to the peptide and facilitating the uptake of the peptide by cells.

2. The composition according to claim 1, wherein the transport moiety is the HIV Tat transport moiety.

3. The composition according to claim 1, wherein the linked peptide and transport moiety have a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 66.

4. The composition according to claim 2, wherein the transport moiety is linked to the peptide by a peptide linker.

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