Compositions and methods for inhibiting the activity of LAR family phosphatases

Abstract

Therapeutic agents are provided in a method for inhibiting and / or reducing the activity, signaling and / or function of leukocyte common antigen-related (LAR) family phosphatases in cells of a subject induced by proteoglycans. [Solution] In some embodiments, the LAR family phosphatase 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% homologous to about 10 to about 20 contiguous amino acids of the wedge-shaped domain of PTPσ.

Description

Technical Field

[0001] This application claims priority from U.S. Provisional Patent Application No. 61 / 621,623, filed Apr. 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 disability 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 mature CNS cannot regenerate.

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

[0005] Although CSPG presents a barrier to axonal regeneration, specific receptors for the inhibitory effect of CSPG have not been identified to date. More specifically, CSPG exhibits dramatic upmodulation after nerve injury, both in the extracellular matrix of scar tissue and in the perineuronal network within more distant targets of severed axons. The inhibitory nature of CSPG is reflected not only in the formation of trophic axonal contraction valves that cannot regenerate through the lesion, but also in the limited ability of surviving fibers to sprout. Although sulfated proteoglycans have been known to be a major contributor to the resilience of glial scars for nearly 20 years, the precise inhibitory mechanisms remain largely unknown. Therefore, there is an urgent need for mechanisms that modulate CSPG function. [Overview of the project] [Problems that the invention aims to solve]

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

[0007] In some embodiments, the LAR family phosphatase 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% homologous to about 10 to about 20 consecutive amino acids of the wedge-shaped domain of PTPσ. For example, the therapeutic agent may include a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 33.

[0008] In other embodiments, the LAR family phosphatase is receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent may 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% homologous to the amino acid sequence of SEQ ID NO: 37. The therapeutic peptide may, for example, include conservative substitutions of at least 1, 2, 3, or 4 amino acids in residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37.

[0009] In some embodiments, the cells are nerve cells, glial cells, glial progenitor cells, or neural progenitor cells.

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

[0011] In yet another embodiment, the cells are present in the target to be treated, and the therapeutic agent is administered to the target to be treated locally or systemically.

[0012] In yet another embodiment, the therapeutic peptide is expressed in cells.

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

[0014] In some embodiments, the disease, disorder and / or condition includes at least one disease, disorder and / or condition of the nervous system.

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

[0016] For example, neurological disorders may include at least one of the following: peripheral or cranial nerves, spinal cord or brain, traumatic or toxic injury to the cranial nerves, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury. Neurological disorders may also include at least one of the following: Alzheimer's disease, Alzheimer's disease-related dementia, Parkinson's disease, diffuse Lewy body dementia, 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 damage may be caused by or related to epilepsy, cerebrovascular disease, autoimmune disease, sleep disorder, autonomic nervous system disorder, bladder disorder, abnormal metabolic state, muscular system disorder, infectious and parasitic diseases, tumors, endocrine disorders, nutritional and metabolic disorders, immune disorders, blood and hematopoietic organ disorders, mental disorders, nervous system disorders, sensory organ disorders, circulatory system disorders, respiratory system disorders, digestive system disorders, genitourinary system disorders, skin and subcutaneous tissue disorders, musculoskeletal and connective tissue disorders, congenital abnormalities, or perinatal conditions.

[0018] Further embodiments described herein relate to therapeutic agents for promoting at least one of the proliferation, motility, viability, and plasticity of nerve cells. These include therapeutic peptides 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% homologous to about 10 to about 20 consecutive amino acids of the wedge-shaped domain of PTPσ. For example, a therapeutic agent may include a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 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] [Figure 1] Photograph showing mature sensory DRG neurons in a spot assay after 5 days of exposure to the CSPG gradient. [Figure 2] (A-D) Photographs showing growth cones of mature sensory DRG neurons in a spot assay exposed to the CSPG gradient. [Figure 3]Graph showing PTPσ density in normal and dystrophic axons and growth cones. [Figure 4] Schematic diagram showing transmembrane phosphatases of the LAR family, LAR, RPTPσ, RPTPδ and their signal transduction components. [Figure 5] Photograph showing mature sensory DRG neurons in a spot assay exposed to a CSPG gradient and treated with solvent control or ISP. [Figure 6] Graph showing the crossing of growth cones of mature sensory DRG neurons in a spot assay exposed to a CSPG gradient and treated with solvent control or ISP. [Figure 7] (A - B) Photograph showing the motility of growth cones of sensory DRG neurons in a spot assay exposed to a CSPG gradient and treated with solvent control or ISP. [Figure 8] Graph showing a plot of Basso, Beattie and Bresnahan scores for spontaneous locomotion after spinal cord injury (SCI) in solvent - treated SCI animals and LAR peptide - treated SCI animals between days 1 and 77. [Figure 9] Graph showing Basso, Beattie and Bresnahan scores of the hindlimbs for spontaneous locomotion after SCI in solvent - treated SCI animals and LAR peptide - treated SCI animals at week 11. [Figure 10] Graph showing slip - over / meter in solvent - treated SCI animals and LAR peptide - treated SCI animals. [Figure 11] Graph showing foot - slip / meter in solvent - treated SCI animals and LAR peptide - treated SCI animals. [Figure 12] Graph showing urine frequency and volume in solvent - treated SCI animals and ISP - treated SCI animals. [Figure 13A] Graph showing urine frequency in solvent - treated SCI animals and LAR peptide - treated SCI animals. [Figure 13B]This graph shows the number of urinations in SCI animals treated with solvents and SCI animals treated with LAR peptides. [Figure 14] These are photographs showing the expression (axonal density) of 5HT in the lumbar spinal cord of solvent-treated SCI animals and ISP-treated SCI animals. [Figure 15] This graph shows the expression of 5HT in the lumbar region of SCI animals treated with solvents and SCI animals treated with ISP. [Modes for carrying out the invention]

[0025] The embodiments described herein are not limited to, but may be modified to, specific methodologies, protocols, reagents, etc. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention as defined solely by the claims. Except in the examples of operation or as indicated, all numbers representing quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term “about”.

[0026] All specified patents and other publications are clearly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications, for example, which may be used in connection with the present invention. These publications are simply made available for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment that the inventors are not granted priority rights to such disclosures on the grounds of prior invention or for any other reason. All references to dates or expressions relating to the contents of these documents are based on information available to the applicant and do not constitute an acknowledgment of the accuracy of the dates or contents of these documents.

[0027] Unless otherwise defined, scientific and technical terms used herein should have meanings generally understood by those skilled in the art. Furthermore, unless required by context, singular forms should include plurals and plural forms should include singulars. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are well known and commonly used 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 inclusive.

[0029] As used herein, the term “administer” to a patient includes distributing, delivering, 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 (for example, to contact a desired cell, such as a desired neuron), including administration into the cerebrospinal fluid or across the blood-brain barrier, delivery by parenteral or oral routes, intramuscular injection, subcutaneous or transdermal injection, intravenous injection, intrabuccal administration, transdermal delivery, and administration by rectal, colonic, vaginal or respiratory routes. The formulation may be administered, for example, by intravenous injection to an unconscious, anesthetized or paralyzed subject, or by intravenous injection to a pregnant subject to stimulate fetal axonal growth. Specific routes of administration may include topical administration (e.g., eye drops, creams or erosive formulations applied under the eyelids, intraocular injection into aqueous or vitreous fluids, injection into the outer layers of the eye via subconjunctival or subspheric injection), parenteral or oral routes.

[0030] As used herein, the term “antibody” includes human and animal mAbs, and synthetic antibodies, including polyclonal antibodies, recombinant antibodies (antisera), humanized antibodies, anti-idiotype antibodies, and chimeric antibodies, including their derivatives. A portion or fragment of an antibody refers to a region of the antibody that retains at least some of its ability to bind to a specific epitope (binding specificity and affinity). The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an antibody paratope 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 typically 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 an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, see 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 terms “growth” or “extension” of an axon (also referred to herein as “neuronal extension”) include the process by which an axon or dendrite extends from a neuron. Extension occurs in new neurites or in the expansion of pre-existing cellular processes. Axonal extension may include the linear expansion of axonal processes up to 5 cells in diameter or more. Neuronal growth processes, including neurite generation, can be evidenced by the expression of GAP-43, which can be detected by methods such as immunohistochemistry. “Stimulating axonal growth” means promoting axonal extension.

[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 regression" refers to the regression of an axon resulting from trauma to the axon.

[0034] As used herein, “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 (e.g., a portion of the polypeptide) that is exogenous and substantially non-homologous to the domain of the first polypeptide. The chimeric protein may present an exogenous domain and may be found in organisms (albeit as different proteins), and may also be expressed as the first protein, or it may be an “interspecies,” “intergenetic,” or other fusion of protein structures expressed by different species of organisms.

[0035] As used herein, the terms “contacting neurons” or “treating neurons” refer to any form of delivery or “administration” of an agent to a cell or whole organism in which the agent can exert its pharmaceutically effective effect in neurons. “Contacting neurons” includes methods of bringing the agent of the present invention into the vicinity of neurons, both in vivo and in vitro. A preferred form of administration can be determined by those skilled in the art, and such forms of administration may vary among agents. For example, when stimulating the axonal growth of neurons in vitro, the agent may be administered, for example, by transfusion, lipofection, electroporation, infection with a viral vector, or by addition to a growth medium.

[0036] As used herein, an effective dose of an agent or therapeutic peptide is an amount sufficient to achieve the desired therapeutic or pharmacokinetic effect, for example, an amount capable of activating neuronal growth. As defined herein, an effective dose of an agent may vary depending on factors such as the state of the disease in the subject, age and weight, and the agent's ability to elicit the desired response in the subject. The administration plan may be adjusted to provide an optimal therapeutic response. An effective dose is also one in which the therapeutic benefits outweigh the toxic or adverse effects of the active compound. As used herein, the term “therapeutically effective dose” means an amount effective in the doses and time required to achieve the desired therapeutic outcome. Therapeutic outcomes may include, for example, a reduction in symptoms, an extension of survival, or improved mobility. Therapeutic outcomes do not necessarily have to be “cure.”

[0037] As used herein, the term “expression” refers to the process by which nucleic acids are translated into peptides or transcribed into RNA that can be translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression includes, where a suitable eukaryotic host cell or organism is selected, mRNA splicing. For non-homologous nucleic acids expressed in a host cell, they must first be delivered to the cell, then enter the cell, and finally reside in the nucleus.

[0038] As used herein, the term “gene therapy” includes the transfer of non-homologous DNA into mammalian cells, particularly human cells, that are affected by a disorder or condition requiring treatment or diagnosis. The DNA is introduced into selected target cells in such a manner that the non-homologous DNA is expressed and that a therapeutic product encoded by it is produced. Alternatively, the non-homologous DNA may in some way intervene in the expression of the DNA encoding the therapeutic product; it may encode a product such as a peptide or RNA that in some way directly or indirectly intervenes in the expression of the therapeutic product. Gene therapy may be used to deliver nucleic acids 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 introduced nucleic acid may encode therapeutic compounds that are not normally produced in the mammalian host, or are not produced in therapeutically effective amounts or for therapeutically useful times, such as growth factors or their inhibitors, tumor necrosis factors or their inhibitors, or receptors for them, for example. The non-homologous DNA encoding the therapeutic product may be modified before being introduced into the cells of the affected host to enhance or otherwise alter the product or its expression.

[0039] As used herein, the terms “gene” or “recombinant gene” refer to nucleic acids comprising an open reading frame that encodes a polypeptide containing both exon and (optionally) intron sequences.

[0040] As used herein, the term “non-homologous nucleic acid sequence” typically refers to DNA that encodes RNA and proteins not normally produced in vivo by the cell on which it is expressed, or DNA that intervenes in or encodes mediators that alter the expression of endogenous DNA by affecting transcription, translation, or other regulatory biological processes. Non-homologous nucleic acid sequences are also called exogenous DNA. Any DNA that a person skilled in the art would recognize or consider to be non-homologous or exogenous to the cell on which it is expressed is encompassed herein by non-homologous DNA. Examples of non-homologous DNA include, for example, DNA that encodes traceable marker proteins, such as drugs that confer drug resistance; DNA that encodes therapeutically effective substances, such as anticancer drugs, enzymes, and hormones; and DNA that encodes other types of proteins, such as antibodies. Antibodies encoded by non-homologous DNA may be secreted or expressed on the surface of cells 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. Homologousity can be determined by comparing the positions in each sequence that can be aligned for comparison. If the positions in the compared sequences are occupied by the same base or amino acid, then the molecules are homologous or identical at those positions. 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 “neurological disorder” includes diseases, disorders, or conditions that directly or indirectly affect the normal function or biological structure of the nervous system in question. The term “stroke” as recognized in the art includes the sudden attenuation or loss of consciousness, sensation, and voluntary movement resulting from the rupture or blockage of an artery in the brain (e.g., by a thrombus). “Traumatic brain injury” as recognized in the art includes conditions in which a traumatic blow to the head causes injury to the brain or the connected spinal cord, with or without penetration of the skull. Typically, the initial trauma can result in a dilating hematoma, subarachnoid hemorrhage, cerebral edema, increased intracranial pressure, and cerebral hypoxia, which in turn lead to serious secondary events due to reduced cerebral blood flow.

[0043] As used herein, the term “neuronal migration” refers to the ability of a neuron cell or a neuronal process to migrate, such as axonal migration or dendritic migration.

[0044] As used herein, the terms “parenteral administration” and “administered parenterally” mean, as used herein, a mode of administration by ordinary injection other than intra-intestinal and local administration, which includes, but are not limited to, intravenous, intramuscular, intra-arterial, intraventricular, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, sub-articular, intraspinal, and intrasternal injections and infusions.

[0045] As used herein, the terms “systemic administration” and “administered systemically” mean 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 entire body of the animal and is therefore subject to metabolism and other similar processes, such as subcutaneous administration.

[0046] As used herein, the terms “patient,” “subject,” “animal,” or “host” refer to any mammal. A subject may be a human, but may also be any mammal requiring veterinary treatment, such as a pet (e.g., a dog or cat), livestock (e.g., a cattle, sheep, chicken, pig, or horse), or laboratory animal (e.g., a rat, mouse, or guinea pig).

[0047] As used herein, “neurons of the peripheral nervous system (PNS)” includes neurons that are located outside or extend beyond the central nervous system (CNS). The PNS is intended to include neurons that are generally understood to be classified as part of the peripheral nervous system, including sensory neurons and motor neurons.

[0048] As used herein, the terms “polynucleotide sequence” and “nucleotide sequence” are also interchangeable herein.

[0049] As used herein, the terms “peptide” and “polypeptide” are also used interchangeably herein and refer to compounds consisting of about 2 to about 90 amino acid residues, in which the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond. Peptides can be derived or isolated from native proteins, for example, by enzymatic 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)). "Peptides" may 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, statins), and rare amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). The amino groups, carboxyl groups, and / or other functional groups on the peptide may be free (e.g., unmodified) or 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 peptides can also be derivatized (e.g., alkylated) using methods known in the art.

[0050] Peptides can be synthesized and assembled into libraries containing two, three to numerous distinct molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry and screened as described herein, or by other suitable methods for determining whether the library contains peptides capable of antagonizing the CSPG / PTPσ interaction. Antagonists of such peptides can then be isolated by suitable means.

[0051] As used herein, the term “peptide mimetic” refers to a protein-like molecule designed to mimic a peptide. Peptide mimetic molecules typically arise from modifications of existing peptides or from designing peptide-mimicking systems, such as peptoids and β-peptides. Regardless of the approach, altered chemical structures are designed to favorably modify molecular properties such as stability or bioactivity. These modifications involve alterations to peptides that do not exist in nature (e.g., altered backbones and the incorporation of unnatural amino acids).

[0052] As used herein, the term “progenitor cell” refers to a cell produced during the differentiation of a stem cell that possesses some, but not all, of the characteristics of its ultimately differentiated offspring. For example, a defined progenitor cell, such as “neural progenitor cell,” is related to a lineage but not to a specific or ultimately differentiated cell type.

[0053] As used herein, the term “stem cell” means a cell capable of self-renewal (i.e., offspring with the same differentiation potential) and capable of producing offspring cells that are further constrained in differentiation potential. Within the context of the present invention, stem cells also include further differentiated cells that have been dedifferentiated, for example, by nuclear transfer, by fusion with more primitive stem cells, by introduction of specific transcription factors, or by 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 “regression” refers to the retreat of an axon from the site of injury, for example, from the site where a glial scar is formed. Here, the regenerating axon ends cease to elongate and become malnourished. These malnourished ends may then regress further from the glial scar and the site of injury.

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

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

[0057] As used herein, the term “tissue-specific promoter” refers to a nucleic acid sequence that acts as a promoter, i.e., regulates the expression of a selected nucleic acid sequence operably linked to a promoter, thereby influencing the expression of the selected nucleic acid sequence in specific cells of a tissue, such as epithelial cells. The term also encompasses so-called “leakage” promoters that primarily regulate the expression of a selected nucleic acid in one tissue but similarly cause expression in other tissues. The term “transfer” is used to refer to the uptake of exogenous DNA by a cell. A cell is transfused when exogenous DNA is introduced inside the cell membrane. Numerous methods of transfusion are commonly known in this technique. See, for example, 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 portions, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into suitable host cells. The term encompasses chemical, electrical, and viral translocation procedures.

[0058] As used herein, the term “transcriptional regulatory sequence” is a general term used throughout this specification to refer to nucleic acid sequences such as start signals, enhancers, and promoters that induce or control the transcription of sequences encoding proteins into which they are manipulably linked. In some examples, the transcription of recombinant genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in the cell type in which expression is intended. It will also be understood that recombinant genes may be under the control of transcriptional regulatory sequences that control the transcription of naturally occurring forms of the protein, which may be identical 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 ligated. Preferred vectors are those capable of one or more of the autonomous replication and expression of the nucleic acid to which they bind. Vectors capable of directing the expression of a gene to which they are manipulably ligated are referred herein to as “expression vectors.”

[0060] As used herein, the term “wild-type” refers to a naturally occurring polynucleotide that codes for a protein, or a portion thereof, or a protein sequence, or a portion thereof, as it normally exists in living organisms. As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and, as appropriate, ribonucleic acid (RNA). The terms should be understood to include RNA or DNA equivalents, analogues, single-stranded (sense or antisense) and double-stranded polynucleotides made from nucleotide analogs and applicable to desired embodiments.

[0061] Agents, compounds, compositions, antibodies, etc., used in the methods described herein should be considered to be purified and / or isolated before use. Purified substances are usually "substantially pure," meaning that nucleic acids, polypeptides, or fragments thereof, or other molecules, have been separated from their naturally associated components. A polypeptide is usually substantially pure if it does not contain at least 60%, 70%, 80%, 90%, 95%, or even 99% by weight of the proteins and other organic molecules that it naturally associates with. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by the expression of recombinant nucleic acids in cells that do not normally express that protein, or by chemical synthesis. "Isolated substances" are taken out of their natural location and environment. In the case of isolated or purified domains or protein fragments, the domain or fragment substantially does not contain amino acid sequences adjacent to the protein in the naturally occurring sequence. The term "isolated DNA" means that the DNA substantially does not contain genes adjacent to a given DNA in the naturally occurring genome. Therefore, the term "isolated DNA" includes, for example, cDNA, cloned genomic DNA, and synthetic DNA.

[0062] As used herein, the terms “part,” “fragment,” “mutant,” “derivative,” and “analog” include, when referring to the polypeptide of the present invention, polypeptides that retain at least some of the biological activity (e.g., inhibition of interactions such as binding) as referenced herein. Polypeptides as described herein may include, without limitation, parts, fragments, mutants, or derivative molecules, insofar as the polypeptide still contributes to its function. Polypeptides of the present invention or parts thereof may include proteolytic fragments, deletion fragments, and, in particular, or fragments that more readily reach the site of action when delivered to an animal.

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

[0064] The LAR family of phosphatases consists of three members: LAR itself, the receptor protein tyrosine phosphatase sigma (PTPσ), and the receptor protein tyrosine phosphatase delta (PTPδ). PTPσ and PTPf (LAR itself) are involved as receptors for chondroitin sulfate proteoglycans (CSPGs), which are major components of glial scars and perineuronal networks. The sugar side chains of CSPGs can bind to LAR and PTPσ expressed by cells such as neurons, inhibiting neuronal proliferation, plasticity, regeneration, and germination failure in neurons.

[0065] PTPσ knockout neurons showed reduced inhibition in various CSPG-mediated assays and increased regeneration after nerve injury, such as after spinal cord injury and ophthalmic nerve contusion. Results with LAR knockouts were inconclusive, showing both increased and decreased regeneration phenotypes. Since CSPGs are major inhibitors of regeneration and plasticity in damaged mature nervous systems, functional inhibitors of these LAR family phosphatases can be used as therapeutic agents to promote neuronal proliferation, plasticity, regeneration, and sprouting.

[0066] Accordingly, some embodiments described herein relate to methods for promoting the proliferation, motility, viability, and / or plasticity of cells expressing LAR family phosphatases that are activated and / or potentially activated by proteoglycans such as CSPG (i.e., neurons, neural progenitor cells, neural stem cells, or endothelial cells). The methods may include administering to cells an amount of a therapeutic agent effective in inhibiting the catalytic activity, signaling, and / or function of LAR family phosphatases. By inhibiting the activity, signaling, and / or function of LAR family phosphatases, the proliferation, motility, viability, and plasticity of these cells can be promoted.

[0067] In certain embodiments, cells expressing LAR family phosphatases include neurons and glial cells. Other examples of cells include endothelial cells. Yet another example of cells expressing LAR family phosphatases that can be activated by proteoglycans can be readily screened using known assays.

[0068] The activity, signaling, and / or function of LAR family phosphatases can be suppressed, inhibited, and / or blocked by several methods, including, for example, direct inhibition of the activity of the intracellular domain of LAR family phosphatases (e.g., by using small molecules, protein mimes, or dominant inhibitory polypeptides); activation of genes and / or proteins that inhibit one or more of the activity, signaling, and / or function of the intracellular domain of LAR family phosphatases (e.g., by increasing the expression or activity of genes and / or proteins); inhibition of genes and / or proteins that are downstream mediators of LAR family phosphatases (e.g., by blocking the expression and / or activity of mediator genes and / or proteins); induction of genes and / or proteins that negatively regulate one or more of the activity, signaling, and / or function of LAR family phosphatases (e.g., by using recombinant gene expression vectors, recombinant viral vectors, or recombinant polypeptides); or gene substitution with low-phenotype variants of LAR family phosphatases (e.g., by homologous recombination, recombinant gene expression, or overexpression using viral vectors, or by mutagenesis).

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

[0070] Neurobeneficial effects may include beneficial responses or outcomes to the health or function of neurons, parts of the nervous system, or the nervous system in general. Examples of such effects include improvements in the ability of neurons or parts of nerves to withstand, regenerate, maintain desired function, proliferate, or survive damage. Neurobeneficial effects may also include producing or achieving such responses or improvements in the function or resilience of components of the nervous system. Examples of producing neurobeneficial effects include stimulating axonal elongation after injury to neurons; making neurons resistant to apoptosis; making nerves resistant to toxic compounds such as 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 mitigating axonal regression, and / or promoting neuronal sprouting.

[0071] One possible mechanism for the regulation, modulation, and / or inhibition of LAR family phosphatases involves the dimerization of the intracellular portion of LAR family phosphatases. In contrast to receptor tyrosine kinases, which are active as dimers and inactive as monomers, some protein tyrosine phosphatases (PTPσ) are inactive in the dimerized state and active as monomers. These include PTPα, PTP1B, and CD45. Each of these molecules can be crystallized in both the active monomeric and inactive dimeric forms. In addition, LAR and CD45 exhibit homobinding 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. Therefore, mimicking dimerization through intracellular targeting therapies allows for the direct inactivation of LAR family phosphatases without altering the intracellular matrix or other ligands.

[0072] We found that peptide mimetic forms of the intracellular portions of LAR family 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 was found to promote neuronal elongation, including restoration of growth cone motility, projection elongation, sprouting, and enhanced neuronal survival and plasticity, as well as inhibiting neuronal axonal retraction.

[0073] In one embodiment, therapeutic agents that inhibit or reduce one or more of the activity, signaling, and / or function of LAR family phosphatases include therapeutic peptides or small molecules that bind to and / or form a complex with the intracellular domain of at least one LAR family phosphatase to inhibit the activity, signaling, and / or function of LAR family phosphatases. Therefore, therapeutic peptides or small molecules that bind to and / or form a complex with the intracellular domain of at least one LAR family phosphatase in nerve cells can be used to promote cell proliferation, motility, viability, and plasticity of these cells.

[0074] In some embodiments, therapeutic agents can be peptide mimes of the wedge-shaped domain (i.e., wedge domain) of the intracellular catalytic domain of LAR family phosphatases. Structural and sequence analysis reveals that all members of the LAR family contain a conserved 24-amino acid wedge-shaped helical / loop / helical motif in a first intracellular catalytic domain that can potentially mediate allogeneic / heterogeneous receptor interactions. Table 1 lists the amino acid sequences of the intracellular portions of LAR family phosphatase members containing the wedge domain. The 24-amino acid wedge domains of these intracellular portions of LAR family phosphatases are identified by underlining. While the specific structure of the wedge domain is conserved across most LAR family wedge domains, the exact amino acids constituting the wedge domain vary between individual proteins and subfamilies. [Table 1]

[0075] The wedge-shaped domains of specific LAR family members were found to be involved in homogeneous interactions or binding with other members of that particular LAR family. For example, in a pull-down assay, the wedge-shaped domain of LAR could specifically interact with full-length LAR but not with other family members such as PTPσ. In addition, in vitro binding assays showed that PTPmu and the wedge-shaped domain peptide of LAR (wedge-shaped domain + HIV-TAT) aggregated homogeneously and specifically, rather than binding randomly to each other. Particularly interesting is the inability of the wedge-shaped domain of LAR to bind to sigma, indicating specificity even among members of similar families.

[0076] Therefore, when expressed in cells (e.g., nerve cells) or conjugated to intracellular transport regions, peptide mimes of these wedge-shaped domains of LAR family phosphatases can be used to eliminate LAR family signaling in CSPG-activated nerve cells, thereby promoting cell proliferation, motility, and viability. The binding of these therapeutic peptides to the intact wedge-shaped domain of the specific PTP potentially (i) interferes with the PTP's ability to interact with target proteins such as phosphatase targets; (ii) interferes with the activity that promotes intermolecular interactions between the PTP and other domains contained in the PTP, such as the catalytically inactive second phosphatase domain D2; prevents protein access to the active phosphatase site; (iii) competes with normal interactors of the wedge-shaped domain; and / or sterically inhibits phosphatase activity.

[0077] In some embodiments, the peptide mimetic (i.e., therapeutic peptide) comprises about 10 to about 20 amino acids and may consist of and / or 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% homologous to the amino acid sequence of about 10 to about 20 consecutive amino acid portions of the wedge-shaped domain of a LAR family phosphatase.

[0078] In other embodiments, the peptide mimetic may consist of approximately 10 to approximately 20 amino acids and / or have an amino acid sequence that is at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or 100% homologous to the approximately 10 to approximately 20 consecutive amino acid parity of the wedge-shaped domain of PTPσ. We found that peptide mimetic (e.g., therapeutic peptides) that correspond to or are substantially homologous to the wedge-shaped domain of PTPσ with a cytoplasmic carrier can remove CSPG-mediated inhibition, allowing neurons to proceed on the CSPG substrate instead of typical inhibition. This effect was dose-dependent and depended on the responding cell expressing PTPσ. Astrocytes that do not express PTPσ at the protein level do not respond to peptide inactivation, while satellite glia expressing PTPσ do respond to the peptide. Furthermore, this peptide can be administered systemically to promote plasticity and functional recovery after severe spinal cord injury.

[0079] As shown in Table 2, the wedge-shaped domain sequence of PTPσ is highly conserved among higher mammals, and a single amino acid change (from threonine to methionine at position 6) prevents 100% homology between mice and rats. [Table 2] TIFF0007829511000003.tif124132

[0080] As shown in Table 2, the first α-helix of the wedge-shaped domain of PTPσ contains amino acids 1-10, the turn region contains amino acids 11-14, and the second α-helix contains amino acids 15-24. For example, the first α-helix of the wedge-shaped domain of human PTPσ has the amino acid sequence DMAEHTERLK (SEQ ID NO: 67), the turn has the amino acid sequence 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 appear to be necessary for the overall structure of the wedge-shaped domain. The conserved amino acid is 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 wedge size and structure.

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

[0083] These conserved substitutions can be present in non-unique domains in the α-helix or turn, particularly at positions 1-3 and 7-10 in the first α-helix; positions 12 and 13 in the turn; and at positions 15, 16, and 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 wedge-shaped binding to PTPσ.

[0084] It has been found that unique amino acids for PTPσ, particularly those differentially expressed in PTPσ and LAR, are necessary for the specificity of wedge-shaped domain binding. These include the EH domain at positions 4 and 5 of the first α-helix, and subsequently at position 6, where threonine or methionine (substitutions in rats and mice) are present. In the turn, there is a unique serine at position 14 in all higher mammals. Finally, there is a unique leucine at position 17 in the second α-helix. The possible roles of these unique amino acids are discussed below.

[0085] The serine residue at the 14th position is particularly interesting due to its location in the wedge-shaped domain. Located in the α-helix, this amino acid extends slightly from the general secondary and tertiary structures of PTPσ, making it available for bonding interactions. In addition, serine is known to facilitate several homogeneous and heterogeneous bonding events, such as hydrogen bonding 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 potential need for its specificity. Focusing on the wedge-shaped domain turn, it is possible that small peptides containing a conserved serine could offer greater stability along with similar functionality. Such peptides could be synthesized as loops with either cysteine ​​terminology 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, this sequence or one of its components is likely required for the wedge specificity of PTPσ.

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

[0088] Accordingly, in other embodiments, the therapeutic peptide may consist of or comprise essentially 14 to 20 amino acids, and comprises the amino acid sequence EHX1ERLKANDSLKL (SEQ ID NO: 37), where X1 is T or M. The therapeutic peptide comprising SEQ ID NO: 37 may contain 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% homologous 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. For 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 may be subject to various other modifications, substitutions, insertions, and deletions, such modifications may provide specific advantages in their use. In this regard, therapeutic peptides that bind to and / or complex with the wedge-shaped domain of LAR family phosphatases may be equivalent to, or substantially homologous to, a sequence of a cited polypeptide that has undergone one or more modifications and which retains the ability to inhibit or reduce one or more of the activity, signaling, and / or function of LAR family phosphatases.

[0091] Therapeutic peptides can be any of various forms of polypeptide derivatives, including amides, protein conjugates, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides, and analogous derivatives.

[0092] It will be well understood that conservative substitutions may also include using a chemically derivatized residue instead of a non-derivatized residue, provided that such a peptide exhibits the required binding activity.

[0093] A "chemical derivative" refers to a polypeptide of a subject having one or more residues that have been chemically derivatized by the reaction of a functional group. Examples of such derivatized molecules include 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 may be derivatized to form a salt, methyl ester, or ethyl ester, or other types of esters or hydrazides. A free hydroxyl group may be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine may be derivatized to form N-im-benzenehistidine. 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 with proline; 5-Hydroxylysine may be substituted with lysine; 3-Methylhistidine may be substituted with histidine; homoserine may be substituted with serine; and ornithine may be substituted with lysine. The polypeptides described herein also include any polypeptides having one or more additions and / or deletions or residues to the sequence of the polypeptides shown herein, provided that the required activity is maintained.

[0094] In yet another embodiment, the therapeutic agent may be a mimetic or competitive inhibitor of downstream proteins activated by LAR family phosphatases. Several downstream proteins and pathways have been shown to act downstream of the LAR family outside of phosphatase activity. Of these, Caskin (Ckn) and LAR-interacting proteins (lipin-α) play roles in both synapse formation and axonal guidance.

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

[0096] Accordingly, in some embodiments, the therapeutic agent may be a peptide mimetic or competitive inhibitor of Ckn that inhibits phosphatase / Ckn binding of the LAR family and mitigates downstream LAR family signaling. The peptide may 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% homologous to the approximately 10 to about 30 consecutive amino acid sequences of the mCkn1 and mCkn2 portions. Examples of peptides having amino acid sequences substantially homologous to the approximately 10 to about 30 consecutive amino acid sequences of the mCkn1 and mCkn2 portions are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0097] In other embodiments, the therapeutic agent may be a peptide mimetic or competitive inhibitor of liprin-α that inhibits LAR family phosphatase / liprin-α binding and mitigates downstream LAR family signaling. Liprin family members play a crucial role in synaptic development and maintenance. Liprin-α may act in signaling downstream of LAR phosphatases. Yeast 2-hybrid interaction screening suggests a primary SAM domain of liprin-α family members as a binding site for LAR phosphatase family members. The peptide may have an amino acid sequence substantially homologous to about 10 to about 30 consecutive amino acids of a subset of liprin-α. An example of a peptide having an amino acid sequence substantially homologous to about 10 to about 30 consecutive amino acids of a subset of liprin-α is SEQ ID NO: 41.

[0098] Similar to the therapeutic peptides described above that bind to or complex with wedge-shaped domains, therapeutic polypeptides that are mimics or competitive inhibitors of mCkn1, mCkn2, or liprin-α can be subject to a variety of modifications, substitutions, insertions, and deletions that provide specific advantages in their use. For example, therapeutic polypeptides can be any of the various forms of polypeptide derivatives, including amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides, and analogous derivatives.

[0099] One or more of the therapeutic peptides described herein can be modified by natural processes, such as post-translational processing and / or chemical modification methods known in the art. Modifications may be present in the peptide backbone, amino acid side chains, and peptides comprising the amino or carboxyl terminus. It will be understood that the same type of modification may be present at several sites of a given peptide to the same or different degrees. Modifications include, but are not limited to, amino acid addition to transfer RNA-mediated proteins such as acetylation, acylation, addition of acetomidomethyl (Acm) groups, ADP-ribosylation, amidation, covalent bonding to flavin, covalent bonding to heme moiety, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and ubiquitination (see, for example, Protein-structure and molecular properties, 2nd Ed., TE Creighton, WH Freeman and Company, New York, 1993).

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

[0101] The therapeutic polypeptides described herein may be prepared by methods known to those skilled in the art. The peptides and / or proteins may be prepared using recombinant DNA. For example, one preparation may involve culturing a host (bacteria or eukaryotic cells) under conditions that provide expression of the peptides and / or proteins in cells.

[0102] Polypeptide purification can be performed by affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, or other purification methods commonly used for protein purification. The purification process can be carried out under non-denaturing conditions. Alternatively, if a denaturation step is required, the protein can be restored using techniques known in that technology.

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

[0104] The linker of an amino acid residue is usually at least one residue, can be 40 or more, and more often 1 to 10 residues. Typical amino acid residues used for linking include glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid. In addition, the polypeptide of the subject can be differentiated by sequences modified by terminal-NH2 acylation, e.g., by acetylation, or by terminal carboxylamidation, e.g., by amidation of thioglycolic acid by terminal modifications such as ammonia and methylamine. Terminal modifications are, as is well known, useful in reducing sensitivity to proteinase digestion, and thus help to extend the half-life of polypeptides in solutions where proteases may be present, especially in biological fluids. In this respect, cyclization of polypeptides is also a useful terminal modification and is particularly preferred in terms of the stable structure formed by cyclization and the bioactivity observed in such cyclic peptides as described herein.

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

[0106] In some embodiments, therapeutic agents comprising the therapeutic peptides described herein may be provided in the form of a conjugating protein or drug delivery construct comprising at least a transport subdomain or portion (i.e., a transport portion) linked to the therapeutic peptide. The transport portion can facilitate the uptake of the therapeutic polypeptide into mammalian (i.e., human or animal) tissues or cells (e.g., nerve cells). The transport portion can be covalently linked to the therapeutic polypeptide. Covalent bonds may include peptide bonds and unstable bonds (e.g., bonds that are easily cleaved or subjected to chemical changes in the internal target cell environment). Furthermore, the transport portion can be crosslinked to the therapeutic polypeptide (e.g., chemical crosslinking, UV crosslinking). The transport portion can also be linked to the therapeutic polypeptide together with the linking polypeptides described herein.

[0107] The transport region can be repeated more than once in the therapeutic polypeptide. Repeating the transport region may affect (e.g., increase) the uptake of the peptide and / or protein by the desired cells. The transport region may be located in either the amino-terminus or carboxyl-terminus region of the therapeutic peptide, or both.

[0108] In one embodiment, the transport portion may include at least one transport peptide that, once ligated to the transport portion, allows the therapeutic polypeptide to penetrate the cell 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 may each have the amino acid sequences of SEQ ID NOs: 42-66 and 70-74, respectively.

[0109] Other known examples of transport portions, subdomains, etc., are described, for example, in Canadian Patent Document No. 2,301,157 (Conjugates containing the homeodomain of Antennapedia), all of which are incorporated herein by reference as a whole, and in U.S. Patents Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604 (Conjugates containing the TatHIV protein; amino acids of the herpes simplex virus-1 DNA-binding protein VP22; histidine tags ranging in length from 4 to 30 histidine repeats, or mutant derivatives or analogs thereof capable of promoting the uptake of the active cargo portion in a receptor-independent manner).

[0110] The 16-amino acid region of the third α-helix of the homeodomain of Antennapedia has been shown to enable the protein (created 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 portion may include polypeptides having a basic amino acid-rich region covalently bonded to the activator portion (e.g., a fragment inhibitor peptide containing an intracellular domain). As used herein, the term “basic amino acid-rich region” refers to a region of protein with a high content of basic amino acids such as arginine, histidine, asparagine, glutamine, and lysine. A “basic amino acid-rich region” may, for example, contain 15% or more basic amino acids. In some cases, a “basic amino acid-rich region” may contain less than 15% basic amino acids but still function as a transporter region. In other examples, the basic amino acid region may contain 30% or more basic amino acids.

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

[0113] In one embodiment, the therapeutic peptides described herein can be non-covalently bound to a signaling medium. An example of a non-covalently bound polypeptide signaling medium is the Chariot protein delivery system (see U.S. Patent No. 6,841,535, J Biol. Chem. 274(35):24941-24946; and Nature Biotec. 19:1173-1176, all of which are incorporated herein by reference in their entirety).

[0114] In other embodiments, therapeutic peptides can be expressed in cells treated with gene therapy, thereby inhibiting LAR family signaling. Gene therapy can use vectors containing nucleotides encoding the therapeutic peptide. A “vector” (sometimes also called a “medium” for gene delivery or gene transport) refers to a macromolecule or molecular complex containing polynucleotides delivered to cells. The delivered polynucleotides may include the coding sequence in gene therapy. Examples of vectors include viral vectors (e.g., adenoviruses (Ad), adeno-associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can intervene in the delivery of polynucleotides to target cells.

[0115] Vectors may also contain other components or functional groups that further modulate gene delivery and / or gene expression, or provide beneficial properties to target cells. Such other components include components that affect cell binding or targeting (including components that mediate cell type or tissue-specific binding); components that affect the uptake of vector nucleic acids by cells; components that affect the localization of polynucleotides within cells after uptake (e.g., agents that mediate nuclear localization); and components that affect polynucleotide expression. Such components may also include markers, such as detectable and / or selectable markers, which can be used to select and detect cells that have taken up and expressed nucleic acids delivered by the vector. Such components may be provided as innate features of the vector (e.g., the use of components that mediate binding and uptake or specific viral vectors with functionality), or the vector may 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, including bifunctional (i.e., positive / negative) markers (see, for example, WO92 / 08796 by Lupton, S., published May 29, 1992; and WO94 / 28143 by Lupton, S., published December 8, 1994), have been described. Such marker genes can provide additional criteria for regulation, which can be advantageous in the context of gene therapy. A wide variety of such vectors are known in this art and are generally available.

[0117] Vectors for use herein include viral vectors, lipid vectors, and other nonviral vectors capable of delivering nucleotides encoding the therapeutic peptides described herein to target cells. The vectors may be targeted vectors, particularly those that preferentially bind 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 the therapeutic peptide.

[0118] Examples of viral vectors are derived from adenoviruses (Ad) or adeno-associated viruses (AAV). Both human and non-human viral vectors can be used, and recombinant viral vectors may be replication-deficient in humans. If the vector is an adenovirus, it may contain polynucleotides with promoters that are manipulably linked to a gene encoding a therapeutic peptide, and these are replication-deficient in humans.

[0119] Other viral vectors that can be used herein include vectors based on herpes simplex virus (HSV). HSV vectors deleting one or more pre-initial genes (IEs) are advantageous because they are generally non-toxic, survive in a latent-like state, and provide efficient transduction to target cells. Recombinant HSV vectors can incorporate approximately 30 kb of non-homologous nucleic acids.

[0120] Retroviruses such as type C retroviruses and lentiviruses may 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 vectors may contain non-homologous (therapeutic) DNA up to 5 kb instead of viral genes. The non-homologous DNA may contain nucleic acids encoding tissue-specific promoters and therapeutic peptides. In the method of delivery to nerve cells, it may also encode ligands for tissue-specific receptors.

[0121] Additional retroviral vectors that may be used include replication-deficient lentiviral vectors, including those based on 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 can infect both actively dividing and non-dividing cells.

[0122] Lentiviruses for use in this application may be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include a tissue-specific promoter that is manipulably ligated to a nucleic acid encoding a therapeutic peptide, as well as the nucleic acid sequence required for vector replication. These former may include the viral LTR, primer binding site, polypurine sequence, att site, and capsid formation site.

[0123] In some embodiments, lentiviral vectors can be employed. Lentiviruses have been shown to be transductionable into different types of CNS neurons (Azzouz et al., (2002) J Neurosci. 22: 10302-12), and their large cloning capacity makes them suitable for use in some embodiments.

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

[0125] Alphaviral vectors, such as those derived from Semliki Forest Fever virus (SFV) and Sindbis virus (SIN), may also be used in this application. The use of alphaviruses is described in Lundstrom, K., Intervirology, 43:247-257, 2000 and Perri et al., Journal of Virology, 74:9802-9807, 2000.

[0126] Recombinant replication-deficient alphavirus vectors are advantageous because they allow for high levels of non-homologous (therapeutic) gene expression and can infect a wide range of target cells. Alphavirus replicons can be targeted to specific cell types by displaying functional non-homologous ligands or binding domains on their virion surface, enabling selective binding to target cells expressing their homologous partners. Alphavirus replicons can establish latency, allowing for long-term expression of non-homologous nucleic acids in target cells. Replicons can also exhibit transient expression of non-homologous nucleic acids in target cells.

[0127] In many viral vectors conforming to the method of this application, more than one promoter can be introduced into the vector, enabling more than one non-homologous gene to be expressed by the vector. Furthermore, the vector may include sequences encoding signal peptides or other portions that promote the expression of therapeutic peptides from target cells.

[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, NY, or in numerous laboratory manuals discussing recombinant DNA techniques. Cells can be transduced using a double-stranded AAV genome in an adenovirus capsid containing a combination of AAV and adenovirus ITR. In other mutations, the AAV vector can be encapsulated in a “underpowered,” “helper-dependent,” or “high-capacity” adenovirus vector. 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 adenoviruses can be integrated into the genome of target cells, potentially achieving stable gene expression.

[0129] Further consideration should be given to other nucleotide sequence elements that facilitate the expression of therapeutic peptides and the cloning of vectors. For example, the presence of an enhancer upstream of the promoter or a terminator downstream of the coding region may, for instance, enhance expression.

[0130] According to another embodiment, a tissue-specific promoter can be fused to a nucleotide encoding a therapeutic peptide described herein. By fusing such a tissue-specific promoter within an adenovirus construct, the expression of the transgene is limited to a specific tissue. The recombinant adenovirus system of this application can be used to determine the degree of efficacy and specificity of gene expression provided by the tissue-specific promoter. 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 viral vector-based methods, non-viral methods can be used to introduce nucleic acids encoding therapeutic peptides into target cells. An overview of non-viral gene delivery methods is provided in Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. An example of a non-viral gene delivery method relating to this application involves using plasmid DNA to introduce nucleic acids encoding therapeutic peptides into target cells. Plasmid-based gene delivery methods are generally known in this art.

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

[0133] In addition, pre-formed cationic liposomes or cationic lipids can be mixed with plasmid DNA to create a complex for translocation into cells. Methods involved in the formation of cationic lipids are outlined in Felgner et al., Ann. NYAcad. Sci. 772:126-139, 1995, and Lasic and Templeton, Adv. Drug Delivery Rev. 20:221-266, 1996. Regarding gene delivery, DNA can also be bound to amphipathic cationic peptides (Fominaya et al., J. Gene Med. 2:455-464, 2000).

[0134] Methods comprising virus-based and non-virus-based components may be used in accordance with this application. For example, a plasmid based on Epstein-Barr virus (EBV) for therapeutic gene delivery is described by Cui et al., Gene Therapy, 8:1508-1513, 2001. Furthermore, a DNA / ligand / polycationic adduct conjugated to an adenovirus is described by Curiel et al., Nat. Immun. 13:141-164, 1994.

[0135] Furthermore, nucleic acids encoding therapeutic peptides can be introduced into target cells by transtransferring them using electroporation. Electroporation is a well-known method and can be used to facilitate transtransfer of plasmid DNA into cells.

[0136] If necessary, the vector encoding the expression of the therapeutic peptide can be delivered in vivo to target cells in the form of an injectable formulation containing a pharmaceutically acceptable carrier, such as physiological saline. Other pharmaceutical carriers, formulations, and preparations may also be used in accordance with this application.

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

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

[0139] A therapeutic dose is the amount capable of producing a medically desirable outcome in the treated animal or human. As is well known in medical technology, the dose for one animal or human depends on numerous factors, including the size of the subject, body surface area, age, the specific composition administered, sex, time and route of administration, general condition, and other drugs administered concurrently. Specific doses of proteins and nucleic acids can be readily determined by those skilled in the art using the experimental methods described below.

[0140] The therapeutic agents described herein may be further modified (e.g., chemically modified). Such modifications may be devised to facilitate molecular manipulation and purification, increase molecular solubility, facilitate administration, target desired sites, and increase or decrease half-life. Numerous such modifications are known in the art and can be applied by skilled practitioners.

[0141] In some embodiments, the therapeutic agents and pharmaceutical compositions comprising the therapeutic agents described herein may be delivered to neurons of the central nervous system (CNS) and / or the primary nervous system (PNS). Such neurons may be damaged or diseased. Alternatively, such neurons may be healthy and undamaged. Such neurons may be located at the site of injury or associated with injury. The neurons targeted for therapeutic administration, delivery / contact of the agents and compositions described herein would be those in which neuronal extension is considered to be beneficial to the subject. Such determination is within the capabilities of a skilled practitioner through a small amount of routine experimentation.

[0142] The therapeutic agents and therapeutic pharmaceutical compositions described herein may also be delivered to non-neuronal cells of the CNS and / or PNS, such as non-neuronal cells that provide support to nerve cells. Such cells include, but are not limited to, glial cells (e.g., astrocytes, oligodendritic cells, ependymal cells, radial glial cells in the CNS; and Schwann cells, satellite glial cells, and intestinal glial cells in the PNS).

[0143] In the therapeutic methods disclosed herein, a therapeutically effective amount of the therapeutic agent is administered to the subject. In one embodiment, the formulation containing the therapeutic agent is administered to the subject within a period of about 100 hours after the injury occurs, for example, within 24, 12, or 6 hours from the time of injury.

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

[0145] When therapeutic agents are delivered to a target, they can be administered by preferred routes, including, for example, orally (e.g., in capsules, suspensions, or tablets), systemically, or parenterally. Parenteral administration includes, for example, intramuscular, intravenous, intra-articular, intra-arterial, subarachnoid, subcutaneous, or intraperitoneal administration. The agents can also be administered orally, perdermally, topically, by inhalation (e.g., intratracheal, intranasal, oral inhalation, or nasal spray), or rectally. Administration can be topical or systemic, where indicated.

[0146] Both local and systemic administration are considered herein. Desirable features of local administration include achieving an effective local concentration of the therapeutic agent as well as avoiding adverse side effects that may arise from systemic administration of the therapeutic agent. In one embodiment, the therapeutic agent can be administered by introduction into the cerebrospinal fluid of the target. In certain embodiments, the therapeutic agent can be introduced into the ventricles, lumbar region, or cisterna magna. In other embodiments, the therapeutic agent can be introduced into the site of nerve or spinal cord injury, into the site of pain or neurodegeneration, or into the eye in contact with neuroretinal 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, the therapeutic agent may be administered subarachnoidally to the target area. As used herein, the term “subarachnoid administration” is intended to include direct delivery of the therapeutic agent to the cerebrospinal fluid of the target area by techniques including injection into the lateral ventricle via a burr hole, cisterna magna puncture, or lumbar puncture (as described in Lazorthes et al., 1991 and Ommaya, 1984, which are incorporated herein by reference). The term “lumbar region” is intended to include the region between the third and fourth lumbar vertebrae (lumbar). The term “cisterna magna” is intended to include the region where the skull ends at the occipital region and the spinal cord begins. The term “ventricle” is intended to include the cavity leading to the central canal of the spinal cord. Administration of the therapeutic agent to any of the above-mentioned sites can be achieved by direct injection of the therapeutic agent or by the use of an infusion pump. Implantable or external pumps or catheters may be used.

[0149] For infusion, therapeutic agents can be formulated in liquid solutions, typically physiologically compatible buffers such as Hanks' solution or Ringer's solution. In addition, therapeutic agents can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms are also included. Infusion can be, for example, in the form of a bolus injection of the therapeutic agent or a continuous infusion (e.g., using an infusion pump).

[0150] In one embodiment, the therapeutic agent can be administered by lateral ventricular injection into the target brain within typically 100 hours (e.g., within 6, 12, 24, or 100 hours from the time of injury) of the injury (resulting in a condition characterized by abnormal elongation of axons of neurons in the central nervous system). The injection can be administered through a trepanation hole made in the target skull. In another embodiment, the therapeutic agent can be administered by a surgically inserted shunt into the target ventricles within typically 100 hours (e.g., within 6, 12, or 24 hours from the time of injury). The injection can be administered to larger lateral ventricles, although injections into the third and fourth cerebellar ventricles may also be possible. In yet another embodiment, the therapeutic agent can be administered by injection into the cisterna magna or lumbar region of the target within 100 hours (e.g., within 6, 12, or 24 hours from the time of injury).

[0151] Additional means of administration to intracranial tissues include application to the olfactory epithelium, followed by transmission to the olfactory bulb and further transfer to nearby parts of the brain. Such administration is possible by atomized or sprayed formulations.

[0152] In another embodiment, the therapeutic agent may be administered to the subject at the site of injury within typically 100 hours of the injury (for example, within 6, 12, or 24 hours from the time of injury).

[0153] In further embodiments, ophthalmic compositions of therapeutic agents described herein are used to prevent or mitigate damage to retinal and optic nerve head tissues, as well as to improve functional recovery after damage to ophthalmic tissues. Ophthalmic conditions that can be treated include, but are not limited to, retinopathy (including diabetic retinopathy and posterior lens fibrosis), macular degeneration, ophthalmic ischemia, and glaucoma. Other conditions treated by the methods of the present invention include injuries related to damage to ophthalmic tissues, such as ischemic perfusion injury, photochemical injuries, and injuries related to ophthalmic surgery, particularly injuries to the retina or optic nerve head due to exposure to light or surgical instruments. The ophthalmic compositions may also be used as adjuncts to ophthalmic surgery, for example, by intravitreal or subconjunctival injection following ophthalmic surgery. The therapeutic agents may be used for the acute treatment of transient conditions, or chronically, particularly in the case of degenerative diseases. The ophthalmic compositions may 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 a target over a long period to induce optimal axonal elongation or sprouting and / or inhibit axonal regression. For example, sustained contact with the active compound can be achieved by repeated administration of the active compound over periods such as one week, several weeks, or more than one month. A pharmaceutically acceptable formulation used to administer the therapeutic agent can also be formulated to provide sustained delivery to the target. For example, the formulation may deliver the active compound for at least one, two, three, or four weeks following the initial administration to the target. For example, a target treated according to the present invention may be treated with the active compound for at least 30 days (by either repeated administration or the use of a sustained delivery system, or both).

[0155] Sustained delivery of a therapeutic agent can be demonstrated, for example, by the continuous therapeutic effect of the agent over a long period of time (for example, sustained delivery of a drug can be demonstrated by the continuous extension of axons of CNS neurons in the subject). Alternatively, sustained delivery of a therapeutic agent can be demonstrated by detecting the presence of the therapeutic agent in the body over a long period of time.

[0156] Approaches to sustained delivery include the use of polymer capsules, minipumps for delivering the formulation, biodegradable implants, or transgene-transplanted 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 (marketed by Alza Corporation) are commercially available and known in this technology. Another mode of administration is via an implantable, externally programmable infusion pump. Infusion pump systems and reservoir systems are also described in U.S. Patents No. 5,368,562 and No. 4,731,058.

[0157] Vectors encoding therapeutic peptides are often administered less frequently than other types of therapeutic agents. For example, the effective dose of such vectors ranges from approximately 0.01 mg / kg to approximately 5 or 10 g / kg and is administered daily, weekly, bi-weekly, monthly, or even less frequently.

[0158] The ability to deliver or express therapeutic peptides enables the regulation of cellular activity in numerous different cell types. These therapeutic peptides can be expressed, for example, in cardiac cells via a cardiac-specific promoter that modulates cardiac contraction (or excitability), in the spinal cord via the HB9 promoter that modulates motor neuron activity after spinal cord injury, and in neurons in brain regions affected by degenerative diseases 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 exposed to a therapeutic agent in vitro to promote axonal elongation. Thus, neurons can be isolated from a subject, propagated in vitro using techniques well known in this art, and then treated to regulate axonal elongation. Briefly, neuronal cultures can be obtained by allowing neurons to migrate from fragments of nerve tissue attached to a suitable substrate (e.g., a culture dish), or by mechanically or enzymatically dissociating the tissue to produce a suspension of neurons. For example, enzymes such as trypsin, collagenase, elastase, hyaluronidase, DNA-degrading enzymes, 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 edition, 1994). Such cells can then be exposed to the therapeutic agent in the amounts and for the durations described above. Once regulation of axonal elongation is achieved in neurons, these cells can be reintroduced to the target cells, for example, through transplantation.

[0160] The ability of an agent to promote nerve regeneration in a subject can be evaluated using any of the various known procedures and assays. For example, the ability of an agent to restore nerve connectivity and / or function after injury can be determined histologically (by preparing sections of neuronal tissue and observing neuronal branching or by showing cytoplasmic transport of pigments). An agent can also be evaluated by monitoring its ability to completely or partially restore the electroretinogram after injury to the neuroretina or optic nerve, or to completely or partially restore the pupillary reflex to light in the injured eye.

[0161] Other tests that may be used include standard tests of neurological function in human subjects or animal models of spinal cord injury (e.g., standard reflex tests, urinary tract tests, urodynamic tests, deep and superficial pain tests, hind limb proprioception tests, gait tests, and evoked potential tests). In addition, nerve impulse conduction can be measured in subjects, for example, by measuring conduction action potentials as an indicator of the production of beneficial neurological effects.

[0162] Animal models that can be used herein include partially treated rat models to investigate how well compounds can enhance the survival and sprouting of the remaining intact fragments of a nearly completely transversely severed spinal cord. Thus, after administration of the candidate agent, these animals can be evaluated for the recovery of specific functions, for example, how well the rat can manipulate a food pellet with its forelimb (the relevant spinal cord is 97% severed).

[0163] Another animal model that can be used in assays is a rat model of stroke. By administering agents to these animals, it is possible to evaluate whether a given compound, route of administration, or dosage provides a neurogenic effect, such as an increase in the level of function, an increase in the rate of function recovery, or an increase in the degree of function retention in the test animals.

[0164] The ability of agents to produce neurologically beneficial effects in subjects can be evaluated using standard neurological assessments used to assess progression in human patients after stroke. Such standard neurological assessments 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, therapeutic agents can be used to treat diseases, disorders, or conditions related to elements of the nervous system, including the central nervous system components, somatic components, autonomic components, sympathetic and parasympathetic components, sensory tissue in the eyes, ears, nose, mouth, or other organs, as well as glial tissue related to the cells and structures of neurons. Neurological disorders can be caused by damage to neurons, such as mechanical damage or damage by toxic compounds, by abnormal proliferation or development of neurons, or by mismodulation, such as downmodulation of neuronal activity. In one embodiment, the therapeutic agent can be applied to a damaged nerve, the site of nerve damage, or the site of nerve damage repair. In some embodiments, the therapeutic agent is applied to the site of primary nerve repair. Damage to a nerve may be equivalent to nerve transection (nerve rupture), in which the nerve is partially or completely severed, or a small area is damaged and surgically removed.

[0166] Neurological disorders can adversely affect the functions of the nervous system, for example, sensory functions (the ability to perceive changes inside and outside the body); integrative functions (the ability to interpret changes); and motor functions (the ability to respond to interpretations by initiating actions such as muscle contraction or granule secretion).

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

[0168] The therapeutic agents described herein also treat neuropsychiatric disorders such as depression, schizophrenia, schizoaffective disorder, Korsakoff psychosis, mania, anxiety or phobic disorders, learning or memory impairments (e.g., amnesia and age-related amnesia), hyperactivity syndrome, dysthymia, major depressive disorder, mania, obsessive-compulsive disorder, neuroactive substance use disorder, anxiety disorders, pathophobias, panic disorder, bipolar disorder, psychogenic pain syndrome, and eating disorders. Other examples of neurological disorders include damage to the nervous system due to infectious diseases (e.g., meningitis, high fever of various etiologies, HIV, syphilis, or post-polio syndrome) and damage to the nervous system due to electricity (including contact with electricity or lighting, and complications of conductive psychiatric therapy). Neurological disorders related to ophthalmic symptoms include damage to the retina and optic nerve, glaucoma, and age-related macular degeneration.

[0169] The developing brain, as well as during pregnancy, is a target for neurotoxicity in developing the central nervous system at many stages of infancy and early childhood, and the methods of the present invention may be used to prevent or treat neurological defects in embryos or fetuses in the womb, premature infants, or young children requiring such treatment, including those with birth defects of nerves. 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 herein may be used in methods to treat medical conditions associated with nerve damage. Medical conditions can include any motor disorders, epilepsy, cerebrovascular diseases, autoimmune diseases, sleep disorders, autonomic nervous system disorders, bladder disorders, abnormal metabolic conditions, muscular system disorders, infectious and parasitic diseases, tumors, endocrine disorders, nutritional and metabolic diseases, immune disorders, diseases of the blood and blood-forming organs, mental disorders, nervous system disorders, sensory organ disorders, circulatory system disorders, respiratory system disorders, digestive system disorders, genitourinary system disorders, skin and subcutaneous tissue disorders, musculoskeletal and connective tissue disorders, birth defects, certain conditions originating perinatal, and symptoms, signs and undefined conditions.

[0171] Treatable cerebrovascular disorders can result from conditions including, but are not limited to, aneurysms, strokes, arrhythmias, myocardial infarctions, ischemic perfusion injuries, and cerebral hemorrhages.

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

[0173] The sleep disorders treatable by this application may be caused by conditions including, but not limited to, sleep apnea and parasomnias.

[0174] Autonomic disorders treatable by this application may result from gastrointestinal disorders, autonomic dysfunction, excessive epiphoresis, excessive rhinorrhea, and cardiovascular diseases, including but not limited to cardiac pulse failure and arrhythmias, hypertension, and carotid sinus disease.

[0175] Bladder disorders treatable by this application may result from conditions including, but not limited to, spinal cord injury and spastic or flaccid bladder.

[0176] Abnormal metabolic conditions treatable by this application may be caused by conditions including, but not limited to, hyperthyroidism or hypothyroidism. Muscular disorders treatable by this application may include, but are not limited to, muscular dystrophy and upper respiratory tract and facial spasms.

[0177] With therapeutic agents, migraines with aura, migraines without aura, menstrual migraines, migraine deformities, variant migraines, migraines with complications, hemiplegic migraines, transforming migraines, chronic daily migraines, transient tension headaches, analgesic rebound headaches, transient cluster headaches, chronic cluster headaches, cluster deformities, chronic paroxysmal migraines, persistent hemiplegia, post-traumatic headaches, post-traumatic neck pain, post-herpetic neuralgia including the head and face, pain resulting from spinal fractures associated with osteoporosis, spinal arthritis pain, pain associated with cerebrovascular disorders and strokes, headaches due to vascular disorders, reflex sympathetic dystrophy, anterior neck pain (which can result from a variety of causes including, but not limited to, arthritis, positional, muscular, intervertebral disc-derived, or degenerative causes including displacement), glossodynia, carotid artery tenderness, cricoid cartilage pain We can also treat neuropathic pain resulting from conditions including, but not limited to, pain, earache due to middle ear lesions, stomachache, sciatica, maxillary neuralgia, laryngosoreness, neck muscle pain, trigeminal neuralgia (sometimes called painful tics), headache after lumbar puncture, headache due to low-pressure cerebrospinal fluid, temporomandibular joint disorders, heterofacial pain, ciliary neuralgia, paratrigeminal neuralgia (sometimes called Leaders syndrome), pyramidal neuralgia, Eagle syndrome, idiopathic intracranial hypertension, orofacial pain, myopoulosplenic pain syndrome including the head, neck and shoulders, cluster headache, cervical headache, paratrigeminal nerve palsy, SPG neuralgia (sometimes also called lower hemigraine, lower facial pain syndrome, Sulder's neuralgia, and Sulder's syndrome), carotid artery tenderness, Bidian's neuralgia, burning pain, and / or combinations of the above.

[0178] As used herein, the term “headache” includes migraine, tension headache, cluster headache, trigeminal neuralgia, secondary headache, tension-type headache, chronic and transient headache, drug-induced headache / rebound headache, chronic paroxysmal migraine, persistent hemiparoxysmal headache, post-traumatic headache, post-herpes headache, vascular headache, reflex sympathetic dystrophy-related headache, cervical headache, internal carotid artery aneurysm headache, sciatica headache, trigeminal neuralgia headache, occipital headache, maxillary headache, drowsy headache, paratrigeminal neuralgia headache, pyramidal headache, Sulder's headache, and Bidian's headache. This can refer to low-CSF pressure headache, TMJ headache, burning headache, pleuritic headache, all primary headaches (e.g., primary stabbing headache, primary cough headache, primary exertional headache, primary headache associated with sexual activity, sleep headache, and new daily persistent headache), trigeminal autonomic headache (e.g., transient paroxysmal hemicalcemia, SUNCT, all possible TACs, and SUNA), chronic daily headache, occipital neuralgia, heterofacial pain, neuropathic trigeminal neuralgia, and other types of headache.

[0179] In further embodiments, the therapeutic agent can be used to promote the viability, plasticity, and / or proliferation of stem cells or progenitor cells. The stem cells may include neural stem cells or progenitor cells that express LAR phosphatase receptors. The therapeutic agent can be administered to stem cells or progenitor cells in vitro, in vitro, or in vivo. When administered to stem cells or progenitor cells in vitro or in vitro, the stem cells or progenitor cells can be transplanted for therapeutic application.

[0180] For neural stem cells / progenitor cells, methods commonly used in the field of regenerative medicine for transplanting neural stem cells / progenitor cells into a desired area may be employed in conjunction with the administration of therapeutic agents to the cells or area. More specifically, for example, a method can be exemplified in which neural stem cells / progenitor cells are suspended in phosphate-buffered saline together with a therapeutic agent, and the resulting cell suspension is added to / injected into the area to transplant the neural stem cells / progenitor cells.

[0181] In other embodiments, the therapeutic agents described herein can be applied to nerve grafts. Examples of grafts include tissues intended for implantation in humans or animals. Various types of grafts, such as autologous grafts, syngeneic grafts, allogeneic grafts, and xenografts, are considered 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 range from about 1 centimeter to about 10 centimeters or exceed about 10 centimeters. The diameter of the nerve graft can be matched to the diameter of the damaged nerve or portion of the nerve as needed. A nerve graft can be a structurally complete nerve fragment to bridge a gap along the length of the recipient nerve or to replace its distal end, i.e., for interterminal grafting. Alternatively, a nerve graft can be a partial nerve fragment or an eccentrically shaped fragment (e.g., a nerve flap) intended to reconstruct a torn nerve that has some structural damage but retains its physical continuity.

[0182] When the 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 one go. This one-go treatment can be applied to raw (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 the therapeutic agent is applied to the damaged nerve, for example, the agent can be applied to the area of ​​the damaged nerve that promotes the repair of the damaged nerve, either at or adjacent to the site of injury.

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

[0184] In other embodiments, spinal cord injuries such as unilateral C2 transection are known to result in elevated inhibitory proteoglycans at the level of the diaphragmatic motor nucleus in the extracellular matrix and pericellular network ipsilateral to the unilateral transection, but distal to the spinal cord lesion. Treatment with chondroitinase ABC (chABC) strongly inhibits the degradation of these matrix molecules, as discussed in U.S. Patent Application 10 / 754,102 incorporated herein by reference.

[0185] It is considered herein that enzymatic modification (via chondroitinase (chABC)) of the inhibitory extracellular matrix in motor neurons surrounding the PNN in conjunction with the administration of therapeutic agents can maximize the sprouting ability and functional effects of the remaining nerve fibers. It is further considered that enhancing and / or resulting in the total sprouting of a much larger number of fibers, combined with increasing the physiological output of the neurons themselves, may act synergistically to improve spinal cord injury. Thus, in another embodiment, the subject may be administered chondroitinase ABC in addition to the therapeutic agents described herein, resulting in 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 may further include administering or contacting cells with an agent that blocks regeneration inhibitors, such as a compound that inhibits myelin-derived blockade of neurogenesis. Known inhibitors of neuronal extension (e.g., regeneration at CNS injury sites) include myelin-derived inhibitors (e.g., Nogo-A, MAG, OMgp, Ehprin B3, Sema4D, and Sema5A), astrocyte-derived inhibitors (e.g., CSPG, KSPG, Ephrin B2, and Slit), and fibroblast-derived inhibitors (e.g., Sema3A). A second agent may be an antagonist to any of these inhibitors. In one embodiment, cells are further contacted with one or more such agents. In one embodiment, the agent inhibits myelin inhibitors of nerve regeneration (e.g., myelin-related glycoproteins (MAG), Nogo, and oligodendritic cell myelin glycoprotein (OMgp)). MAG inhibitors are disclosed in U.S. Patent No. 5,932,542. Nogo inhibitors are disclosed in U.S. Patent Application Publication No. 2009 / 215691. OMgp inhibitors are disclosed in U.S. Patent Application Publication No. 2008 / 188411. Cells can be exposed to the agent that inhibits the interaction of CSPG with PTPσ before, after, and / or simultaneously with this agent.

[0187] In some embodiments, cells may be exposed to agents that activate neuronal proliferation pathways (e.g., the CNS). Some of these agents include, but are not limited to, neurotrophic factors such as inosine, mannose, growth, or glucose-6-phosphate, as described in 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 in TGF#946 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 neuronal elongation further comprises exposing neurons (e.g., CNS) to a cAMP modulator and / or polyamine that increases the concentration of intracellular cAMP (e.g., cAMP) (Cai et al., 35 Neuron 711 (2002)). For example, the ability of mature rat ganglion cells to respond to mannose requires elevated cAMP levels (Li et al., 2003).

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

[0189] This example demonstrates that long-term exposure to CSPG disrupts, stabilizes, and hyperattaches growth cones. In this example, the spot assay used to measure CSPG induced stabilization in mature sensory neurons. method

[0190] Sensory dorsal root ganglion (DRG) neurons from mature female rats were obtained and grown on a gradient of chondroitin sulfate proteoglycan agrecan as previously described. Delta T stop-motion dishes with a glass bottom were coated with poly-L-lysine (PLL) and incubated overnight at room temperature. Spots were created by dissolving 2 mg / ml of agrecan in Hanks equilibrium salt solution (HBSS) without calcium and magnesium. Eight 2 μl dots were placed on half of each dish and allowed to dry on the glass with ample time. Finally, the dishes were coated with 10 μg / ml laminin at 37°C for 3 hours. After incubation, 6000 mature dissociated DRG neurons were added to each dish in neurobasal medium A supplemented with Glutamax, Penn / strep, and B27. Cells were grown for 4–6 days.

[0191] Immediately before starting the stop-motion animation, the Delta T dish was sealed with a glass coverslip. While warming the object and sample stage to maintain the cells at 37°C, 100x microscope stop-motion animation was performed. Images were acquired every 30 seconds and closed together to create a stop-motion film. The dynamics of the growth cone and filopopods were tracked and manually quantified using Metamorph. result

[0192] Figures 1 and 2 (A-D) show that mature sensory neurons exposed to a CSPG agrecan gradient stabilize in specific areas of the spot's edge and excessively adhere to the substrate, leading to growth cone formation, filopopod elongation, 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 demonstrates that LAR expression is higher in stabilized growth cones than in motile cones. In this example, a spot assay was performed on glass coverslips, with a slight modification to the technique used in microscopy time-lapse. After PLL treatment, the coverslips were dried and coated with a small amount of nitrocellulose to enhance the adhesion interactions necessary for spot formation. After drying the nitrocellulose, four spots were created on each coverslip (one set of four) using 700 μg / ml agrecan and 5 μg / ml laminin dissolved in HBSS. After drying, the coverslips were coated with 5 μg / ml laminin at 37°C for 3 hours. 2,000 isolated mature dorsal root ganglion neurons were added to each coverslip in neurobasal medium A supplemented with Glutamax, Penn / strep, and B27. In addition, peptides were added at the concentration required for the time to be seeded on the plate. After growing the cells for 5 days, they were fixed with 4% paraformaldehyde.

[0194] Slides were fixed and stained with goat anti-PTPσ (1:100, R&D Systems) and mouse anti-B3 tubulin (1:500, Invitrogen). Axons and growth cones were imaged at 100x magnification. For each dystrophic and non-dystrophic neuron, the density of PTPσ in the growth cone and axonal segment was analyzed using ImageJ (N=40).

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

[0196] As schematically illustrated in Figure 4, the leukocyte common antigen-associated (LAR) family of phosphatases consists of three members: LAR itself, the receptor protein tyrosine phosphatase sigma (RPTPσ), and the receptor protein tyrosine phosphatase delta (RPTPδ). Recent studies have shown binding interactions between LAR or RPTPσ and the sugar side chain of chondroitin sulfate proteoglycan (CSPG), a molecule highly inhibitory to nerve growth, plasticity, and regeneration. Furthermore, crystallographic and sequence analyses 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 helical / loop / helical motif in the first intracellular catalytic domain that mediates homogeneous / heterogeneous receptor interactions. Using a peptide mimetic of this wedge-shaped domain tagged to a cytoplasmic TAT sequence, we successfully eliminated LAR activity in the neurotrophic factor signaling paradigm. We used NIH's BLAST to identify the orthologous sequences of RPTPσ and RPTPδ, and designed wedge-shaped domain peptides for each target. The peptides were newly created intracellular LAR-blocking peptides (ILP), intracellular sigma-blocking peptides (ISP), and intracellular delta-blocking peptides (IDP). Interestingly, the high conservation of this domain in higher vertebrates indicates its functional importance.

[0197] Wedge-shaped PTPσ in rats and mice: DMAEHMERLKANDSLKLSQEYESI (SEQ ID NOs: 20 and 21)

[0198] The wedge shape of human PTPσ: DMAETMERLKANDSLKLSQEYESI (Sequence ID 33)

[0199] Peptides as tags HIV-TAT It is conjugated to create a functional blockade peptide. HIV-TAT

[0200] NH2 GRKKRRQRRRC DMAEHMERLKANDSLKLSQEYESI-NH2PTPσ mouse / rat (SEQ ID NO: 53)

[0201] NH2 GRKKRRQRRRC DMAETMERLKANDSLKLSQEYESI-NH2PTPσ Human (Sequence ID 54)

[0202] NH2 GRKKRRQRRRC DLADNIERLKANDGLKFSQEYESI-NH S LAR (Sequence ID 55)

[0203] NH2 GRKKRRQRRRCE LADHIERLKANDNLKFSQEYESI-NH2PTP 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 culture medium when seeding neurons into plates.

[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 nerve cell bodies at each individual spot. result

[0207] Figures 5–7 show that the wedge-shaped domain peptide of PTPσ(ISP) enables neurons to extend processes across the CSPG gradient. Treatment was dose-dependent, with the optimal transposition being 2.5 μm. In addition, LAR peptide (ILP) also enabled neurons to transpose the CSPG (Figure 6). Neither the solvent control nor the mixed ISP / TAT allowed neurons to extend across the gradient. Time-lapse photography showed that ISP treatment stabilized axons and prevented excessive attachment, allowing growth cones to extend filopopods and maintain motility. 65% of the analyzed growth cones remained motile after 4–6 days in vitro, in contrast to 8% under control conditions. Example 4

[0208] This example describes a method for generating moderate / severe spinal cord injury (SCI) by Infinite Horizon device in mature female Sprague-Dolly rats for spinal cord injury (SCI) assays used in Examples 5-8. Briefly, rats were deeply anesthetized with a ketamine / xylazine cocktail. Immediately under anesthesia, the backs were shaved and disinfected with iodine and ethanol. Entering from the dorsal side, the 7th-10th lumbar vertebrae were exposed by skin incision, and laminectomy was performed at the 8th and 9th segments to expose the intact spinal cord. The vertebral column and spinal cord were fixed in a positional frame prior to the blunt impact. Finally, the rats received a 250kd Infinite Horizon contusion aligned to the midline without dwell time. After suturing the muscles and closing the skin, the animals were placed on a 37°C hot pad and given free food and water upon awakening from surgery. Pain was monitored, and animals in pain were given a low dose of marcaine at the injection site. Saline solution and gentamicin (antibiotic) were administered for 5 days post-surgery to prevent bladder infection. This experiment was performed three 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 day 1 post-injury and continuing for 7 weeks, each animal received a daily subcutaneous administration of either the solvent, 5 μM ISP, or 5 μM ILP on the back of the lesion (49 treatments, 11 μg / rat / day). Example 5

[0210] This example demonstrates hindlimb movement and spontaneous movement patterns according to the Basso-Beaty-Bresnahan scale for spontaneous movement after SCI in solvent-treated SCI animals and LAR peptide-treated SCI animals. method

[0211] Animals were allowed to move freely on a table for 3 minutes, while their hind limb movements and spontaneous movement patterns were scored by blinded observers according to the Basso-Beaty-Bresnahan scale for spontaneous movement after SCI (Basso et al, 1995). Behavioral examinations were performed weekly on days 1, 4, and 7 after injury, and then weekly for a further 10 weeks. Statistical analysis was performed using two-way repeated measures ANOVA.

[0212] Figures 8 and 9 show that, following the initial period of spinal cord impact, all treatment groups recovered basic hindlimb movement by two weeks post-injury, with an average BBB score of 9 (supporting weight on the hindlimbs without stomping). Over the next 10 weeks, both solvent-treated and LAR peptide-treated animals recovered only slightly beyond this point, on average regaining the ability to walk with occasional weight bearing. On average, ISP-treated animals continued to recover, reaching a score of 12 at week 6 (consistent stomping with occasional hind-forelimb coordination) and exceeding 13 by week 7 (between frequent and consistent coordination). Individually, one animal reached a score of 19, which was nearly perfect spontaneous movement with a raised tail, consistent toe clearance in stomping, and correct foot placement. Additional animals reached near-normal scores of 18.5 and 18. Seven of the 15 animals regained at least frequent coordinated stomping. Example 6

[0213] This example shows the results of lattice walking tests on solvent-treated SCI animals and LAR peptide-treated SCI animals. method

[0214] 12 weeks after spinal cord injury, wire grid (100cm x 75cm, 1cm wire) 2 Animals were allowed to roam freely on a grid (gap). An overhead camera tracked their movements and calculated the total distance traveled (Ethovision), while a blinded observer manually measured the number of missteps. The data is expressed as the total number of left and right missteps per meter traveled. The grid walking test was performed only once to prevent the animals from training and artificially improving (rehabilitation phenomenon). result

[0215] The sensorimotor coordination and balance recovery were measured using the grid walking test. Figures 10 and 11 show that solvent-treated animals averaged 6 missteps per meter moved on the grid. ILP treatment resulted in a very modest, non-significant improvement in missteps in the grid walking test. ISP treatment resulted in significantly fewer missteps on average than solvent-treated and ILP-treated animals. In addition, the fact that several animals misstepped less than 3 times suggests a near-complete recovery of this behavior. Example 7

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

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

[0218] In the final experiment, animals were anesthetized with urethane 14 weeks after injury. This anesthesia prevented excessive movement while protecting the bladder reflex. A catheter was inserted into the bladder via the urethra to allow slow perfusion with saline solution. 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 in metabolic cages. Animals were placed in metabolic cages where urination was measured by force transducers during dark cycles at 4, 8, and 12 weeks post-injury. Figure 12 shows that no significant recovery was observed on average at 4 or 8 weeks, but ISP led to a significant increase in urination frequency 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 frequency, 1 time every 2 hours, with a significantly increased volume / urination. ISP treatment significantly increased post-injury urination frequency by up to 2 times on average, and several animals reached normal (untreated) urination frequency levels.

[0220] To determine whether the animals had complete control over the contractility of the bladder muscles and sphincter, they underwent terminal uromechanical analysis 14 weeks post-injury. Under uromechanical and slow saline perfusion into the bladder, untreated animals contracted their bladder muscles, resulting in a sharp rise in bladder pressure (see trace above). The decrease in pressure corresponds to the release of pressure from the external urethral sphincter, aiding urination in the animals. Both of these behaviors are completely lost after spinal cord injury, in which case the gentle rise in bladder pressure eventually reaches a maximum value, and saline leaks out. The external urethral sphincter occasionally releases pressure, but this does not correlate with the contraction of the detrusor muscle of the bladder, which leads to inadequate urination. Figure 13(A-B) shows that after ISP treatment, many animals regained patterned external urethral sphincter release (marked by red arrows) and bladder contraction in harmony.

[0221] Superior recovery in solvent-treated animals was defined as being better than twice the standard deviation of the solvent mean (urination and BBB). Animals were placed in each group. Of the 15 animals, 13 showed significant behavioral improvement, and 4 were within a range of significant function in 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] The animals were perfused transcardially with 4% paraformaldehyde, and the vertebral column was resected. One additional day after PFA, the vertebral column was removed and cryoprotected with 30% sucrose for 3–7 days. The corresponding segments L1–L3 were embedded, and 20 μm transverse sections were placed on slides.

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

[0225] For axon density analysis, the gray matter contour was drawn, and the average pixel intensity was calculated using ImageJ. Since minimal staining was observed in the posterior column of the spinal cord, the pixel intensity of this region was subtracted as the internal background for each individual section. Once sections were randomly selected, they were analyzed every 200 μm over a total distance of 2 cm (10 segments). The highest and lowest pixel intensities were excluded, and the remaining 8 were averaged. result

[0226] 5HT is a key neurotransmitter in the spinal cord whose role is to control motor network acquisition and excitability. When used in combination with other therapies, 5HT agonists can significantly enhance motor behavior after spinal cord injury. We stained for 5HT expression in the lumbar spinal cord and several segments below the level of injury. The lumbar spinal cord contains pacemakers for hindlimb spontaneous movement and bladder control, and motor neurons for the muscles of the feet and bladder. Figure 14 shows that in untreated animals, 5HT expression or axonal density was very high in the left and right gray matter with a uniform staining pattern. At high magnification, fibers penetrating the white matter can be seen. Fourteen weeks after spinal cord injury, 5HT expression was significantly lower in solvent-control animals, with only two or three small patches remaining in the gray matter. ISP treatment resulted in a dramatic increase in 5HT staining throughout the gray matter. The staining was remarkably robust and varied greatly from section to section with heterogeneous patches scattered throughout the gray matter. This pattern suggests the sprouting and / or regeneration of 5HT in the evaded motor output center. Since the two ISP non-responders did not show a dramatic increase in 5HT expression, the increase in 5HT expression correlated well with behavioral recovery. Quantitative analysis again showed a significant increase in 5HT expression throughout the gray matter (Figure 15). Example 9

[0227] We identified several proteins and pathways acting downstream of the LAR family outside of phosphate activity (Figure 4). Of these, Caskin (Ckn) and LAR-interacting protein α (lipin-α) play important roles in both synapse formation and axonal guidance. In the yeast 2-hybrid interaction system, mCkn1 directly binds mLAR and mPTPRδ, while mCkn2 directly binds mLAR and mPTPσ. The interaction of Ckn with LAR family phosphatases was mapped to regions containing two sterile α motif (SAM) domains that constitute the phenotype. We created homology maps for Ckn in Drosophila, mouse, rat, and human using BLAST, which aligns the protein acceptance sequences 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 that we hypothesize acts as a small molecule competitor to LAR family phosphatase / Ckn binding and moves downstream in LAR family signaling. Work in the Drosophila system identifies the C-terminal region of dCkn as necessary for downstream signaling events. We designed 20-amino acid peptides for both mCkn1 and mCkn2, which are targets but may also act as small molecule competitors downstream of the targets. [Table 3]

[0229] Lipin family members play crucial roles in synaptic development and maintenance (we hypothesize that lipin-α may act in downstream signaling of LAR phosphatases). Yeast 2-hybrid interaction screening has linked the first SAM domain of lipin-α family members as a binding domain 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 lipin-α family (Table 4). It is intriguing that the four orthologues of lipin-α have retained this identical region throughout evolution, suggesting its functional importance. We hypothesize that this small molecule competitor can disrupt LAR / lipin-α1-4 interactions and thus disrupt downstream signaling of LAR phosphatase family members. [Table 4]

[0230] While the present invention has been specifically shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications can be made therein in form and detail 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 incorporated herein by reference in their entirety.

[0231] This application also encompasses the following inventions. (1) A method for inhibiting and / or reducing the activity, signaling, and / or function of leukocyte-common antigen-associated (LAR) family phosphatases induced by proteoglycans in target cells, A method comprising administering to cells a therapeutic agent that inhibits one or more of the catalytic activity, signal transduction, and function of LAR family phosphatases without inhibiting the binding of proteoglycans to or the activation thereof of LAR family phosphatases. (2) The method according to (1), wherein the LAR family phosphatase is the 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-shaped domain of PTPσ. (3) The method according to (1), wherein the LAR family phosphatase is the 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-shaped domain of PTPσ. (4) The method according to (1), wherein the LAR family phosphatase is the 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 approximately 85% homologous to approximately 10 to approximately 20 consecutive amino acids of the wedge-shaped domain of PTPσ. (5) The method according to (1), wherein the LAR family phosphatase is the 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% homologous to about 10 to about 20 consecutive amino acids of the wedge-shaped domain of PTPσ. (6) The method according to (1), wherein the LAR family phosphatase is the 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 according to (1), wherein the LAR family phosphatase is the receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide consisting of SEQ ID NO: 37. (8) The method according to (1), wherein the LAR family phosphatase is the receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide which is at least about 65% homologous to SEQ ID NO: 37. (9) The method of (8) wherein the therapeutic peptide comprises a conservative substitution of at least one amino acid in residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37. (10) (2) to (8) any method wherein the cells are nerve cells, glial cells, glial progenitor cells, or neural progenitor cells. (11) A method (2) to (8) wherein the therapeutic agent is linked to a therapeutic peptide and includes a transport portion that facilitates the uptake of the therapeutic peptide by cells. (12) The method in which the transport portion is the HIV Tat transport portion (11). (13) A method in which cells are present in the target to be treated, and the therapeutic agent is administered systemically to the target to be treated (11). (14) A method in which cells are the target of treatment, and the therapeutic agent is administered locally to the cells (11). (15) One of the following methods (2) to (8) is used to express the therapeutic agent in cells. (16) A method for treating diseases, disorders and / or conditions related to the activation and signaling of LAR family phosphatases, A method comprising administering a therapeutic agent to target cells expressing LAR family phosphatases, which inhibits one or more of the catalytic activity, signaling, and / or function of LAR family phosphatases without inhibiting the binding of proteoglycans to or activation thereof of LAR family phosphatases. (17) (16) A method in which a disease, disorder and / or condition comprises at least one disease, disorder and / or condition of the nervous system. (18) A method of (17) relating to a disease, disorder and / or condition of the nervous system comprising at least one of neuropathy, neuropsychiatric disorder, nerve injury, neurotoxic disorder, neuropathic pain, and neurodegenerative disorder. (19) A method of neurological disorders comprising at least one of peripheral nerves or cranial nerves, spinal cord or brain, traumatic or toxic injury to cranial nerves, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury (18). (20) A method wherein the neuropathy includes at least one of Alzheimer's disease, Alzheimer's disease-related dementia, Parkinson's disease, diffuse Lewy body dementia, senile dementia, Huntington's disease, Tourette syndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditary motor and sensory neuropathy, diabetic neuropathy, progressive supranuclear palsy, epilepsy, or Jacob-Creutzfeldt disease (18). (twenty one) (18) The method by which nerve damage is caused by or related to at least one of epilepsy, cerebrovascular disease, autoimmune disease, sleep disorder, autonomic nervous system disorder, bladder disorder, abnormal metabolic state, muscular system disorder, infectious and parasitic disease, tumor, endocrine disorder, nutritional and metabolic disorder, immune disorder, blood and hematopoietic organ disorder, mental disorder, nervous system disorder, sensory organ disorder, circulatory system disorder, respiratory system disorder, digestive system disorder, genitourinary system disorder, skin and subcutaneous tissue disorder, musculoskeletal and connective tissue disorder, congenital anomaly, or perinatal condition. (twenty two) The method is characterized in that the LAR family phosphatase is the 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-shaped domain of PTPσ (18). (twenty three) The method wherein the LAR family phosphatase is the 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-shaped domain of PTPσ (18). (twenty four) The method wherein the LAR family phosphatase is the 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 approximately 85% homologous to approximately 10 to approximately 20 consecutive amino acids of the wedge-shaped domain of PTPσ (18). (twenty five) The method is characterized in that the LAR family phosphatase is the 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% homologous to about 10 to about 20 consecutive amino acids of the wedge-shaped domain of PTPσ (18). (26) A method (18) in which the LAR family phosphatase is the 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) A method (18) in which the LAR family phosphatase is the receptor protein tyrosine phosphatase sigma (PTPσ), and the therapeutic agent comprises a therapeutic peptide consisting of SEQ ID NO: 37. (28) A method comprising a therapeutic peptide (18) in which the LAR family phosphatase is the receptor protein tyrosine phosphatase sigma (PTPσ) and the therapeutic agent is at least about 65% homologous to SEQ ID NO: 37. (29) A method (28) in which the therapeutic peptide comprises a conservative substitution of at least one amino acid in residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37. (30) Any method (22) to (29) wherein the cells are nerve cells, glial cells, glial progenitor cells, or neural progenitor cells. (31) A method (22) to (29) wherein the therapeutic agent is linked to a therapeutic peptide and includes a transport portion that facilitates the uptake of the therapeutic peptide by cells. (32) The method in which the transport portion is the HIV Tat transport portion (31). (33) A method in which the therapeutic agent is administered systemically to the subject being treated (31). (34) A method in which the therapeutic agent is administered locally to cells (31). (35) The therapeutic agent is expressed in cells by one of the following methods (22) to (19). (36) A method for treating nerve damage in the subject, A method comprising administering a therapeutic agent to damaged nerve cells of a target, which inhibits one or more of the catalytic activity, signaling, and / or function of LAR family phosphatases without inhibiting the binding of proteoglycans to or the activation thereof. (37) A method of nerve injury comprising at least one of peripheral nerves or cranial nerves, spinal cord or brain, traumatic or toxic injury to cranial nerves, traumatic brain injury, stroke, cerebral aneurysm, and spinal cord injury (36). (38) A method comprising a therapeutic agent comprising a therapeutic peptide, wherein 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-shaped domain of PTPσ (36). (39) A method comprising a therapeutic agent comprising a therapeutic peptide, wherein 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-shaped domain of PTPσ (36). (40) A method comprising a therapeutic agent comprising a therapeutic peptide, wherein the therapeutic peptide has an amino acid sequence that is at least approximately 85% homologous to approximately 10 to approximately 20 consecutive amino acids of the wedge-shaped domain of PTPσ (36). (41) A method comprising a therapeutic agent comprising a therapeutic peptide, wherein the therapeutic peptide has an amino acid sequence that is at least about 95% homologous to about 10 to about 20 consecutive amino acids of the wedge-shaped domain of PTPσ (36). (42) A method (36) comprising a therapeutic agent comprising a therapeutic peptide selected from the group consisting of SEQ ID NOs: 9 to 33. (43) A method (36) comprising a therapeutic agent comprising a therapeutic peptide consisting of Sequence ID No. 37. (44) A method (36) comprising a therapeutic peptide in which the therapeutic agent is at least about 65% homologous to SEQ ID NO: 37. (45) The 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) The method according to any one of (38)-(45), wherein the therapeutic agent is linked to the therapeutic peptide and comprises a transport moiety that facilitates the uptake of the therapeutic peptide by cells. (47) The method according to (36), wherein the transport moiety is the HIV Tat transport moiety. (48) The method according to (36), wherein the therapeutic agent is administered systemically to the subject to be treated. (49) The method according to (36), wherein the therapeutic agent is administered locally to cells. (50) The method according to any one of (38)-(45), wherein the therapeutic agent is expressed in cells. (51) A therapeutic agent that promotes at least one of neuronal cell 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: 9-33. (52) The therapeutic agent according to (51), further comprising a transport moiety that is linked to the therapeutic peptide and facilitates the uptake of the therapeutic peptide by cells. (53) The therapeutic agent according to (52), wherein the transport moiety is the HIV Tat transport moiety. (54) A therapeutic agent that promotes at least one of neuronal cell 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) The 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) The therapeutic agent according to (54), wherein the therapeutic peptide comprises SEQ ID NO: 37. (57) A therapeutic agent comprising (54)-(56) a transport portion linked to a therapeutic peptide to facilitate the uptake of the therapeutic peptide by cells. (58) A therapeutic agent whose transport portion is the HIV TAT transport portion (57). (59) A pharmaceutical composition, A pharmaceutical composition comprising a therapeutic agent including a synthetic therapeutic peptide which is at least approximately 65% ​​homologous to SEQ ID NO: 37, and a transport portion linked to the therapeutic peptide to facilitate the uptake of the therapeutic peptide by cells. (60) A pharmaceutical composition comprising a therapeutic peptide comprising a conservative substitution of at least one amino acid in residues 4, 5, 6, 7, 9, 10, 12, or 13 of SEQ ID NO: 37 (59). (61) A pharmaceutical composition comprising a therapeutic peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-33 and 37 (59). (62) A pharmaceutical composition in which the transport portion is the HIV Tat transport portion (59). (63) A pharmaceutical composition in which the therapeutic agent is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-66 and 70 (59).

Claims

1. A composition for treating nerve injury or neurological disorder, comprising a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33, and a transport portion linked to the peptide to facilitate the uptake of the peptide by cells.

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

3. The composition according to claim 1 or 2, wherein the transport portion is linked to the peptide by a peptide linker.

4. The composition according to any one of claims 1 to 3, wherein the linked peptide and the transport portion have an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 66.

Images