Methods and compositions for inducing neural plasticity

Abstract

A method for promoting compensatory plasticity of spared neural cells after neural injury, comprising contacting the spared neural cells with an effective amount of a therapeutic agent comprising a therapeutic peptide, wherein the therapeutic peptide comprises an amino acid sequence having at least 70% identity to SEQ ID NO:32.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 727,420, filed on September 5, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Notes on sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated into this specification by reference. The name of the text file containing the sequence listing is CWR026868WOORD.txt. The text file is 24KB; created on August 28, 2019; and submitted via EFS-Web with the submission of this specification. Background Art

[0005] Neurological damage results in dysfunction or death of neural tissue, manifesting as a variety of symptoms and effects. The damage may be due to external events, such as traumatic brain injury or ischemic conditions, internal events, such as stroke, aneurysm, cerebral hemorrhage, thrombosis or embolism, or more chronic neurodegenerative diseases, such as multiple sclerosis.

[0006] As an illustrative example, a stroke occurs when there is an interruption in blood flow to the brain, resulting in death of neural tissue and focal neurological deficits. Signs and symptoms may vary depending on the location and extent of the stroke. There are almost 800,000 strokes of all types each year in the United States, and ischemic strokes account for approximately 80% of these strokes. Roger et al. (2011) Circulation 123(4):e18-e209. In Europe, the estimated annual stroke events exceed 1.1 million, of which a similar percentage of approximately 80% are ischemic strokes. Heuschmann et al. (2009) Stroke 40(5):1557-1563.

[0007] Guidelines for the evaluation and treatment of patients with acute stroke focus on reperfusion therapies and factors that may worsen stroke or complicate the clinical course. The diagnosis of acute ischemic stroke is made by a combination of history and physical examination consistent with focal ischemia and resulting neurologic deficits. Brain imaging with computed tomography (CT) or magnetic resonance imaging (MRI) is used to exclude hemorrhage and other focal pathology and to document early signs of ischemia.

[0008] Stroke can also be considered a chronic disease. Manifestations of chronic stroke disease include cognitive deficits, dysphagia, and gait disturbances, which may acutely worsen in the form of decompensated swallowing or gait or delirium. As with all chronic diseases, patients with chronic stroke disease are at increased risk for further acute stroke, with the risk of recurrent stroke being approximately six times the risk of a first stroke in the general population of the same age and sex.

[0009] Many treatments for nerve damage adopt a repair strategy to restore the function of damaged neurons. However, the time window for intervention before complete loss of function and / or neuronal death is short. Therefore, despite progress in the art in addressing many nerve injuries, there is still a need for effective and flexible treatments to restore function and / or mitigate negative effects after nerve damage. The present disclosure addresses these and related needs. Summary of the invention

[0010] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0011] The present disclosure generally relates to agents, compounds and methods for treating neural damage in a subject in need thereof, e.g., neural damage caused by traumatic brain injury (TBI), ischemia, stroke (e.g., ischemic stroke and / or chronic stroke disease), aneurysm, cerebral hemorrhage, thrombosis, embolism, multiple sclerosis (MS) or Alzheimer's disease, and also relates to methods for treating diseases or conditions associated with glial scar formation and / or chondroitin sulfate proteoglycans (CSPGs) in the nervous system of a subject.

[0012] In one aspect, the present disclosure provides a method for promoting compensatory plasticity of spared neural cells after neural injury. The method comprises contacting the spared neural cells with an effective amount of a therapeutic agent that inhibits one or more catalytic activities, signal transduction or functions of PTPσ in the spared neural cells. In some embodiments, the therapeutic agent comprises a therapeutic peptide, wherein the therapeutic peptide comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 32, or having at least 70% identity to SEQ ID NO: 33.

[0013] In another aspect, the present disclosure provides a method for treating neural injury in a subject. The method comprises promoting compensatory plasticity of spared neural cells after neural injury by administering to the subject an effective amount of a therapeutic agent, the therapeutic agent comprising a therapeutic peptide, wherein the therapeutic peptide comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 32 or having at least 70% identity to SEQ ID NO: 33.

[0014] In various embodiments of these aspects, the spared neural cell can be a neural stem cell, can include oligodendrocyte progenitor cells (OPC) and / or glial precursor cells (GPC), or can be a neuron. In some embodiments, compensatory plasticity can be manifested in the neurite growth of the spared neural cell, such as axon sprouting (sprouting) or dendrite sprouting or bifurcation (branching). In some embodiments, compensatory plasticity can be manifested in the compensatory migration of the spared neural cell towards neural injury. In some embodiments, neural injury is located in the central nervous system, such as brain. In some embodiments, neural injury is due to traumatic brain injury (TBI), multiple sclerosis (MS), Alzheimer's disease, ischemia, stroke, aneurysm, cerebral hemorrhage, thrombosis or embolism.

[0015] In some embodiments, the therapeutic agent further comprises a transport moiety attached to the therapeutic peptide and promotes uptake of the therapeutic peptide by cells. In some embodiments, the transport moiety is an HIV Tat transport moiety. In some embodiments, the therapeutic agent is administered systemically, intrathecally, or intravitreally to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above aspects of the present invention and the many advantages that come with it will become more readily understood and better appreciated by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0017] Figure 1 An example of a method for achieving cell-specific deletion of PTPσ in an animal model is schematically shown.

[0018] Figures 2A-2C : Figure 2A A T2-weighted MRI scan image is shown. Figure 2B Shown is a graph showing vehicle and ISP treated groups having equivalent infarct size 18 hours after stroke, stroke was surgically induced by transient proximal middle cerebral artery occlusion (tMCAO), treatment started 24 hours after stroke. Figure 2C The graph shows that continuous ISP treatment after stroke increased the survival rate of stroke animals over time (67.742%) compared to the survival rate of vehicle-treated stroke animals over time (44.828%).

[0019] Figures 3A-3C The graph shows the results of computer-automated locomotor empty field analysis, showing that mice treated with continuous ISP after stroke had enhanced locomotor activity in terms of parameters of total distance, horizontal activity, and vertical activity, respectively, from 2 to 4 weeks after stroke. n=7-12, *<p<0.05, **p<0.01, ANOVA.

[0020] Figure 4 Graph showing that post-stroke ISP treatment improves sensorimotor function of the stroke-affected limb as measured by the adhesion removal test.

[0021] Figure 5A-5B The graph shows the time to find the target hole in the Barnes maze ( Figure 5A ) and the number of trials and errors ( Figure 5B ) measured by post-stroke ISP treatment improved cognitive function in stroke mice.

[0022] Figures 6A-6C Figure 3 shows computer-automated motion field analysis showing differences in total distance ( Fig. 6A ), horizontal activities ( Figure 6B ) and vertical activities ( Figure 6C Enhanced locomotor activity of ISP-treated mice in terms of parameters of post-stroke delay (7 days after stroke). n=7 per group, *, p<0.05, and **, p<0.01, ANOVA.

[0023] Figures 7A-7F ISP treatment was shown to enhance neuroblast formation and axon sprouting in the corticospinal tract. Figures 7A-7C are images and graphs showing that post-stroke ISP treatment enhances DCX+ neuroblasts near the lateral ventricle and adjacent striatal tissue in post-stroke mice. *p<0.05, n=4. Scale bar=100 μm. Figure 7D-7F Cartoon, image and figure, respectively, showing that ISP treatment after stroke enhances axonal sprouting from the contralateral corticospinal tract. CST fibers crossing at the cervical spinal cord (arrows) are labeled by contralateral cortical BDA tracing. (p<0.01, Student's t-test, n=3 per group).

[0024] Figures 8A-8F Shown is the generation and characterization of NSC-specific deletion of the PTPσ gene and simultaneous labeling of a tomato reporter in conditional (inducible) KO mice. Fig. 8A Schematic representation of the procedure for making an inducible / conditional KO (cKO) of the PTPσ gene with simultaneous labeling. Figure 8B and 8CElectrophoresis images are shown, demonstrating conditional and recombinant alleles with or without induction. PTPσ gene recombination is only observed in adult NSC+nestrate ( Figure 8B ) and enriched primary adult NSC neurospheres ( Figure 8C ) was observed. Figures 8D-8F is an image showing that in the striatum ( Fig.8D ) and DG( Fig. 8E ) and their processes to the CA3 region in the hippocampus ( Fig.8F ). Scale bar = 100 mm.

[0025] Figures 9A-9H A strategy for evaluating PTPσ deficiency on axonal sprouting mechanisms for nerve repair is shown. Fig.9A Schematic illustration of the procedure to generate AAV-mediated adult neuron-specific deletion of the PTPσ gene in conditional PTPσ mice and simultaneous labeling of a tomato reporter. Fig. 9B are images of exemplary electrophoresis analyses demonstrating conditional and recombined alleles in tissues of test mice. Fig. 9C is an image of PTPσ-induced loss of tomato reporter markers, showing cortical neurons (labeled in d; in Fig.9D tomato reporter markers in the striatum (marked in e; in Fig.9E middle), spanning the corpus callosum (marked as f; in Fig.9F to the contralateral cortex (marked as g; in Figure 9G (enlarged in). Figure 9H The corticospinal tract (CST) and the image showing that the tomato reporter also successfully labels the corticospinal tract (CST). Scale bar = 50 mm.

[0026] Figures 10A-10E The formation and characterization of adult neural stem cell cultures is shown. Fig. 10A Schematic representation of the formation of adult neural stem cell (NSC) cultures from wt or cKO mice. Fig. 10B and 10C It is confirmed that adult NSC produces CSPG ( Fig. 10B ) and is neuroepithelial stem cell protein positive ( Fig. 10C ) images. Fig. 10D is an image from a gradient spot experiment, where wt NSCs (neuroepithelial stem cell protein positive; arrows) are unable to penetrate the outer edge of CSPGs visualized by CS56 immunostaining (green). Fig.10Eare images showing that cKO NSCs can penetrate the CSPG rim, indicating loss of PTPσ function in them. Scale bar = 100 um.

[0027] Figures 11A-11E It is shown that an aggrecan substrate coating results in decreased migration of adult NSCs, and that deletion of the PTPσ gene in cKO NSC cells (compared to wt) results in enhanced migration both at basal levels (without aggrecan coating) and with an aggrecan coating. Fig.11A Representative images of wild-type NSCs in the absence of aggrecan substrate coating. Fig. 11B are representative images of cKO NSCs in the absence of an aggrecan substrate coating. Given that NSCs themselves produce CSPGs, it explains why the absence or inhibition of PTPσ enhances NSC migration in the absence of an aggrecan substrate coating. In the ischemic brain, because reactive astroglial cells produce additional CSPGs within the substrate surrounding the injury, the migration of NSCs in the presence of an additional aggrecan substrate coating was also tested. Fig. 11C are images of representative wild-type NSCs in the presence of an aggrecan basal coating, showing that additional aggrecan coating inhibits the migration of WT NSCs and that loss of PTPσ in cKO cells can enhance the migration of cKO cells even in the presence of additional aggrecan coating. Fig.11D are images of representative cKO NSCs in the presence of aggrecan substrate coating. Scale bar = 50 um. Quantification of migration is shown in (E). ** and *** indicate p < 0.01 and p < 0.001 compared to wt cells, # and ### indicate p < 0.05 and p < 0.001 compared to no aggrecan coating conditions (two-way ANOVA, Tukey post-test comparison).

[0028] Fig. 12A and 12B Pharmacological inhibition of the CSPG-PTPσ pathway by ISP was shown to display similar results as genetic PTPσ deficiency. Fig. 12A is a series of images showing the migration of control cells and ISP-treated cells cultured with or without aggrecan coating. As shown, aggrecan coating leads to reduced migration of adult NSCs, and ISP treatment alleviates CSPG inhibition of NSC migration. Fig. 12B The diagram shows Fig. 12A Normalized migration index of cells under various conditions shown in . Two-way ANOVA, ** and *** indicate p<0.01 and p<0.001 compared to control treated NSCs, # indicates p<0.05 compared to the condition without aggrecan coating. Scale bar = 100 um.

[0029] Figures 13A-13C Primary PTPσ cKO adult NSCs were shown to have increased neurite outgrowth compared to wild-type NSCs on an aggrecan substrate. Fig.13A and 13B Representative images of MAP2 immunostaining in wild-type and cKO NSCs differentiated in vitro for 5 days, respectively. Scale bar = 50 μm. Fig. 13C Quantification of neurite length by unbiased imaging and quantification of 50 differentiated neuronal cells are shown. ** indicates p<0.01, Student's t-test.

[0030] Fig.14A and 14B It is shown that ISP treatment enhances neurite outgrowth in WT primary NSC cells. Figure 14 is a series of representative images of MAP2 immunostaining in wild-type NSCs differentiated for 5 days in vitro. Quantification of neurite length was performed by unbiased imaging and quantified on 50 differentiated neuronal cells per condition. Fig. 14B The graph shows the observed neurite length for control, scrambled peptide treated cells and ISP peptide treated cells. There was no significant difference between control and scrambled peptide treated cells and ** indicates p < 0.01 in ISP treated cells compared to control or scrambled peptide treated cells. One-way ANOVA. DETAILED DESCRIPTION

[0031] The present disclosure generally relates to agents, compounds and methods for treating neural damage in a subject in need thereof, e.g., neural damage caused by traumatic brain injury (TBI), ischemia, stroke (e.g., ischemic stroke and / or chronic stroke disease), aneurysm, cerebral hemorrhage, thrombosis, embolism, multiple sclerosis (MS) or Alzheimer's disease, and also to methods for treating diseases or disorders associated with glial scarring and / or chondroitin sulfate proteoglycans (CSPGs) in the nervous system of a subject. In some embodiments, the present disclosure relates to promoting compensatory plasticity of spared neural cells after neural injury.

[0032] The present disclosure is based in part on the inventor's discovery that chondroitin sulfate proteoglycans (CSPGs) can accumulate or regulate in glial scars in the peri-infarct region throughout the chronic stage in animals and human stroke patients. This accumulation of CSPGs at the peri-infarct region during the chronic stage of stroke has been closely associated with the inhibition of post-stroke neuronal plasticity remodeling (which includes the formation of new local circuits, interhemispheric connections, and corticospinal tract axon sprouting), potentially leading to limited recovery of stroke animals and human patients. As described in more detail below, the inventors have discovered a systemic peptide therapy (e.g., intracellular sigma peptide (ISP) therapy) that inhibits or modulates PTPσ catalytic activity, signal transduction, and / or function in nerve cells, overcomes the CSPG barrier, and significantly improves multiple aspects of functional recovery in murine stroke models, including general motor function, specific upper limb sensory motor function, and cognitive function. During the study of the recovery mechanism, it was determined that ISP modulation of PTPσ signaling in nerve cells promotes recovery in several aspects by inducing migration and sprouting activity of nerve cells (e.g., undamaged neurons) that survived the injury to allow functional compensation for the existence of damaged neurons. This ultimately allows the induction of an ameliorative effect even when ISP treatment is delayed after nerve injury. In addition, the systemic peptide treatment inhibits or modulates PTPσ catalytic activity, signaling and / or function, resulting in a reduction in chronic atrophy of the brain after stroke. Therefore, this confirms that the ability to promote the plasticity of spared (e.g., undamaged) nerve cells proximal and distal to nerve injury is highly relevant to therapies that promote recovery and survival after injury (e.g., malignant stroke).

[0033] According to the above, in one aspect, the present disclosure provides a method for promoting compensatory plasticity of spared neural cells after neural injury. The method comprises contacting the spared neural cells with an effective amount of a therapeutic agent, which inhibits or modulates one or more catalytic activities, signal transduction or functions of PTPσ in the spared neural cells. This method may be applicable to other methods for treating neural injury in a subject, which are to promote compensatory plasticity of spared neural cells after neural injury by administering an effective amount of a therapeutic agent or composition of the present disclosure to the subject, which is also covered by the present disclosure. In some embodiments, the therapeutic agent comprises a therapeutic peptide. The therapeutic peptide may comprise an amino acid sequence having at least 70% identity with SEQ ID NO: 32, or having at least 70% identity with SEQ ID NO: 33.

[0034] The term "treatment" as used herein refers to the medical management of a disease, disorder or condition (e.g., nerve damage) of a subject (e.g., a human or non-human mammal, such as another primate, horse, dog, pig, mouse, rat, guinea pig, rabbit, etc.). Treatment can encompass any indicator of successful treatment or improvement of a disease or condition (e.g., nerve damage), including any parameter such as elimination, alleviation, reduction of symptoms, or making the disease or condition more tolerable to the patient, slowing the rate of degeneration or decline, or making degeneration less attenuating. Treatment or improvement of symptoms can be based on subjective or objective parameters, including the results of a doctor's examination. For example, the "NIHSS scale" mentioned herein is a commonly used scale for measuring the level of damage caused by stroke (Kasner S E. Lancet Neurol. 2006; 7: 603-12). Therefore, the term "treatment" includes administering the composition of the present disclosure to alleviate or prevent or inhibit the development of symptoms or conditions associated with a disease or condition (e.g., nerve damage). In addition, the term includes: conservative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition or disorder; preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy intended to improve the relevant disease, pathological condition or disorder.

[0035] The term "therapeutic effect" means the overall improvement, reduction, or elimination of a disease or condition, a symptom of a disease or condition, or a side effect of a disease or condition in a subject.

[0036] The term "therapeutically effective" means an amount of a composition that can be readily determined to produce a therapeutic effect (e.g., inducing compensatory plasticity of spared neurons and / or improving motor function, sensorimotor function, or cognition). The effective amount of a drug as defined herein can vary according to factors such as the disease state, age, and weight of the subject, and the ability of the drug to elicit a desired response in the subject. The dosing regimen can be adjusted to provide the optimal therapeutic response. An effective amount is also an amount in which the therapeutically beneficial effects of the active compound outweigh any toxic or deleterious effects thereof.

[0037] The term "compensatory plasticity" refers to the induction of phenotypic changes in healthy neural cells that contribute to neural function and that compensate for the loss or degeneration of damaged neural cells. Healthy neural cells are referred to as "spare" neural cells, meaning that they are not directly damaged in the neural injury addressed by the present method.

[0038] In some embodiments, the spared neural cells are neural stem cells. Neural stem cells are multipotent undifferentiated neural cells that can eventually differentiate into neurons and glia of the nervous system. In other embodiments, the spared neural cells may include oligodendrocyte progenitor cells (OPC) and / or glial precursor cells (GPC). In other embodiments, the spared neural cells are neurons. Neurons or neural cells are cells in neural tissue that transmit electrical impulses along parts of their bodies (e.g., along axons) to communicate with other cells by releasing neurotransmitters into small synaptic gaps that separate cells. Neurons, as encompassed by the present disclosure, can be distinguished from neural stem cells by expressing certain cell markers. Specifically, neurons are typically neuroepithelial stem cell protein-(nestin-) and DCX+ (for immature neurons), or neuroepithelial stem cell protein- and NeuN+ or apt2+ (for mature neurons). In contrast, neural stem cells are typically neuroepithelial stem cell protein+ and DCX-.

[0039] As described in more detail below, it is determined that the inhibition of the catalytic activity, signal transduction and / or function of PTPσ induces neurite outgrowth in the spared nerve cells after nerve injury (e.g., stroke). Therefore, in some embodiments, the therapeutic agent / peptide induces compensatory neurite growth in the spared nerve cells. Neurite growth can be manifested in axon sprouting in the spared nerve cells. Axon sprouting is the extension of axons (i.e., nerve fibers) from the cell body to develop or otherwise grow. The typical function of axons is to conduct electrical impulses from the nerve cell body toward another cell. In other embodiments, neurite growth can be manifested in dendrite sprouting in the spared nerve cells. Dendrites are branched protoplasmic extensions of nerve cells that propagate the electrochemical stimulation received from other nerve cells to the cell body. In either embodiment of the two, neurite sprouts, whether axon sprouts or dendrite sprouts or bifurcations, all lead to increased synaptic contacts with adjacent, surrounding or even distant nerve cells. Without being limited to a specific theory, the increased synaptic contacts compensate for the synaptic contacts lost due to damage to other cells. In the case of compensatory synaptic addition via neurite sprouting, alternative or bypass connections can be established, thereby rerouting signaling pathways and thus can serve to restore function following damaged nerve cell loss or degeneration.

[0040] In some embodiments, the neurite growth described herein can occur in spared nerve cells, which are at the proximal end of the nerve injury site or at the distal end of the injury site. The term "proximal" or "distal end" means a relative distance and can represent different distances depending on the background. In some cases, the spared nerve cells at the proximal end of the nerve injury site are cells near or otherwise close to the injury site, which, for example, directly contact the damaged nerve cells or within a distance that can be measured by cell length or width of the damaged cells. On the contrary, the spared nerve cells at the distal end of the nerve injury site can be far away in different tissues or regions such as the brain, or even in other regions of the central nervous system, such as in the spinal cord. For example, in some embodiments, the therapeutic agent / peptide induces neurite growth in the surviving neurons at the distal end of the nerve injury site (e.g., a position in the contralateral corticospinal tract).

[0041] In other embodiments, the therapeutic agent / peptide induces compensatory migration of spared neural cells toward neural injury. In some embodiments, the spared neural cells exhibiting compensatory migration are neural stem cells. In other embodiments, the spared neural cells exhibiting compensatory migration are oligodendrocyte progenitor cells (OPC) and / or glial precursor cells (GPC). In some embodiments, the therapeutic agent / peptide induces compensatory migration of spared neural cells proximal to the neural injury site. Although the present disclosure encompasses embodiments in which the compensatory migration of spared neural cells causes the spared neural cells to enter the neural injury site, the present disclosure is not so limited. The present disclosure also encompasses embodiments in which the compensatory migration of spared neural cells causes the spared neural cells to be closer to the neural injury site than they were before the administration of the therapeutic agent / peptide. In some embodiments, the migrated spared neural cells cross the CSPG ring.

[0042] The method of the present disclosure addresses the neural damage that occurs in the central nervous system. In some embodiments, the neural damage is located in the brain. As described in more detail below, the stroke model is used to determine the induced plasticity of the neural cells that survived the neural damage. In view of the enhanced plasticity effect that occurs in the spared neural cells (including the neural cells at the distal end of the injury site), it will be understood by those of ordinary skill in the art that the present disclosure is not limited to the situation of stroke, but also encompasses other forms of neural damage. Therefore, neural damage may be attributed to: traumatic brain injury (TBI), such as concussion; neurodegenerative diseases, such as multiple sclerosis (MS); Alzheimer's disease, ischemia (such as focal ischemia or global ischemia); stroke (such as ischemic stroke and / or chronic stroke disease); aneurysm, cerebral hemorrhage, thrombosis, embolism; and such as such in which neural cells are damaged.

[0043] The term "ischemia", also referred to herein as "cerebral ischemia", "brain ischemia" or "cerebrovascular ischemia", is a condition in which blood flow to the brain is insufficient to meet metabolic needs. This results in poor oxygen supply or cerebral hypoxia, which leads to death of brain tissue or cerebral infarction, also known as "ischemic stroke". "Ischemic stroke" is a subtype of stroke and is usually the result of interruption of blood supply to the brain due to obstruction of a cerebral artery. The terms "cerebral ischemia" and "ischemic stroke" may be used interchangeably herein.

[0044] There are two types of cerebral ischemia: "focal ischemia", which is limited to a specific area of ​​the brain; and "global ischemia", which covers a wide area of ​​brain tissue. Typically, cerebral ischemia is characterized by patients showing one or more of the following symptoms: difficulty talking and understanding, paralysis or numbness of the face, arms or legs, difficulty seeing in one or both arms, headaches and difficulty walking. In order to determine the type of stroke and the most appropriate treatment, the emergency team needs to evaluate the type of stroke and the area of ​​the brain affected by the stroke. The emergency team also needs to rule out other possible causes of the symptoms, such as brain tumors or drug reactions. There are several tests that are generally used to determine the type of stroke: physical examination, blood tests (glucose level, blood cell count, serum electrolytes such as sodium, potassium or calcium, cholesterol, total lipids, HDL, LDL or coagulation factors such as antithrombin III, protein C, protein S; factor VIII; activated protein C resistance; particularly relevant are coagulation factor and platelet assays), computed tomography (CT) scans, magnetic resonance imaging (MRI), carotid ultrasound, cerebral angiography and echocardiography. For a review of the diagnosis of ischemic stroke, see Am Fam Physician., 2015, 91(8): 528-36. The acute stage of ischemic stroke is referred to herein as "acute ischemic stroke" and is defined as within 4 hours of onset. The chronic stage of stroke or ischemic stroke is referred to herein as chronic stroke disease and occurs after the acute stage of ischemic stroke.

[0045] Inhibition of one or more catalytic activities, signaling, or functions of PTPσ in spared neural cells, and therapeutic agents for this purpose, will now be described.

[0046] The activity, signaling and / or function of PTPσ can be suppressed, inhibited and / or blocked in several ways including: directly inhibiting the activity of the intracellular domain of PTPσ (e.g., by using small molecules, peptide mimetics, antibodies, intracellular antibodies or dominant negative polypeptides); activating (e.g., by increasing the expression or activity of genes and / or proteins) genes and / or proteins (which genes and / or proteins inhibit one or more activities, signaling and / or functions of the intracellular domain of PTPσ); inhibiting genes and / or proteins that are downstream mediators of PTPσ (e.g., by blocking the expression and / or activity of mediator genes and / or proteins); introducing (e.g., by using recombinant gene expression vectors, recombinant viral vectors or recombinant polypeptides) genes and / or proteins (which genes and / or proteins negatively regulate one or more activities, signaling and / or functions of PTPσ); or replacing the gene with, for example, a hypomorphic mutant of PTPσ (e.g., by homologous recombination, overexpression using recombinant gene expression or viral vectors, or mutagenesis).

[0047] Therapeutic agents that inhibit or reduce one or more activities, signal transduction and / or functions of PTPσ may include agents that reduce and / or inhibit the activity, signal transduction and / or function of PTPσ. Such agents may be delivered intracellularly and once delivered intracellularly enhance at least one of the motor function, sensorimotor function or cognition of the subject.

[0048] In some embodiments, the therapeutic agent that inhibits or reduces one or more activities, signal transduction and / or functions of PTPσ comprises a therapeutic peptide or small molecule that binds and / or complexes with the intracellular domain of PTPσ (specifically the intracellular wedge domain) to inhibit the activity, signal transduction and / or function of PTPσ. Therefore, therapeutic peptides or small molecules that bind and / or complex with the intracellular domain of PTPσ of spared neural cells (e.g., neural stem cells, OPCs, GPCs, neurons and / or glial cells) can be used to promote compensatory cell growth, motility (e.g., migration), neurite outgrowth, survival, or other features that promote compensatory plasticity of these cells.

[0049] In some embodiments, the therapeutic agent may be a peptide mimetic of the wedge domain (i.e., wedge domain) of the intracellular catalytic domain of PTPσ, such as described in WO 2013 / 155103A1, which is incorporated herein by reference in its entirety. The peptide mimetic of the wedge domain of PTPσ, when expressed in cells (e.g., neurons and / or glial cells) or conjugated to an intracellular transport moiety, can bind and / or camera with the wedge domain expressed in surviving neural cells, resulting in the elimination of PTPσ signaling in the spared neural cells, thereby promoting cell growth, motility and survival. For example, binding of these therapeutic peptides to the intact wedge domain of PTPσ can: (i) interfere with the ability of PTPσ to interact with a target protein (e.g., a phosphatase target); (ii) interfere with the activity of promoting intermolecular interactions between PTPσ and another domain contained in PTPσ (e.g., the catalytically inactive second phosphatase domain D2); (iii) prevent the protein from approaching the active phosphatase site; (iv) compete for normal interactors beyond the wedge domain; and / or (v) sterically inhibit phosphatase activity.

[0050] As described above, in some embodiments, the therapeutic agent may comprise, consist essentially of, and / or consist of a therapeutic peptide comprising an amino acid sequence of about 10 to about 20 amino acids having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% identity to an about 10 to about 20 contiguous amino acid portion of an amino acid sequence of a wedge domain of PTPσ. In some embodiments, the about 10 to about 20 contiguous amino acid portion includes the N-terminal alpha helix and the contiguous amino acids of the 4 amino acid turn of the wedge domain. In some embodiments, the reference wedge domain of PTPσ is the human PTPσ sequence. In some embodiments, the therapeutic peptide comprises an amino acid sequence having at least about 70%, at least about 78%, at least about 85%, at least about 92%, or 100% identity to SEQ ID NO: 32. In other embodiments, the therapeutic peptide comprises an amino acid sequence that is at least about 70%, at least about 78%, at least about 85%, at least about 92%, or 100% identical to SEQ ID NO:33.

[0051] As disclosed below, peptides (e.g., therapeutic peptides) having a cytosolic carrier corresponding to or substantially identical to the wedge domain of PTPσ can alleviate CSPG-mediated inhibition of post-stroke neuronal plasticity remodeling, enhancing at least one of motor function, sensorimotor function, or cognition. Advantageously, the therapeutic peptide can be administered systemically.

[0052] As shown in Table 1, the wedge domain sequence of PTPσ is highly conserved among higher mammals, with only a single amino acid change (threonine to methionine at position 6) from human to mouse and rat, preventing 100% homology.

[0053]

[0054]

[0055] As shown in Table 1, the first α helix of the wedge domain of PTPσ comprises amino acids 1-10, the turn region comprises amino acids 11-14, and the second α helix comprises amino acids 15-24. For example, the first α helix of the wedge domain of human PTPσ (SEQ ID NO: 25) has the amino acid sequence of DMAEHTERLK (SEQ ID NO: 29), the turn has the amino acid sequence of ANDS (SEQ ID NO: 30), and the second α helix has the amino acid sequence of LKLSQEYESI (SEQ ID NO: 31).

[0056] The wedge domain also shares sequence homology with other members of the LAR family, LAR and PTPδ. These amino acids are likely required for the overall structure of the wedge domain. Conserved amino acids include an alanine at position 13, which marks the end of the first alpha helix and the beginning of the turn, making it likely required for the overall wedge size and structure.

[0057] Because the general secondary and tertiary structures of the wedge domain remain consistent in most receptor PTPσ, several conservative substitutions can be made to therapeutic peptides targeting the PTPσ wedge domain to achieve similar results. Examples of conservative substitutions include: substitution of one non-polar (hydrophobic) residue (e.g., isoleucine, valine, leucine, or methionine) for another, substitution of one polar (hydrophilic) residue for another, such as substitutions between arginine and lysine, substitutions between glutamine and asparagine, substitutions between glycine and serine, substitution of one basic residue (e.g., lysine, arginine, or histidine) for another, and / or substitution of one acidic residue (e.g., aspartic acid or glutamic acid) for another.

[0058] These conservative substitutions can occur in non-unique domains in alpha helices or turns, specifically at positions 1-3 and 7-10 in the first alpha helix; positions 12 and 13 in the turn; and positions 15, 16, 18-24 in the second alpha helix. These amino acids may be required for the overall structure of the wedge domain, but are not required for the specific binding of the wedge to PTPσ.

[0059] Unique amino acids of PTPσ, particularly those differentially expressed in PTPσ compared to LAR, were found to be required for specific binding of the wedge domain. These include the EH domain at positions 4 and 5 in the first α-helix, followed by a threonine or methionine (rat and mouse substitution) at position 6. In all higher mammals, in the turn, there is a unique serine at position 14. Finally, there is a unique leucine at position 17 in the second α-helix. The potential roles of these unique amino acids are discussed below.

[0060] The serine residue at position 14 in the turn is of particular interest due to its location in the wedge domain. This amino acid, located in the turn between the α helices, is slightly extended from the general secondary and tertiary structure of PTPσ, making it available for binding interactions. In addition, serine, due to its hydroxyl group and the polarity it contains, is known to promote several homoaffinity and heteroaffinity binding events, such as hydrogen bonding between adjacent serines. It is also known that serine undergoes various modifications, such as phosphorylation, making it more likely to be necessary for specificity. Smaller peptides concentrated in the turns in the wedge domain and containing conserved serines may provide higher stability with similar functions. Such peptides can be synthesized as rings in which disulfide bonds are created with cysteines on both ends.

[0061] Unique amino acids in the first alpha helix include glutamic acid at position 4, histidine at position 5, and either threonine or methionine at position 6. Although histidine participates in the consensus wedge domain, it is not present in LAR, PTPδ, PTPμ, or CD45. Because all three amino acids are either charged or polar, this sequence or one of its components is required for PTPσ wedge specificity.

[0062] In addition, the second α-helix contains a unique leucine at position 17. Leucine has been shown to be a key adhesive molecule for the three-dimensional structure of the leucine zipper. In these molecules, which are structurally similar to the wedge domain, the leucine located at approximately 7 intervals of the opposing α-helix interacts with the hydrophobic region of the opposing α-helix. Because there is also a leucine at position 9 in the first α-helix, this unique leucine is believed to be required for the overall three-dimensional structural integrity of the PTPσ wedge.

[0063] Thus, in some embodiments, the therapeutic peptide can comprise, consist essentially of, or consist of about 14 to about 20 amino acids and comprise the amino acid sequence EHX1ERLKANDSLKL (SEQ ID NO:32), wherein X1 is T or M. The therapeutic peptide comprising SEQ ID NO:32 can comprise at least one, at least two, at least three, at least four, or at least five conservative substitutions such that the therapeutic peptide has an amino acid sequence that is at least about 70%, at least about 78%, at least about 85%, at least about 92%, or 100% identical to SEQ ID NO:32.

[0064] In some embodiments, conservative substitutions may be with amino acid residues 4E, 5R, 6L, 7K, 9N, 10D, 12L, or 13K of SEQ ID NO: 32. As non-limiting examples, amino acid residue 4E may be replaced with D or Q, amino acid residue 5R may be replaced with H, L, or K, amino acid residue 6L may be replaced with I, V, or M, amino acid residue 7K may be replaced with R or H, amino acid residue 9N may be replaced with E or D, amino acid residue 10D may be replaced with E or N, amino acid residue 12L may be replaced with I, V, or M, and / or amino acid residue 13K may be replaced with R or H. Any one or more of the substitutions may be implemented in any combination, as long as the resulting sequence achieves the same above parameters.

[0065] Description of many aspects of therapeutic peptides is generally set forth herein in the context of SEQ ID NO: 32, and the present disclosure also encompasses embodiments in which the therapeutic peptide comprises a variant of a wedge domain as set forth in SEQ ID NO: 33. It should be understood that all other aspects and features of the therapeutic peptides described herein also apply to this variant embodiment unless otherwise expressly stated. Thus, in other embodiments, the therapeutic peptide may comprise, consist essentially of, or consist of about 14 to about 20 amino acids, and comprise the amino acid sequence DMAEHX1ERLKANDS (SEQ ID NO: 33), wherein X1 is T or M. The therapeutic peptide comprising SEQ ID NO: 33 may comprise at least one, at least two, at least three, at least four, or at least five conservative substitutions such that the therapeutic peptide has an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 33.

[0066] In some embodiments, conservative substitutions may be with amino acid residues 7E, 8R, 9L, 10K, 12N, or 13D of SEQ ID NO: 33. As an example, amino acid residue 7E may be substituted with D or Q, amino acid residue 8R may be substituted with H, L, or K, amino acid residue 9L may be substituted with I, V, or M, amino acid residue 10K may be substituted with R or H, amino acid residue 12N may be substituted with E or D, and amino acid residue 13D may be substituted with E or N.

[0067] In some embodiments, the therapeutic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-25, 32, and 33.

[0068] The therapeutic peptides described herein may undergo other various changes, substitutions, insertions and deletions, wherein such changes provide certain advantages in their use. In this regard, the therapeutic peptide that binds and / or complexes with the wedge domain of PTPσ may correspond to or be substantially, but not completely, identical to the sequence of the polypeptide, in which one or more changes are made, and which retains the ability to inhibit or reduce one or more activities, signaling and / or functions of PTPσ.

[0069] The therapeutic peptide can be any of various forms of polypeptide derivatives, including amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides and the like derivatives.

[0070] It will be understood that conservative substitutions may also involve the use of chemically derivatized residues in place of non-derivatized residues, provided that the peptide exhibits the requisite binding activity.

[0071] As used herein, the term "chemical derivative" or its grammatical variants refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those in which free amino groups have been derivatized to form amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazines. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Chemical derivatives also include those polypeptides that contain one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine. The polypeptides described herein may also include any polypeptides having one or more additions and / or deletions or residues relative to the sequence of the polypeptides whose sequences are shown herein, as long as the necessary activity is maintained.

[0072] One or more of the therapeutic peptides described herein may also be modified by natural processes (e.g., post-translational processing) and / or by chemical modification techniques known in the art. Modifications may occur in the peptide, including at the peptide backbone, amino acid side chains, and amino or carboxyl termini. It will be appreciated that the same type of modification may be present at several sites of a given peptide to the same or varying extent. Modifications include, for example, and without limitation, acetylation, acylation, addition of acetamidomethyl (Acm), ADP-ribosylation, amidation, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment to a nucleotide or nucleotide derivative, covalent attachment to a lipid or lipid derivative, covalent attachment to phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, γ-carboxylation, glycosylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination (for reference, see Protein-structure and molecular properties, 2nd ed., TECreighton, WH Freeman and Company, New-York, 1993).

[0073] The peptides and / or proteins described herein may also include, for example, biologically active mutants, variants, fragments, chimeras and analogs; fragments of an amino acid sequence comprising truncated one or more amino acids, wherein the truncation may start at the amino terminus (N-terminus), carboxyl terminus (C-terminus) or interior of the protein. Analogs of the present invention include insertion or replacement of one or more amino acids. Variants, mutants, fragments, chimeras and analogs may act as inhibitors of LAR family phosphatases (without limitation to this example).

[0074] The therapeutic polypeptides described herein can be prepared by methods known to those skilled in the art. Peptides and / or proteins can be prepared using recombinant DNA. For example, a preparation can include culturing host cells (bacteria or eukaryotic organisms) under conditions that allow expression of the peptides and / or proteins in the cell.

[0075] The purification of polypeptides can be carried out by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity or other purification techniques typically used for protein purification. The purification steps can be carried out under non-denaturing conditions. On the other hand, if a denaturing step is required, the protein can be reactivated using techniques known in the art.

[0076] In some embodiments, the therapeutic peptides described herein may contain additional residues that may be added at either terminus of the polypeptide to provide a "linker" by which the polypeptide can be conveniently linked and / or attached to other polypeptides, proteins, detectable moieties, labels, solid matrices, or carriers.

[0077] The amino acid residue linker is typically at least one residue, and can be 40 or more residues, more often 1 to 10 residues. The typical amino acid residue for connection is glycine, tyrosine, cysteine, lysine, glutamic acid and aspartic acid or the like. In addition, the subject polypeptide can be modified sequence and different by terminal-NH2 acylation (for example acetylation or thioglycolic acid amidation), terminal-carboxamidation (for example with ammonia, methylamine) and similar terminal modifications. As known, terminal modifications can be used for reducing the sensitivity to protease digestion, and therefore for extending the half-life of polypeptide in solution (particularly the biological fluid in which protease may be present). In this regard, because the stable structure formed by cyclization and in view of the biological activity shown in cyclic peptides as herein described, polypeptide cyclization is also an effective terminal modification, and is still particularly preferred.

[0078] In some embodiments, the joint can be a flexible peptide joint, which connects the therapeutic peptide polypeptide to other polypeptides, proteins, and / or molecules such as detectable moieties, labels, solid substrates, or carriers. Flexible peptide joints can be about 20 or less amino acid lengths. For example, a peptide joint can comprise about 12 or less amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some cases, a peptide joint comprises two or more of the following amino acids in any combination: glycine, serine, alanine, and threonine. In some embodiments, a peptide joint does not comprise a cysteine ​​residue.

[0079] In some embodiments, the therapeutic agent comprising the therapeutic peptide described herein can be provided in the form of a conjugated protein or a drug delivery construct, which comprises at least one transport subdomain or portion (i.e., transport moiety) connected to the therapeutic peptide. The transport moiety can promote the ingestion of the therapeutic polypeptide into a mammal (i.e., human or animal) tissue or cell (e.g., nerve cell). The transport moiety can be covalently linked to the therapeutic peptide. The covalent link can include a peptide bond or an unstable bond (e.g., a bond that is easily cleaved or chemically changed in the internal target cell environment). In addition, the transport moiety can be cross-linked to the therapeutic polypeptide (e.g., chemical cross-linking, UV cross-linking). The transport moiety can also be connected to the therapeutic polypeptide using a connecting polypeptide as described herein.

[0080] The transport moiety can be repeated more than once in the therapeutic agent. The repetition of the transport moiety can affect (e.g., increase) the uptake of the peptide and / or protein by the desired cell. The transport moiety can also be located at the amino terminal region of the therapeutic peptide or at its carboxyl terminal region or at both regions.

[0081] In some embodiments, the transport moiety may comprise at least one transport peptide sequence that allows the therapeutic polypeptide to penetrate into the cell by a receptor-independent mechanism once attached to the transport moiety. An example of a Tat sequence encompassed by the present disclosure is shown in SEQ ID NO: 34. For example, in an illustrative, non-limiting embodiment, the transport peptide is a synthetic fusion peptide comprising at least one Tat-mediated protein delivery sequence and a sequence having at least 70% identity to one of SEQ ID NOs: 1-25, 32, and 33. In a specific illustrative embodiment, wherein the Tat-mediated protein delivery sequence is linked to a wedge domain sequence of one of SEQ ID NOs: 1-25, 32, and 33 with a one-residue peptide linker, the fusion peptide may have an amino acid sequence of SEQ ID NOs: 35-61, respectively. The Xaa shown in each of SEQ ID NOs: 35-59 (and the first Xaa shown in each of SEQ ID NOs: 60 and 61) may be any amino acid residue, such as glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, or the like. In some embodiments, the linker amino acid (expressed as Xaa) shown is glycine, serine, alanine or threonine. In some embodiments, the linker amino acid (expressed as Xaa) shown is not cysteine. Although the sequences disclosed in SEQ ID NO: 35-61 each have a single linker amino acid residue, it should be understood that other embodiments with longer (comprising two or more amino acid residues) peptide linkers are also encompassed by the present disclosure. Longer peptide linkers are described above.

[0082] Other examples of known transport moieties, subdomains, and the like that are encompassed by the present disclosure are described, for example, in Canadian Patent Document No. 2,301,157 (conjugates containing homeodomain of antennapedia) and U.S. Patent Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604, all of which are incorporated herein by reference in their entirety. Such examples include conjugates comprising amino acids of the Tat HIV protein; Herpes simplex virus-1 DNA binding protein VP22, a histidine tag ranging from 4 to 30 histidine repeats in length, or variants, derivatives, or homologs thereof that are capable of promoting uptake of the active cargo moiety by a receptor-independent process.

[0083] A 16 amino acid region of the third α-helix of the Antennapedia homeodomain has also been shown to enable proteins (prepared as fusion proteins) to cross cell membranes (PCT International Publication No. WO 99 / 11809 and Canadian Application No. 2,301,157. Similarly, the HIVTat protein has been shown to be able to cross cell membranes.

[0084] In addition, (multiple) transport moieties can comprise polypeptides having a basic amino acid enriched region covalently linked to an active agent portion (e.g., a fragment inhibitor peptide comprising an intracellular domain). The term "basic amino acid enriched region" as used herein relates to a region of a protein having a high content of basic amino acids (e.g., arginine, histidine, asparagine, glutamine, lysine). A "basic amino acid enriched region" can have, for example, 15% or more basic amino acids. In some cases, a "basic amino acid enriched region" can have less than 15% basic amino acids and still be used as a transporter region. In other cases, a basic amino acid region will have 30% or more basic amino acids.

[0085] The transport moiety(ies) may further comprise a proline-rich region. The term proline-rich region as used herein refers to a region in which a polypeptide has 5% or more (up to 100%) proline in its sequence. In some cases, a proline-rich region may have between 5% and 15% proline. In addition, a proline-rich region refers to a region of a polypeptide that contains more proline than is normally observed in naturally occurring proteins (e.g., proteins encoded by the human genome). Such use of a proline-rich region may serve as a transporter region.

[0086] In one embodiment, the therapeutic peptides described herein can be non-covalently linked to a transduction agent. An example of a non-covalently linked polypeptide transduction agent is the Chariot protein delivery system (see U.S. Pat. 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).

[0087] The therapeutic agents described herein may be modified (e.g., chemically modified). Such modifications may be designed to facilitate control or purification of the molecule, improve the solubility of the molecule, facilitate targeted desired location administration, increase or decrease half-life. Many such modifications are known in the art and may be applied by a skilled artisan.

[0088] In the therapeutic method embodiments disclosed herein, a therapeutically effective amount of a therapeutic agent is administered to a subject suffering from a chronic function associated with a chronic stroke disease. The formulation comprising the therapeutic agent can be administered to the subject one or more times from the time of, for example, detection or onset of a stroke to days, weeks, months, and / or years after detection or onset of a stroke.

[0089] Therapeutic agents can be delivered to the subject by any suitable path, including, for example, local and / or systemic administration. Systemic administration can include, for example, parenteral administration. The phrase "parenteral administration" means a mode of administration other than enteral administration and topical administration, with injection as a typical example, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In some embodiments, systemic administration includes: intramuscular, intravenous, intraarticular, intraarterial, intrathecal, intravitreal, subcutaneous or intraperitoneal administration. Therapeutic agents can also be administered by oral, transdermal, local, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or by rectum. In some embodiments, therapeutic agents can be administered to the subject via intravenous administration by utilizing an infusion pump, thereby delivering the dosage of therapeutic agents every day or weekly.

[0090] Desirable features of local administration may include achieving effective local concentrations of the therapeutic agent, and avoiding potential adverse side effects of systemic administration of the therapeutic agent. In one embodiment, the therapeutic agent may be introduced directly into the brain of a subject.

[0091] Pharmaceutically acceptable formulations of therapeutic agents can be suspended in an aqueous carrier and introduced through a conventional hypodermic needle or using an infusion pump.

[0092] For injection, the therapeutic agent can be formulated in a liquid solution, typically in a physiologically compatible buffer such as Hank's solution or Ringer's solution. Alternatively, the therapeutic agent can be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also provided. Injection can be in the form of, for example, a single dose injection or a continuous infusion (e.g., using an infusion pump) of the therapeutic agent.

[0093] It should be understood that the amount, volume, concentration and / or dosage of the therapeutic agent administered to any animal or human subject depends on many factors, including the subject's size, body surface area, age, specific composition to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously. Specific variations of the above amounts, volumes, concentrations and / or dosages of the therapeutic agent can be readily determined by those skilled in the art using the following experimental methods.

[0094] In some embodiments, therapeutic agents, such as therapeutic peptides described herein, can be administered locally and / or systemically to a subject in need thereof at a dose or amount of about 0.1 μmol, about 1 μmol, about 5 μmol, about 10 μmol or more; or about 0.0001 mg / kg, about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg or about 1 mg / kg to about 5 mg / kg or 10 mg / kg of the subject being treated. The therapeutic agent can be administered daily, weekly, biweekly, monthly, or less frequently until maximum recovery of motor, sensorimotor, and / or cognitive deficits is achieved.

[0095] Therapeutic agents can be administered with a fixed unit dose between 1-1000mg IV (e.g., between 100-600mg IV, e.g., between 200 and 400mg IV, e.g., about 300mg IV). When administered subcutaneously, therapeutic agents are usually administered with a dosage between 1mg-100mg SC (e.g., 75mg). Therapeutic agents can also be administered with a dosage between 1 and 10mg / kg (e.g., about 6.0, 4.0, 3.0, 2.0, 1.0mg / kg). In some cases, continuous administration can be performed, for example, via a subcutaneous pump.

[0096] In some embodiments, the spared neural cells are contacted with the therapeutic agent within 7 days of injury, such as within about 1, about 2, about 3, about 4, about 5, about 6, and about 7 days after the neural injury occurs.

[0097] In embodiments of treating a subject, a therapeutic agent is administered to a subject within 7 days after injury, such as within about 1, about 2, about 3, about 4, about 5, about 6, and about 7 days after the occurrence of neural damage. For example, in some embodiments, a therapeutic agent, such as a therapeutic peptide, is administered to a subject 12 hours or more, such as 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, or 24 hours or more after the onset of neural damage, such as stroke. For example, a therapeutic agent can be administered after acute stroke or about 12 hours or more after the onset of stroke.

[0098] In another embodiment, the therapeutic agent can be administered systemically by intravenous injection to the subject, or locally at the site of injury, typically about 24 hours, about 48 hours, about 100 hours, or about 200 hours or more after a neural injury, such as a stroke.

[0099] In other embodiments, the pharmaceutically acceptable formulation for administering (multiple) therapeutic agents can also be formulated to provide sustained delivery of active compounds to the subject. For example, the formulation can deliver active compounds during at least one week, two weeks, three weeks or four weeks (inclusive) after the initial administration to the subject. For example, the subject treated according to the methods described herein can be treated with a therapeutic agent during at least 30 days (by repeated administration or by using a sustained delivery system or both).

[0100] Methods of sustained delivery include the use of polymer capsules, micropumps for delivery of formulations, biodegradable implants, or implanted transgenic autologous cells (see U.S. Pat. No. 6,214,622). Implantable infusion pump systems (e.g., INFUSAID pumps (Towanda, PA); see Zierski et al., 1988; Kanoff, 1994) and osmotic pumps (sold by Alza Corporation) are commercially available or otherwise known in the art. Another mode of administration is by an implantable externally programmable infusion pump. Infusion pump systems and reservoir systems are also described in, for example, U.S. Pat. Nos. 5,368,562 and 4,731,058.

[0101] The pharmaceutical composition can be administered to any subject who may experience the beneficial effects of compensatory neuroplasticity, such as plasticity manifested in enhanced motor function, sensorimotor function and / or cognition. Among these animals, the most important is human, but the embodiments described herein are not intended to be limited to this.

[0102] As described, the therapeutic agent can be used in a method for treating neural damage in a subject. The method can include administering a therapeutically effective amount of a therapeutic agent described herein to a subject in need thereof. The therapeutically effective amount can include an effective amount (dose) that enhances compensatory plasticity of spared neural cells, for example, plasticity can be manifested in at least one of motor function, sensorimotor function, or cognition in the subject.

[0103] In some embodiments, an effective amount of a therapeutic agent described herein can be administered to enhance the generation of neural cells, such as NSCs, neurons, and / or glial cells in the central nervous system of a subject, and the amount of neurons and / or glial cells generated is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% compared to the amount of neural cells, such as NSCs, neurons, and / or glial cells in a subject not administered the therapeutic agent. %, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000%.

[0104] In some embodiments, the subject treated by the methods described herein has suffered from an acute middle cerebral artery (MCA) ischemic event or stroke, such as an ischemic stroke. Ischemic stroke is a rapidly progressive loss of brain function due to ischemia (lack of glucose and oxygen supply) caused by thrombosis (e.g., venous thrombosis), embolism, or systemic hypoperfusion that interferes with the blood supply to the brain. As a result, the affected area of ​​the brain cannot function, resulting in an inability to move one or more limbs on one side of the body, an inability to understand or express speech, or an inability to see a visual field on one side.

[0105] Symptoms of an acute middle cerebral artery (MCA) ischemic event or ischemic stroke include, for example, hemiparesis, decreased sensation and muscle weakness in the face, numbness, decreased sensation or vibration perception, altered smell, taste, hearing or vision (total or partial), drooping eyelids (ptosis) and eye muscle weakness, decreased reflexes, balance problems and nystagmus, altered breathing and heart rate, inability to turn the head to one side due to weakness of the sternocleidomastoid muscle, tongue weakness (inability to stick out and / or move from side to side), aphasia, apraxia, visual field loss, memory deficits, hemi-neglect, disorganized thinking, confusion, hypersexual posturing, agnosia, difficulty walking, altered motor coordination and vertigo and / or imbalance.

[0106] The time of onset of an ischemic event or stroke, such as an ischemic stroke, can be determined by any available method. For example, a subject can be questioned by, for example, a physician, about various symptoms of a stroke (as described herein) to determine the approximate time of stroke onset. In some cases, the time of stroke onset is difficult to determine, such as when the subject awakens with a stroke or if the onset of symptoms is otherwise undetectable. In such cases, stroke onset can be determined by determining the time when the subject was last known to be healthy, such as last known to be normal (LKN). In some cases, MRI of the brain can be used to determine the time of onset and / or duration of a stroke in a subject (see, e.g., Petkova et al.; Radiology (2010) MR imaging helps predict time from symptom onset in patients with acute stroke: implications for patients with unknown onset time, 257 (3): 782-92, incorporated herein by reference in its entirety).

[0107] In some embodiments, as described herein, the method for treating nerve damage in a subject in need thereof further comprises administering an additional therapy for nerve damage. It is otherwise stated that the treatment methods disclosed herein can be part of a combination therapy for addressing nerve damage. For example, additional therapies for treating stroke can also include, for example, thrombolysis (e.g., tissue plasminogen activator (tPA)), thrombectomy, angioplasty and stents, therapeutic hypothermia and drugs (e.g., aspirin, clopidogrel and dipyridamole). In some embodiments, additional therapies are, for example, thrombolytics, neuroprotectants, anti-inflammatory drugs, steroids, cytokines or growth factors. The thrombolytics used can be tissue plasminogen activator or urokinase. The neuroprotective agent used may be an agonist of a receptor selected from the group consisting of N-methyl-D aspartate receptor (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA), glycine receptor, calcium channel receptor, bradykinin B2 receptor and sodium channel receptor, or selected from the group consisting of bradykinin B1 receptor, α-aminobutyric acid (GABA) receptor and adenosine A1 receptor. The anti-inflammatory drug used may be a member of the interleukin-1 and tumor necrosis factor family.

[0108] Standard tests for neurological recovery (e.g., National Institute of Health Stroke Scale (NIHSS), Barthel index, modified Rankin scale (mRS), Glasgow Outcome scale, Montreal Cognative Assessment (MoCA), Stroke Impact Scale (SIS-16)) can be used by those skilled in the art to determine efficacy. NIHSS classifies stroke severity based on the subject's ability to answer questions and perform activities related to level of consciousness, language, visual field loss, extraocular movements, motor intensity, ataxia, dysarthria, sensory loss, and elimination and inattention. There are 15 items, and the grade of each item is scored on a 3 to 5 scale, where for all items, 0 is normal and 42 is the maximum severity score. NIHSS scores of 1-4 represent mild strokes; scores of 5-15 represent moderate strokes, scores of 16-20 represent moderate to severe strokes: and scores of 21-42 represent severe strokes.

[0109] General Definition

[0110] Unless specifically defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. For definitions and terminology in the art, the skilled artisan specifically refers to Ausubel, FM, et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (2010); Coligan, JE, et al. (Eds.), Current Protocols in Immunology, John Wiley & Sons, New York (2010); Mirzaei, H. and Carrasco, M. (Eds.), Modem Proteomics - Sample Preparation, Analysis and Practical Applications in Advances in Experimental Medicine and Biology, Springer International Publishing, (2016); Comai, L, et al. (Eds.), Proteomic: Methods and Protocols in Methods in Molecular Biology, Springer International Publishing, (2017); Alberts, B., et al. Molecular Biology of the Cell, WW Norton & Company; 6th Edition (2014); and Kandel, ER, et al. Principles of Molecular Biology. ofNeural Science, McGraw-Hill Education / Medical; 5th ed. (2012).

[0111] For convenience, certain terms used herein, in the specification, examples and claims are provided here. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is defined solely by the claims.

[0112] Unless the context requires otherwise, singular terms will include pluralities and plural terms will include the singular. The words "a" and "an" when used with the word "comprising" in a claim or specification mean one or more unless expressly stated otherwise. The term "or" is used in the claims to mean "and / or" unless expressly stated to mean only alternatives or that the alternatives are mutually exclusive, although the present disclosure supports a definition of only alternatives and "and / or".

[0113] Unless the context clearly requires otherwise, throughout this specification and claims, the words "comprising," "including," and the like should be interpreted as inclusive, as opposed to exclusive or exhaustive, and should mean "including, but not limited to." Words using the singular or plural also include the plural and singular, respectively. The term "consisting essentially of means that the referenced composition may include additional elements, variations, and / or sequences, but that the additional elements, variations, and / or sequences do not significantly contribute to the functionality of the composition.

[0114] The word "about" refers to a number that is within a range of a minimum difference above or below a reference number (e.g., an amount, level, value, number, frequency, percentage, measurement, size, amount, weight, or length). For example, "about" can refer to a number that is within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above or below a reference number.

[0115] As used herein, the term "polypeptide" or "protein" refers to a polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used, with the L-isomer being preferred. As used herein, the term polypeptide or protein also encompasses any amino acid sequence and includes modified sequences such as glycoproteins. The term polypeptide is expressly intended to cover naturally occurring proteins, as well as those produced recombinantly or synthetically. The term "peptide" simply refers to a relatively short polypeptide polymer, for example, up to about 20, about 30, about 40, about 50, about 60, about 70, about 80 or about 90 amino acids in length. The term "chimeric" or "fused" in the context of a protein or peptide refers to a first amino acid sequence encoding a polypeptide fused to a second amino acid sequence defining a domain (e.g., a polypeptide portion) that is foreign and substantially non-homologous relative to the domain of the first polypeptide. A chimeric protein may present foreign domains that are present in an organism that also expresses the first protein (but in a different protein), or it may be an "interspecies," "intergenic," etc. fusion protein of protein structures expressed by different species of organisms.

[0116] The skilled artisan will recognize that individual substitutions, deletions or additions to a peptide, polypeptide or protein sequence (which alters, adds or deletes a single amino acid or a percentage of amino acids in the sequence) are "conservatively modified variants" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitution tables provide functionally similar amino acids and are well known to those skilled in the art. The following six groups are examples of amino acids that are considered conservative substitutions for each other:

[0117] (1) Alanine (A), serine (S), threonine (T),

[0118] (2) Aspartic acid (D), Glutamic acid (E),

[0119] (3) Asparagine (N), Glutamine (Q),

[0120] (4) Arginine (R), Lysine (K),

[0121] (5) isoleucine (I), leucine (L), methionine (M), valine (V), and

[0122] (6) Phenylalanine (L), tyrosine (Y), and tryptophan (W).

[0123] Sequence identity refers to the similarity of two polymer sequences such as protein sequences. The determination of sequence identity can be easily completed by those of ordinary skill in the art using accepted algorithms and / or techniques. Sequence identity is typically determined by comparing two optimally aligned sequences on a comparison window, wherein the portion of a peptide or polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the consistent amino acid residues or nucleic acid bases are located in the two sequences, dividing the number of positions matched by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Various software-driven algorithms are easily available, such as BLAST N or BLAST P to perform this comparison.

[0124] The term "wild type" (or "wt") refers to a naturally occurring polynucleotide sequence encoding a protein or a portion thereof, or a protein sequence or a portion thereof, respectively, as it normally occurs in vivo.

[0125] Drugs, compounds, compositions, etc. used in the methods described herein are considered to be purified and / or isolated prior to their use. Purified material is generally "substantially pure," meaning that a nucleic acid, polypeptide, or fragment thereof, or other molecule has been separated from components that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99% free by weight from proteins and other organic molecules with which it is naturally associated. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express the protein, or by chemical synthesis. "Isolated material" has been removed from its natural location and environment. In the case of an isolated or purified domain or protein fragment, the domain or fragment is substantially free of the amino acid sequence that flanks the protein in the naturally occurring sequence.

[0126] When referring to polypeptides, the terms "portion", "fragment", "variant", "derivative" and "analog" include any polypeptide that retains at least part of the biological activity described herein (e.g., inhibiting an interaction such as binding). The polypeptides described herein may include, without limitation, portions, fragments, variants or derivative molecules, as long as the polypeptide still functions. The polypeptides of the present invention or portions thereof may include proteolytic fragments, deletion fragments, specifically, or fragments that are more easily accessible to the site of action when delivered to an animal.

[0127] Disclosed are materials, compositions and components, which can be used for the methods and compositions of the present disclosure, can be used in conjunction with the methods and compositions of the present disclosure, can be used to prepare the compositions of the present disclosure, or are products and compositions of the methods of the present disclosure. It should be understood that when the combination, subset, interaction, group, etc. of these materials are disclosed, even if each individual combination and arrangement of these compounds are not explicitly disclosed (specifically mentioned), each combination in various individual and common combinations is also specifically contemplated. This concept applies to all aspects of the present disclosure, including but not limited to the steps of the method. Therefore, the specific elements of any of the above-mentioned embodiments can be combined or replaced with elements in other embodiments. For example, if there are various executable additional steps, it should be understood that each step in these additional steps can be performed together with any specific method step or combination of method steps of the method of the present disclosure, and each such combination or subset of the combination is specifically contemplated and should be considered to have been disclosed. In addition, it should be understood that the embodiments described herein can be implemented using any suitable material such as those described in other parts of this article or suitable materials known in the art.

[0128] Publications cited herein, and the subject matter to which they are cited, are expressly incorporated herein by reference in their entirety.

[0129] Specific illustrative embodiments are described below, in which the inventors demonstrate that intracellular sigma peptide (ISP) treatment promotes recovery in a neural injury model. Specifically, it has been shown that, for example, by ISP treatment, inhibition of CSPG-induced PTPσ signaling overcomes the CSPG barrier and improves multiple aspects of functional recovery in a murine stroke model, including general motor function, specific upper limb sensory motor function, and cognitive function. The recovery mechanism was also examined. ISP modulation of PTPσ signaling promotes several aspects of recovery by inducing migration and sprouting activity in neural cells that have survived injury (i.e., undamaged neurons) to allow functional compensation of damaged neurons. This ultimately allows the induction of an improvement effect even when ISP treatment is delayed after neural injury.

[0130] First, the efficacy of ISP treatment after stroke in C57BL / 6 mice was tested using the proximal middle cerebral artery occlusion (pMCAO) model. Three groups of mice (n=59) underwent MCAO surgery to induce large strokes in striatal and cortical tissues, mimicking human "malignant" strokes that are often lethal in humans. Stroke mice underwent T2-weighted MRI scans to determine the size of stroke lesions and were blindly divided into two equally distributed groups that received either vehicle (5% DMSO) or ISP (20 mg / mouse / day or 30 mg / mouse / day subcutaneous injection) daily starting 24 hours after stroke onset 6 weeks after stroke onset.

[0131] The mice were characterized by MRI before treatment was initiated 24 hours after stroke. The data showed that there was no difference between the two groups of animals in the extent of ischemic damage as determined by MRI scans (see, e.g. Figure 2A and 2B ISP treatment initiated 24 hours after stroke (compared to the 4.5 hr rtPA treatment window from stroke onset for the only FDA-approved stroke treatment) significantly improved survival in stroke animals (see, e.g. Figure 2C ). This may be due to the effects of anti-CSPGs, which ultimately counteract the inflammatory and swelling response of the brain during the acute phase of stroke.

[0132] In all surviving mice, we observed, by automated empty field analysis using computer monitoring, that post-stroke ISP treatment significantly improved the ability of stroke mice to respond to stroke at 2–4 weeks post-stroke in terms of multiple parameters (i.e., total distance traveled, total horizontal activity, and total vertical activity; see Figure 3A , 3B , 3C) significantly improve motor function.

[0133] Considering that the most common functional deficit after stroke is motor impairment of the contralateral upper limb, and that greater than 90% of human stroke survivors suffer from sensory deficits, the effect of post-stroke ISP treatment on the performance of stroke mice in a sensorimotor behavioral test (i.e., the tape removal test) was also examined. In this test, mice are required to remove tape adhered to their affected and unaffected front paws. The data showed that ISP treatment significantly improved the speed at which mice were able to remove the tape from the affected limb (without any noticeable effect on the unaffected limb). This confirms that the results of ISP treatment are clearly related to the sensory and motor deficits induced by stroke ( Figure 4 ).

[0134] Cognitive decline is also a major cause of disability in stroke survivors. Therefore, the effect of ISP treatment on cognitive function in stroke mice was also examined. The Barnes maze test was used to evaluate the learning / memory function of mice. The data showed that ISP-treated mice used significantly less time and fewer trials and errors to find the escape hole in the Barnes maze 4 weeks after stroke (see Figure 5A ).

[0135] These data demonstrate that systemic ISP treatment improves multiple aspects of functional recovery in stroke mice, including general motor function, specific upper limb sensorimotor function, and cognitive function. These data also suggest that ISP treatment reduces chronic atrophy of the brain after stroke (see Figure 5B The improvements in acute survival and chronic function in surviving mice suggest at least two possible mechanisms that could potentially benefit patients with neurological injuries, thus providing interesting and targetable avenues to increase survival after injuries such as malignant stroke and improve long-term functional recovery in injury survivors.

[0136] In order to test the timing of ISP administration relative to the time of injury and the corresponding effect on recovery, temporary proximal MCAO surgery (35min) was performed on adult C57b1 / 6J female and male mice, as described above. Before stroke and 7 days after stroke, animals were tested for baseline behavior to ensure that there was no difference between the two groups of animals before starting treatment. At the time point when SVZ and SGZ NSC were activated, the effect of delayed post-stroke treatment starting from ISP 7 days after stroke was tested. During 3 consecutive weeks, mice received ISP or vehicle injection (1mg / kg / day) every day. Empty field movement test and tape removal test were performed weekly after stroke until 4 weeks. Figures 6A-6C The delayed ISP treatment modality was shown to provide significant effects in improving performance in multiple parameters (i.e., total distance, horizontal activity, and vertical activity, respectively) over 4 weeks in an open-field exercise test. This has significant clinical translational impact because, as demonstrated, 7 days post-stroke provides a significantly wider treatment window compared to the current FDA-approved tPA treatment window.

[0137] Using a mouse stroke model, it was also shown that ISP treatment enhanced neuroblast formation and axonal sprouting in the corticospinal tract at locations distal to the injury. Figures 7A-7F As shown, these assays demonstrated that post-stroke ISP treatment enhanced DCX+ neuroblasts near the lateral ventricle and adjacent straddle tissue in post-stroke mice. Post-stroke ISP treatment enhanced axonal sprouting from the contralateral corticospinal tract region. This is the first demonstration that post-stroke ISP treatment enhances corticospinal tract projections from the contralateral cortex, showing a mechanism of induced plasticity and spared neuroblasts.

[0138] To further investigate the potential mechanisms of this remarkable functional recovery as described above, an inducible conditional PTPσ knockout model was created from nerve injury induced by ISP treatment. Figure 1 Schematically illustrate the method for implementing the cell-specific deletion of PTPσ. PTPσfloxed mice, neuroepithelial stem cell protein-CreERT2-PTPσ conditional knockout mice (neural stem cell-specific cKO) and cortical neuron-specific cKO (using AAV-hSyn-cre virus injection in PTPσfloxed mice) were produced. After three generations of hybridization, neuroepithelial stem cell protein-CreERT2-PTPσ conditional knockout mice (cKO) were obtained. CKO mice allow the conditional deletion of PTPσ in adult NSC at the desired time. In addition, in these conditional KO mice, AAV-hSynl-cre injection into the contralateral or peri-infarct sites will allow us to specifically make the PTPσ gene in mature sprouting neurons in the contralateral or peri-infarct sites.

[0139] Conditional KO mice were born at the expected Mendelian ratio, confirming that the floxed allele did not affect the normal development and survival of cKO mice without inducing genetic recombination. Cortical neuronal PTPσ mice were generated by injecting AAV-hSyn-cre virus into the motor and somatosensory cortices of PTPσ floxed mice. Successful targeting of the floxed allele and recombination of the allele were confirmed in cKO mice, in NSC-specific cKO, in brain regions containing adult NSCs, and in cortex-specific recombination in AAV-hSyn-cre injected mice (see Figure 1 , 8A -8F and 9A-9H).

[0140] Figures 8A-8F It is shown that conditional knockout mice allow the study of the role of the CSPG-PTPσ pathway in neurogenesis, and its contribution to functional recovery after neuronal injury such as stroke. Figures 9A-9H It is shown that conditional knockout mice allow the study of the effects of PTPσ modulation on axon sprouting mechanisms by injecting AAV-hSyn-cre into the peri-infarct region as well as into the contralateral cortical region, thereby deleting the gene in the mature neurons present at the expected time. In this model, peri-infarct injections and contralateral cortical injections allow the distinction of the contribution of proximal processes (sprouting of peri-infarct neurons) and any distal processes (sprouting of contralateral cortical neurons).

[0141] Because initial results showed that ISP treatment increased the number of DCX+ cells migrating toward the infarct zone (see Figures 7A-7FSVZ NSC migration from wild-type or cKO PTPσ mice was examined as described above. Adult neural stem cell (NSC) neurosphere cultures were prepared from the SVZ of adult wild-type and NSC-cKO mice ( Fig. 10A Migration assays showed that neuroepithelial stem cell protein (+) NSCs produced CSPG ( Fig. 10B and 10C In the CSPG spot assay, it was shown that wild-type NSCs were unable to cross the CSPG ring edge ( Fig. 10D ), consistent with previous studies. In stark contrast, cKO NSCs were able to cross the outer edge of the CSPG ring, indicating that PTPσ function was successfully eliminated in cKO NSCs ( Fig.10E ).

[0142] In addition, CSPG-induced PTPσ signaling was demonstrated for the first time to be critical in regulating migration and neurite outgrowth in adult NSCs. Aggrecan basal coating resulted in reduced migration of adult NSCs, and deletion of the PTPσ gene in cKO NSC cells resulted in decreased migration of adult NSCs at basal levels (without aggrecan coating) and in the presence of aggrecan coating (see Figures 11A-11E ) both enhance migration. Pharmacological inhibition of the CSPG-PTPσ pathway by ISP exhibits similar results to PTPσ gene deletion (see Fig. 12A and 12B ). Furthermore, both genetic deletion of PTPσ in adult cKO NSCs and pharmacological inhibition of PTPσ by ISP peptides consistently resulted in increased neurite outgrowth in differentiated NSCs, whereas scrambled ISP peptides had no effect (see Figures 13A-13C , 14A and 14B).

[0143] In short, these data obtained from PTPσ conditional knockout mice show the clear functional importance of PTPσ in the basic biology of nerve cells (including adult NSC) spared after nerve injury.The observed functions include neuronal differentiation, neurite outgrowth and migration, which are important cellular mechanisms involved in neurogenesis under basic conditions and in damage such as post-stroke plasticity.Significantly, it has been shown that the ISP treatment started even 7 days after stroke still effectively enhances functional recovery.This result is extremely important, because there is currently no available therapeutic therapy beyond the acute treatment window (6 hours pharmacologically and 24 hours surgically) in stroke patients.

[0144] Because neurogenesis is not a major contributor to neural repair in SCI, the role of the CSPG-PTPσ pathway has previously been studied more in damaged neurons in the spinal cord injury (SCI) model. However, neurogenesis and migration of DCX+ neuroblasts have been shown to make functional contributions to post-stroke recovery. The data disclosed herein show that genetic deletion of PTPσ in adult NSCs and pharmacological inhibition of PTPσ by ISPs consistently enhance neurite outgrowth in newly differentiated neuroblasts and migration of these cells.

[0145] Example

[0146] The following examples are provided in this disclosure for purposes of illustration and not limitation.

[0147] Example

[0148] Materials and methods for implementing various embodiments of the present disclosure

[0149] 1. Animals

[0150] C57BL / 6 mice were purchased from Jackson Laboratory and housed in the animal facility of Case Western Reserve University. Mice were maintained on a 12-hour light / dark cycle and were fed ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University. C57BL / 6 male mice aged 10-12 weeks were used in this study.

[0151] 2. Murine Model of Transient Focal Ischemia

[0152] Temporary middle cerebral artery occlusion (tMCAO) was induced in male C57BL / 6 mice (12 weeks old, 25-30 g) by intraluminal occlusion of the left MCA for 45 min with a monofilament coated with silicone rubber (Cat.602212PK10Re and 602312PK10Re, Doccol Corporation). In brief, mice were anesthetized with isoflurane. Body temperature was monitored and maintained at 37 ± 0.5 ° C by a constant temperature blanket control unit (Harvard apparatus). In order to minimize the pain of animals, buprenorphine was injected subcutaneously to mice. A midline incision was made on the skin covering the top of the skull, and the skin was pulled laterally to fix a flexible microtip on the surface of the left top skull of the mouse (0.5 mm behind the bregma and 3.5 mm on the side). Subsequently, a midline neck incision was made to separate the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) of the mouse. The silicone rubber coated monofilament was introduced into the ECA via arteriotomy and slowly advanced through the ICA toward the starting point of the MCA according to the Longa method. To ensure consistent and successful blockade of the MCA, regional cerebral blood flow was monitored in all stroke animals by laser Doppler flowmetry (PeriFlux system 5000, Perimed, Sweden). After incision closure, mice were given 1 ml of warm saline subcutaneously and placed in a heated animal intensive care unit until recovery.

[0153] 3. Magnetic resonance imaging (MRI)

[0154] 23 h after induction of cerebral ischemia, infarct volume was measured using a horizontal biospec 9.4T scanner (Bruker Inc., Billerica, MA) equipped with a 3-cm cage coil. During the MRI scanning procedure, mice were anesthetized with a 1.5% isoflurane / oxygen mixture and placed in a prone position in a cradle. The body temperature of the mice was maintained at 33°C by blowing warm air into the scanner through a feedback control system (SA Instruments, Stony Brook, NY). During the experiment, respiratory rate was also monitored. To quantify the volume of ischemic edema, multi-slice T2-weighted axial images were acquired using a rapid acquisition and relaxation enhancement (RARE) sequence under the following parameters: TE / TR, 15 / 2000ms; RARE factor, 8; NAV, 4; matrix size, 256x256; layer thickness, 1mm; number of layers, 13; field of view, 2.4x2.4cm. Image reconstruction and analysis were performed using MATLAB-based software developed in-house (Natick, MA, USA). The ischemic edema volume and brain tissue ROI were derived from T2-weighted images. Therefore, the percentage of ischemic infarct volume was calculated according to the following formula: ∑(contralateral area-ipsilateral non-infarct area) / ∑contralateral area×100%.

[0155] 4. Peptide Preparation

[0156] The peptides were purchased from CS-Bio (CA, USA) with >98% purity. The lyophilized peptides were dissolved in sterile water and stored at -80°C until use. The peptide sequences are as follows:

[0157] ISP: GRKKRRQRRRCDMAEHMERLKANDSLKLSQEYESI(SEQ ID NO: 62)

[0158] Messy ISP (SISP): GRKKRRQRRRCIREDDSLMLYALAQEKKESNMHES

[0159] (SEQ ID NO: 63)

[0160] 5. Systemic peptide therapy

[0161] First, a 10% DMSO (1.25 ml DMSO in 23.73 ml sterile saline) vehicle solution was prepared for each mouse. Then, the appropriate ISP peptide was added to the vehicle solution, then aliquoted into 1.5 ml Eppendorf tubes (each corresponding to the daily dose for a single mouse) and frozen at -80°C. The final ISP peptide concentration was 0.3 μg / μl. After MRI scanning, ischemic mice were randomly divided into two equally distributed groups according to the size of the stroke lesion. 24 h after ischemia and every afternoon thereafter during 6 weeks, mice were subcutaneously injected with ISP (30 μg / day, 100 μl) or vehicle (5% DMSO / saline, ). The experiment was conducted in a blind manner.

[0162] 6. Quantifying Brain Atrophy in Stroke Animals

[0163] Brain sections (25 μm) at 6 weeks post-stroke were mounted on PLL-coated slides. The sections were rehydrated in KH2PO4 buffer (pH 4.5) for 10 min and then stained in preheated 10% Giemsa solution at 42°C for 30 min. After a quick rinse with KH2PO4 buffer, the sections were dehydrated in absolute ethanol, transparentized in xylene, and then mounted with Histoseal. A series of sections were imaged by Path Scan Enabler IV slide scanner. The contralateral and ipsilateral brain areas were quantified using ImageJ software. The atrophy rate was calculated as follows: ∑(contralateral brain area × ipsilateral brain area) / ∑contralateral brain area × 100%.

[0164] 7. Tracing and quantification of anterograde axonal sprouting

[0165] Four weeks after tMCAO, mice were anesthetized with a 1.5% isoflurane / oxygen mixture and stabilized in a stereotaxic frame. 1.5 μl of biotin dextran amine (BDA, MW10,000; 10% PBS, invitrogen) was injected into three sites in the cortex contralateral to the lesion (coordinates: 1. A / P 0.0mm, M / L-2mm, D / V-1mm; 2. A / P 0.5mm, M / L-1.5mm, D / V-1mm; 3. A / P 0.5mm, M / L-2mm, D / V-1mm,). Two weeks later, the brain and cervical spinal cord were collected after cardiac perfusion with PBS and then 4% paraformaldehyde. After overnight fixation in 4% paraformaldehyde and cryoprotection in 20% and 30% sucrose, coronal brain slices and spinal cord cross-sections were cut into 30 μm thickness. For BDA detection, sections were rinsed in 0.1 M PB and incubated in 0.3% H2O2 for 30 min to inactivate endogenous peroxidase, and then ABC kit (Vector Laboratories, Burlingame, CA, USA) was incubated for 2 hours. The staining was visualized with 2,3' diaminobenzidine tetrahydrochloride (0.5 mg / ml in 0.1 M PB). The number and length of BDA+ fibers crossing the midline were assessed in a blinded manner. Sections were analyzed using ImageJ software.

[0166] 8. Neurobehavioral Assessment

[0167] All behavioral tests were performed in a blinded manner in the light phase. To reduce stress, mice were acclimated to the behavioral testing room for 1 h before the start of the test. All apparatus were cleaned with 75% ethanol to avoid instinctive odors between mice.

[0168] 8.1 Motor Function

[0169] As previously described, mouse locomotor activity was assessed using an Accuscan activity monitor (Columbus, OH, USA) one day before and 3, 7, 14, 21, 35, and 42 days after tMCAO. There are 16 horizontal and 8 vertical infrared sensors (2.5 cm apart) in this monitor. Each mouse was placed in a 42x42x31cm resin glass open box for 1 hour with food and water supply. In order to avoid observer bias, this locomotor test was automatically monitored by computer and software. Locomotor activity was calculated by automated Versamax software (Accuscan, Columbus, OH, USA). The following variables were measured: (A) horizontal activity (the total number of beam interruptions in the horizontal sensor); (B) total distance traveled (cm, the distance traveled by the animal); (C) vertical activity (the total number of beam interruptions in the vertical sensor).

[0170] 8.2 Barnes maze test

[0171] Spatial memory of ischemic mice was examined using a Barnes maze (Stoelting Company, WoodDale, IL, USA) 28 days after tMCAO. The maze consisted of a 91.5 cm diameter circular platform with 20 holes around the perimeter. Mice were prevented from wandering aimlessly around by a blower fan and a strong light above the platform.

[0172] On day 0, after removal of the start chamber, mice were gently guided into the target hole.

[0173] On the first day, mice were trained 4 times in 2 phases to find the escape passage under the target hole. Once the mice entered the target hole, the hole was covered and the mice were allowed to stay therein for 2 min. If the mice could not find the target hole position within 5 min, the mice were guided into the target hole by the observer. On the second day, a test was conducted, and the video was taken until the mice entered the target hole or stopped at 5 mins when the mice could not find the target hole position. The time spent to find the escape hole and the number of errors made by the mice when looking for the hidden hole were measured by the observer in a blind manner.

[0174] 8.3 Tape Removal Test

[0175] In order to check sensory motor deficits, this test was performed 7, 14, 21, 28, 35 and 42 days after stroke. Each mouse was placed in a transparent cylinder (15 cm diameter) during the 1 min adaptation period. Thereafter, two different colored adhesive markers (punched to obtain 2.5 mm diameter, Tough Spots) were applied to the front paws of each mouse with equal pressure. The required time for removing the adhesive markers was measured, up to 2 min. In order to achieve the best performance level, mice should be trained for 4 days before surgery.

[0176] 9. Neural Stem Cell Culture

[0177] Primary stem cells were obtained from 5-week-old C57BL / 6J mice. Immediately after euthanasia, the whole brain was collected and sliced ​​under a microscope to obtain subventricular zone (SVZ) tissue. After mechanical dissociation with a puncture knife, the tissue fragments were treated with trypsin and 10 4 cells / cm 2 The cells were resuspended at 4% saturation as single cells in neurobasal medium (NBM-GF) supplemented with growth factors (epidermal growth factor and basic fibroblast growth factor). Subsequent passage of cells was performed every 7 days using (innovative #AT-104, CA, USA) until the cells established a viable cell line and the cell debris naturally decreased after each passage. On day 4 of each passage, the proliferating spheres were fed with NBM-GF. In this study we used neurospheres at passages P3-P8.

[0178] 10. CSPG gradient crossing assay

[0179] CSPG gradients were prepared on coverslips as described previously. Briefly, 24-well glass coverslips were coated with poly-L-lysine and nitrocellulose, and a mixture of 700 ug / ml aggrecan (A1960) and 10 μg / ml laminin (11243217001, Sigma) was spotted on the coated coverslips. After drying, the coated coverslips were incubated with laminin at 37°C for 3 hours. Transfected neural stem cells were plated on 10 4 Cells were plated at a density of 10 cells / coverslip and cultured in NBM-GF medium. After 7 days, wells were fixed with 4% paraformaldehyde for 15 min at room temperature and stored at 4°C in phosphate-buffered saline until staining.

[0180] 11. Migration of Neural Stem Cells on CSPGs

[0181] To determine the effect of CSPG on neural stem cell migration in vitro, flat-bottomed 48-well plates were first coated with poly-L-lysine overnight and then rinsed with water. Aggregate proteoglycan (A1960, sigma) was coated on 48-well plates, diluted in sterile water at concentrations of 1 μg / ml and 10 μg / ml, overnight, and then rinsed with water. Control wells contained only poly-L-lysine. Neurospheres of equal size were seeded in each well in NBM-GF medium containing 2.5 μM ISP peptide or scrambled peptide (n=7 neurospheres in 7 wells, each condition), and the plates were incubated at 37°C for 21 hours. Thereafter, images of each well were taken using a LeicaDMi8 widefield microscope. Migration activity was defined as the total area of ​​the neurosphere divided by the inner area of ​​the neurosphere. The inner area and total area of ​​the neurosphere were measured by ImageJ software. Distance measurements were performed by observers in a blinded manner.

[0182] 12. Neural Stem Cell Differentiation Assay

[0183] Briefly, glass coverslips in untreated 24-well plates were coated with poly-ornithine and laminin. After neurospheres were split during passaging, individual cells were plated at 10 4 cells / cm 2Density seeded in 500 μl NBM-GF. Every other day, 250 μl of culture medium was removed from each well and 300 μl of fresh NBM-GF was added. When the attached cells reached approximately 70% confluence (approximately day 5), all NBM-GF in each well was gently removed and immediately replaced with neurobasal medium (without growth factor (NBM), containing ISP peptide (2.5 μM) or scrambled peptide (2.5 μM)). Each well was replenished every day by removing 250 μl of culture medium and adding 300 μl of culture medium (containing ISP or scrambled peptide). On day 5 after the replacement of NBM-GF was completed, the wells were fixed with 4% polyformaldehyde for 15 min at room temperature and stored in phosphate-buffered saline at 4°C until staining.

[0184] 13. Immunohistochemistry

[0185] Mice were anesthetized with Avertin and perfused with PBS and 4% polyoxymaldehyde (PFA). The brain was sliced ​​and postfixed overnight in 4% PFA at 4°C, balanced in 20% sucrose and 30% sucrose. 25 μm thick slices were incubated for 1 hour in 4% BSA / 0.3% Triton-x100. After blocking, the slices were incubated overnight at 4°C with primary antibodies, and then incubated with appropriate secondary antibodies (conjugated with Alexa fluorescence 488 or 594). The following primary antibodies were used: 5-HT (1: 500, Immunostar, Hudson, WI) and CS56 (1: 500, C8035, Sigma). For each staining, at least three separate animals / groups were detected, and images were captured with a fluorescent microscope. Staining was quantified using Image J software (US NationalInstitutes of Health, USA).

[0186] 14. Immunocytochemistry

[0187] Cells cultured on coverslips were fixed in 4% PFA for 15 min, permeabilized in 0.1% Triton-X100 for 10 min, and then incubated in 10% normal goat serum for 1 hour. Thereafter, cells were incubated in diluted primary antibodies at 4°C overnight, followed by incubation in appropriate secondary antibodies goat anti-mouse or anti-rabbit IgM or IgG (conjugated with Alexa Fluor 488 or 594) (1:1000, Invitrogen). Glass coverslips were mounted on microscope slides in Mowiol containing DAPI (Sigma, St. Louis, USA). The following primary antibodies were used: MAP2 (1:500, AB5622, Millipore), neuroepithelial stem cell protein (1:500, NB100-1604, Novus), and CS56 (1:500, C8035, Sigma). Three coverslips were analyzed for each condition. Fields in each coverslip were randomly selected and imaged by Stereo Investigator Software (MBF Bioscience, Williston, VT, USA), and quantitative data were obtained by using NIH ImageJ software.

[0188] 15. Statistical Analysis

[0189] All studies were analyzed in a blinded manner using GraphPad Prism 6.00 software. Data are expressed as mean ± standard deviation. Statistical significance was set at p < 0.05. Statistical analysis was performed by two-tailed unpaired Student's t-test, one-way or one-way ANOVA and post hoc analysis by Tukey's multiple comparison test, Dunnett's multiple comparison test, or Sidak's multiple comparison test. No statistical test was used to predetermine the sample size, but our sample size is similar to that commonly used in the field.

[0190] While exemplary embodiments have been shown and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention. Sequence Listing <110> Cass Western Reserve University <120> Methods and compositions for inducing neural plasticity <130> CWR-026868WO ORD <160> 63 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> PRT <213> African clawed frog (Xenopus borealis) <400> 1 Asp Leu Ala Glu His Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Ser Gln Glu Tyr Glu Ser 20 <210> 2 <211> twenty one <212> PRT <213> Anolis carolinensis <400> 2 Asp His Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln 1 5 10 15 Glu Tyr Glu Ser Ile 20 <210> 3 <211> twenty four <212> PRT <213> Zebrafish (Danio rerio) <400> 3 Glu Leu Ala Glu His Thr Glu Leu Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 4 <211> twenty four <212> PRT <213> Aureus Tilapia (Oreochromis aureus) <400> 4 Glu Leu Ala Glu His Thr Glu Leu Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 5 <211> 24 <212> PRT <213> Gallus gallus <400> 5 Glu Leu Ala Glu His Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 6 <211> 24 <212> PRT <213> Fringilla coelebs <400> 6 Glu Leu Ala Glu His Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 7 <211> 24 <212> PRT <213> Ornithorhynchus anatinus <400> 7 Glu Leu Ala Glu His Thr Asp His Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 8 <211> 24 <212> PRT <213> Tasmanian devil (Sarcophilus harrisii) <400> 8 Glu Met Ala Glu His Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 9 <211> 24 <212> PRT <213> Ferret (Mustela putorius) <400> 9 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 10 <211> 24 <212> PRT <213> Allen's bushbaby (Galago alleni) <400> 10 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 11 <211> 24 <212> PRT <213> Golden-handed tamarin (Callithrix aurita) <400> 11 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 12 <211> 24 <212> PRT <213> Black rat (Rattus rattus) <400> 12 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 13 <211> 24 <212> PRT <213> Mouse (Mus musculus) <400> 13 Asp Met Ala Glu His Met Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 14 <211> 24 <212> PRT <213> Dog (Canis familiaris) <400> 14 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 15 <211> 24 <212> PRT <213> Bearded pig (Sus barbatus) <400> 15 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 16 <211> 24 <212> PRT <213> Bos primigenius <400> 16 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 17 <211> 24 <212> PRT <213> Ovis aries <400> 17 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 18 <211> 24 <212> PRT <213> Orcinus orca <400> 18 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 19 <211> twenty four <212> PRT <213> Squirrel Monkey (Saimiri sciureus) <400> 19 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 20 <211> twenty four <212> PRT <213> Hamadryas Baboon (Papio hamadryas) <400> 20 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> twenty one <211> twenty four <212> PRT <213> Gorilla <400> twenty one Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> twenty two <211> twenty four <212> PRT <213> White-handed gibbon (Hylobates lar) <400> twenty two Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 23 <211> 24 <212> PRT <213> Capped langur (Macaca radiata) <400> 23 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 24 <211> 24 <212> PRT <213> Chimpanzee (Pan troglodytes) <400> 24 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 25 <211> 24 <212> PRT <213> Human (Homo sapiens) <400> 25 Asp Met Ala Glu His Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys 1 5 10 15 Leu Ser Gln Glu Tyr Glu Ser Ile 20 <210> 26 <211> 24 <212> PRT <213> Black rat (Rattus rattus) <400> 26 Asp Leu Ala Asp Asn Ile Glu Arg Leu Lys Ala Asn Asp Gly Leu Lys 1 5 10 15 Phe Ser Gln Glu Tyr Glu Ser Ile 20 <210> 27 <211> 24 <212> PRT <213> Black rat (Rattus rattus) <400> 27 Glu Leu Ala Asp His Ile Glu Arg Leu Lys Ala Asn Asp Asn Leu Lys 1 5 10 15 Phe Ser Gln Glu Tyr Glu Ser Ile 20 <210> 28 <211> 24 <212> PRT <213> Black rat (Rattus rattus) <400> 28 Lys Leu Glu Glu Glu Ile Asn Arg Arg Met Ala Asp Asp Asn Lys Ile 1 5 10 15 Phe Arg Glu Glu Phe Asn Ala Leu 20 <210> 29 <211> 10 <212> PRT <213> Human (Homo sapiens) <400> 29 Asp Met Ala Glu His Thr Glu Arg Leu Lys 1 5 10 <210> 30 <211> 4 <212> PRT <213> Homo sapiens <400> 30 Ala Asn Asp Ser 1 <210> 31 <211> 10 <212> PRT <213> Homo sapiens <400> 31 Leu Lys Leu Ser Gln Glu Tyr Glu Ser Ile 1 5 10 <210> 32 <211> 14 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (3) <223> Xaa is T or M <400> 32 Glu His Xaa Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu 1 5 10 <210> 33 <211> 14 <212> PRT <213> Homo sapiens <220> <221> MOD_RES <222> (6) <223> Xaa is any amino acid residue <400> 33 Asp Met Ala Glu His Xaa Glu Arg Leu Lys Ala Asn Asp Ser 1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 34 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 35 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 35 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Leu Ala Glu His 1 5 10 15 Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys Ser Gln Glu Tyr Glu 20 25 30 Ser <210> 36 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 36 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp His Thr Glu His 1 5 10 15 Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr Glu Ser Ile 20 25 30 <210> 37 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 37 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Leu Ala Glu His 1 5 10 15 Thr Glu Leu Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 38 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 38 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Leu Ala Glu His 1 5 10 15 Thr Glu Leu Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 39 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 39 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Leu Ala Glu His 1 5 10 15 Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 40 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 40 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Leu Ala Glu His 1 5 10 15 Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 41 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 41 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Leu Ala Glu His 1 5 10 15 Thr Asp His Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 42 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 42 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu Met Ala Glu His 1 5 10 15 Thr Glu His Leu Lys Ala Asn Asp Asn Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 43 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 43 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 44 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 44 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 45 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <400> 45 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 46 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 46 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 47 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 47 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Met Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 48 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 48 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 49 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid <400> 49 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 50 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 50 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 51 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <400> 51 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 52 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 52 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 53 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 53 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 54 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> misc_feature <222> (11)..(11) <223> Xaa can be any naturally occurring amino acid <400> 54 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 55 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 55 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 56 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 56 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 57 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 57 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 58 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 58 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 59 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <400> 59 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Thr Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 60 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <220> <221> MOD_RES <222> (14)..(14) <223> Xaa is any amino acid residue <400> 60 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Glu His Xaa Glu Arg 1 5 10 15 Leu Lys Ala Asn Asp Ser Leu Lys Leu 20 25 <210> 61 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (11)..(11) <223> Xaa is any amino acid residue <220> <221> MOD_RES <222> (17) <223> Xaa is any amino acid residue <400> 61 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Xaa Asp Met Ala Glu His 1 5 10 15 Xaa Glu Arg Leu Lys Ala Asn Asp Ser 20 25 <210> 62 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 62 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Cys Asp Met Ala Glu His 1 5 10 15 Met Glu Arg Leu Lys Ala Asn Asp Ser Leu Lys Leu Ser Gln Glu Tyr 20 25 30 Glu Ser Ile 35 <210> 63 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 63 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Cys Ile Arg Glu Asp Asp 1 5 10 15 Ser Leu Met Leu Tyr Ala Leu Ala Gln Glu Lys Lys Glu Ser Asn Met 20 25 30 His Glu Ser 35

Claims

1. Use of a composition for the manufacture of a medicament for treating nerve damage by promoting compensatory plasticity of spared nerve cells after nerve damage, the composition comprising: A therapeutic agent comprising: a therapeutic peptide, wherein the therapeutic peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-25 and 32, and a transport moiety attached to the therapeutic peptide and facilitating uptake of the therapeutic peptide by a cell, wherein the neural injury is located in the brain and is due to focal cerebral ischemia, global cerebral ischemia, malignant stroke, acute ischemic stroke, chronic stroke disease, thrombosis or embolism, and the therapeutic peptide induces compensatory neurite growth of the spared neural cells in the brain.

2. The use according to claim 1, wherein the spared neural cells are neural stem cells.

3. The use according to claim 1, wherein the spared neural cells comprise oligodendrocyte progenitor cells (OPC) and / or glial precursor cells (GPC).

4. The use according to claim 1, wherein the spared neural cells are neurons. The use according to claim 1 , wherein the neurite growth comprises axonal sprouting of the spared nerve cells. The use according to claim 1 , wherein the neurite growth comprises dendritic sprouting or bifurcation of the spared neural cells.

7. The use according to claim 1, wherein the peptide induces compensatory migration of spared neural cells toward the neural injury.

8. The use according to claim 1, wherein the transport moiety is the HIV Tat transport moiety.

9. The use according to claim 1 or 8, wherein the transport moiety is linked to the therapeutic peptide by a peptide linker.

10. The use according to any one of claims 1-7, wherein the therapeutic agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-61.

11. The use according to any one of claims 1 to 7, wherein the spared neural cells are contacted with the therapeutic agent within 7 days after injury.

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