Treatment of glial scarring with CSPG reduction peptides

Abstract

Provided herein are compositions, systems, kits, and methods for treating glial scarring caused by injury to a central nervous system (CNS) site by administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP. In particular embodiments, CNS site is the result of a stroke in a subject (e.g., human subject that has had a stroke). In certain embodiments, the CRP is composed of a cell membrane penetrating domain, a lysosome targeting domain, and a chondroitin sulfate proteoglycan (CSPG) binding domain.

Description

[0001] TREATMENT OF GLIAL SCARRING WITH CSPG REDUCTION PEPTIDES

[0002] The present application claims priority to U.S. Provisional application 63 / 668,559, filed July 8, 2024, which is herein incorporated by reference in its entirety.

[0003] FIELD

[0004] Provided herein are compositions, systems, kits, and methods for treating glial scarring caused by injury to a central nervous system (CNS) site by administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP. In particular embodiments, CNS site is the result of a stroke in a subject (e.g., human subject that has had a stroke). In certain embodiments, the CRP is composed of a cell membrane penetrating domain, a lysosome targeting domain, and a chondroitin sulfate proteoglycan (CSPG) binding domain.

[0005] BACKGROUND

[0006] There are over 795,000 people in the United States suffering strokes every year and 87% of all strokes are ischemic strokes, where blood flow to the brain is blocked (Stroke Facts, CDC). Ischemic strokes is a leading cause of brain damage, which eventually causes long-term disability and death in humans [1]. There are few therapeutic options existed to treat stoke patients who are typically associated with anatomical deficits and functional impairment in the chronic phase of injury. The development of glial scars at the border between spare and damaged tissue is one of the major reasons to limit recovery from stroke in both preclinical animal models [2,3] and human patients [4,5]. The inhibitory gliosis at the injured cortex (gray matter) has been well studied after ischemia stroke [2,3]. In addition to the gray matter injury, stroke-related motor deficit is caused by profound damaged corticospinal tract and associated white matter changes [6,7]. However, there is little information regarding the development of gliosis in the distal damaged corticospinal tract (CST) in the cervical spinal cord that may limit the plasticity of remodeling or axonal regeneration from the ischemic stroke.

[0007] The glial scars, which contain reactive astrocytes and inhibitory extracellular matrix (ECM) proteins such as chondroitin sulfate proteoglycans (CSPGs) [8,9], appears to serve a beneficial function in the acute phase of stroke, by confining the lesion and limiting toxic effects on adjacent tissue

[0010] . However, as stroke becomes chronic, glial scars inhibit axonal growth and, perhaps, other repair processes [11-13], thereby interfering with long-term anatomical and functional recovery.

[0008] SUMMARY

[0009] Provided herein are compositions, systems, kits, and methods for treating glial scarring caused by injury to a central nervous system (CNS) site by administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP. In particular embodiments, CNS site is the result of a stroke in a subject (e.g., human subject that has had a stroke). In certain embodiments, the CRP is composed of a cell membrane penetrating domain, a lysosome targeting domain, and a chondroitin sulfate proteoglycan (CSPG) binding domain.

[0010] In some embodiments, provided herein are methods of treating glial scarring caused by injury to a central nervous system (CNS) site of a subject comprising: administering a composition comprising a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP (e.g., mRNA in a lipid carrier, DNA in an expression vector, etc.), to a subject with glial scarring caused by injury to a CNS site, wherein the CRP comprises: i) a cell membrane penetrating domain, ii) a lysosome targeting domain; and iii) a chondroitin sulfate proteoglycan (CSPG) binding domain.

[0011] In further embodiments, provided herein are kits and systems comprising: a) composition comprising a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP (e.g., mRNA in a lipid carrier, DNA in an expression vector, etc.), wherein the CRP comprises: i) a cell membrane penetrating domain, ii) a lysosome targeting domain; and iii) a chondroitin sulfate proteoglycan (CSPG) binding domain; and b) a device for administering the composition. In certain embodiments, the composition is located inside the device.

[0012] In particular embodiment, the administering at least partially causes reduction in the level of glial scarring at the CNS site (e.g., 5% . . . 10% . . . 20% . .. 30% . . . 40% . . . 50% . . . or more). In other embodiments, the glial scarring reduction is measured by a computerized tomography (CT) scan or an MRI scan.

[0013] In further embodiments, the injury to the CNS site is caused by a stroke. In additional embodiments, the administering causes at least some cognitive ability gain in the subject, and wherein optionally the cognitive ability gain is measured by: Cognitive Assessment Scale for Stroke Patients (CASP); Montreal Cognitive Assessment (MoCA); IQCODE; or short- IQCODE. In further embodiments, the CNS site is located in the spinal cord of the subject. In other embodiments, the CNS site is located in the brain of the subject. In some embodiments, the subject is a human, wherein the CNS site was caused by a stroke, which is optionally a chronic ischemic stroke. In additional embodiments, the administering is conducted within about 12 or 24 or 36 or 48 hours, or between 6 and 24 hours, or between 12 and 24 hours, of the injury to the CNS site. In further embodiments, the administering is conducted after at least three days since the injury to the CNS site. In additional embodiments, the administering is conducted after a week or more, or a month or more, since the injury to the CNS site. In certain embodiments, the administration is accomplished by a method selected from: subcutaneous, intravenous, intracranial, and intrathecal injection or infusion.

[0014] In some embodiments, the cell membrane penetrating domain is located at the N- terminal or C-terminal of the CRP. In other embodiments, the lysosome targeting domain is located at the N-terminal or C-terminal of the CRP. In further embodiments, the CSPG binding domain is located between the cell membrane penetrating domain and the lysosome targeting domain. In additional embodiments, the cell membrane penetrating domain comprises or consists of an amino acid sequence shown in any of SEQ ID NOS: 1 and 5-27 and 59-61, or a sequence with one or two amino acid additions, subtractions, or substitutions. In other embodiments, the lysosome targeting domain comprises or consists of an amino acid sequence shown in any of SEQ ID NOS: 2 or 28-36, or a sequence with one or two amino acid additions, subtractions, or substitutions. In particular embodiments, the CSPG binding domain comprises or consists of an amino acid sequence shown in any of SEQ ID NO: 3 or 37-53 or 62-71, or a sequence with one or two amino acid additions, subtractions, or substitutions. In additional embodiments, the administering is performed by a method selected from: intravenously and subcutaneous injection (e.g., above the cervical spinal cord).

[0015] In some embodiment, provided herein are compositions comprising a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP comprises: a) a cell membrane penetrating domain which comprises or consists of an amino acid sequence shown in any of SEQ ID NOs: 59-61, b) a lysosome targeting domain; and c) a chondroitin sulfate proteoglycan (CSPG) binding domain.

[0016] In further embodiments, provided herein are compositions comprising: a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP comprises: a) a cell membrane penetrating domain, b) a lysosome targeting domain; and c) a chondroitin sulfate proteoglycan (CSPG) binding domain which comprises or consists of an amino acid sequence shown in any of SEQ ID NOs: 62-71. DESCRIPTION OF THE FIGURES

[0017] Figure 1. CRP reduces stroke-increased CSPGs by directing specifically -bound CSPGs to a lysosome for degradation.

[0018] Figure 2. The experimental design and procedures in Example 1 to test efficacy of CRP in reducing CSPGs in rats with chronic ischemia stroke.

[0019] Figure 3. Two- week delayed CRP treatment significantly decreases photothrombotic ischemia-increased GFAP+ astrocytes at the sites surrounding the ischemic lesions in the cortex. The delayed treatment with scrambled peptide or CRP was started at 2 weeks postischemia, twice a day via subcutaneous injection, till 8 weeks post-ischemia. IHC was used to detect GFAP+ astrocytes (magenta) at 8 weeks after photothrombotic ischemia. As shown in the representative confocal images (A-D), significantly increased GFAP was observed in rats treated with scrambled peptide (A, B) in the cortex surrounding the ischemic lesions, as compared to rats with CRP treatment (C, D). Panels B and D were the higher-magnified images boxed in the panels A and C, respectively. Graphs represent mean ± SEM of 5 or 6 animals per group for the percentage intensity change of GFAP to the ipsilateral site (E). **p < 0.01 vs. scrambled peptide-treated group (Mann-Whitney test, two-tailed P value) by Graphpad Prism 8.4.3.

[0020] Figure 4. Two-week delayed CRP treatment significantly decreases photothrombotic ischemia-increased CSA at the sites surrounding the ischemic lesions in the cortex. The delayed treatment with scrambled peptide or CRP was started at 2 weeks post-ischemia, twice a day via subcutaneous injection, till 8 weeks post-ischemia. IHC was used to detect CSA (green) at 8 weeks after photothrombotic ischemia. As shown in the representative confocal images (A-D), significantly increased CSA was observed in rats treated with scrambled peptide (A, B) in the cortex surrounding the ischemic lesions, as compared to rats with CRP treatment (C, D). Panels B and D were the higher- magnified images boxed in the panels A and C, respectively. Graph represents mean±SEM (N=5 or 6 per group) of the percentage intensity change of GFAP to the ipsilateral site (E) and groups were compared using Mann- Whitney test, two-tailed P value: **p < 0.01 vs. scrambled peptide-treated group by Graphpad Prism 8.4.3.

[0021] Figure 5. Two-week delayed CRP treatment significantly decreases photothrombotic ischemia-increased GFAP+ astrocytes in the contralateral corticospinal tract (CST) of the cervical spinal cord. The chronic ischemia-induced increases in GFAP+ astrocytes (green) were especially significant in the contralateral CST of the cervical spinal cord at 8 weeks after photothrombotic ischemia, as compared to ipsilateral CST when treatment with scrambled peptide (A). Such significant increases in GFAP+ astrocytes were significantly decreased by two- week delayed CRP treatment (B). (C) Graph represents mean+SEM (N=5 or 6 per group) and groups were compared using Mann- Whitney test, two-tailed P value: **p < 0.01 vs. scrambled peptide-treated group by Graphpad Prism 8.4.3.

[0022] Figure 6. Two-week delayed CRP treatment significantly decreases photothrombotic ischemia-increased CSA in the contralateral CST of the cervical spinal cord. The chronic ischemia-induced increases in CSA (red) were especially significant in the contralateral CST of the cervical spinal cord at 8 weeks after photothrombotic ischemia when compared to ipsilateral CST when treatment with scrambled peptide (A). Such significant increases in CSA were significantly decreased by two-week delayed CRP treatment (B). (C) Graph represents mean±SEM (N=5 or 6 per group) and groups were compared using Mann- Whitney test, two-tailed P value: **p < 0.01 vs. scrambled peptide-treated group by Graphpad Prism 8.4.3.

[0023] Figure 7. Two-week delayed CRP treatment significantly improves forelimb motor function after chronic ischemia stroke. Cylinder test was conducted every other week, and the results were presented as the individual rat (A), or each group of rats. As shown, two-week delayed CRP treatment (N=6) for additional 4 and 6 weeks significantly increase the use of affected forelimb, as compared to scrambled peptide treated animals (N=5). Data represent mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Bonferroni test, * p<0.05 vs. scrambled peptide treatment.

[0024] DETAILED DESCRIPTION

[0025] Provided herein are compositions, systems, kits, and methods for treating glial scarring caused by injury to a central nervous system (CNS) site by administering a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding the CRP. In particular embodiments, CNS site is the result of a stroke in a subject (e.g., human subject that has had a stroke). In certain embodiments, the CRP is composed of a cell membrane penetrating domain, a lysosome targeting domain, and a chondroitin sulfate proteoglycan (CSPG) binding domain.

[0026] Chondroitin sulfate proteoglycans (CSPGs) are the major components of scar, which are significantly increased after injury / trauma to the central nervous system (CNS). The CSPG reduction peptides (CRPs), and nucleic acid sequences encoding CRPs, provide an effective way to remove CSPGs to reduce glial scarring. In certain embodiments, the CRP has the following sequence:

[0027] N - YGRKKRRQRRR-PKPRVTWNKKGKKVNSQRF-KFERQKILDQRFFE - C (SEQ ID NO:4). One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acids specified in SEQ ID NO:4. The SEQ ID NO: 4 sequence has the following three components:

[0028] Cell membrane penetrating domain: YGRKKRRQRRR (SEQ ID NO:1);

[0029] Lysosome targeting domain : KFERQKILDQRFFE (SEQ ID NO:2); and CSPG binding domain: PKPRVTWNKKGKKVNSQRF (SEQ ID NOG);

[0030] Each of these domains may be constructed with longer, shorter, or mutated versions of the sequences shown in SEQ ID NOS: 1-3 and 5-53 and 62-71. For example, one could change one, two, three amino acids in these sequences. For example, one could make conservative changes to a particular amino acid sequence. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. In certain embodiments, provided herein are peptides that have substantial identity to at least a portion of the amino acid sequences shown in SEQ ID NOs: 1- 58 and 72-77.

[0031] In certain embodiments, the cell-membrane penetrating domain of a CRP comprises an amino acid sequence as shown in SEQ ID NOs: 1 and 5-27 and 59-61 of Table 1 or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 1.

[0032] TABLE 1

[0033] In other embodiments, the cell membrane penetrating peptide is a protein transduction domain (PTD) with the presence of multiple arginine (R) residues as shown in SEQ ID NOs: 1 and 5-27 and 59-61 of Table 1 . In other embodiments, the cell membrane penetrating peptide is a cell penetrating peptide (CPP) from the CPPsite 2.0, which is an updated version of database CPPsite. This site contains around 1700 unique cell penetrating peptides (CPPs) along with their secondary & tertiary structure, and can be found at www. followed by "crdd.osdd.net / raghava / cppsite / ."

[0034] In certain embodiments, the lysosomal targeting domain of a CRP comprises an amino acid sequence as shown in SEQ ID NOs: 2 and 28-36 of Table 2, or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. In other embodiments, the lysosomal targeting domain is a chaperone- mediated autophagy (CMA)-targeting motif (CTM) containing a KFERQ-like motif, such as those shown in SEQ ID NOs: 2 and 28-36 of Table 2. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 2.

[0035] TABLE 2

[0036] In certain embodiments, the CSPG binding domain of a CRP comprises an amino acid sequence as shown in SEQ ID NOs: 3 and 37-53 and 62-71 of Table 3 or such a peptide with one, two, or three (or more) N-terminal or C-terminal additions, subtractions or mutations therein. One of skill in the art could construct a corresponding nucleic acid sequence based on the known codon triplets for the amino acid sequences shown in Table 3.

[0037] TABLE 3

[0038] In certain embodiment, the CRP peptide comprises the sequence of any of SEQ ID NOs:54-58 and 72-77, and variants thereof (e.g., one or two amino acid additions, substitutions, or deletions):

[0039] SEQ ID NO:54 - GRKKRRQRRR-RVTWNKKGKKVNSQR-KFERQKILD

[0040] SEQ ID NO:55 - RKKRRQRRR-NKKGKKVNSQRF-KFERQ

[0041] SEQ ID NO:56 - KFERQKILDQRFFE-PKPRVTWNKKGKKVNSQRF-YGRKKRRQRRR SEQ ID NO:57 - PKPRVTWNKKGKKVNSQRF-KFERQKILDQRFFE-YGRKKRRQRRR SEQ ID NO:58 - KFERQKILDQRFFE-YGRKKRRQRRR-PKPRVTWNKKGKKVNSQRF SEQ ID NO:72 - KFERQKILD-RVTWNKKGKKVNSQR-GRKKRRQRRR

[0042] SEQ ID NO:73 - RVTWNKKGKKVNSQR-KFERQKILD-GRKKRRQRRR

[0043] SEQ ID NO:74 - KFERQKILD- GRKKRRQRRR-RVTWNKKGKKVNSQR

[0044] SEQ ID NO:75 - KFERQ-NKKGKKVNSQRF-RKKRRQRRR

[0045] SEQ ID NO:76 - NKKGKKVNSQRF-KFERQ-RKKRRQRRR

[0046] SEQ ID NO:77 - KFERQ- RKKRRQRRR-NKKGKKVNSQRF

[0047] EXAMPLES

[0048] EXAMPLE 1

[0049] A CSPGs degrading peptide to treat chronic ischemic strokes

[0050] Considering the significant role of the glial scars in limiting recovery from chronic stroke, we have developed a translatable strategy, CSPG reduction peptide (CRP) to target CSPGs, the major components of glial scars (e.g., in order to treat stroke patients in the chronic phase). The CRP in this Example had the following three components: Cell membrane penetrating domain: YGRKKRRQRRR (SEQ ID NO:1); Lysosome targeting domain: KFERQKILDQRFFE (SEQ ID NO:2). CSPG binding domain: PKPRVTWNKKGKKVNSQRF (SEQ ID NO:3)

[0051] The full CRP sequence in this Example is as follows: N - YGRKKRRQRRR-PKPRVTWNKKGKKVNSQRF- KFERQKILDQRFFE - C (SEQ ID NO:4).

[0052] The designed CRP (SEQ ID NO:4) includes a cell membrane-penetrating domain (SEQ ID NO:1), a CSPGs binding domain (SEQ ID NO:3), and a lysosome targeting domain (SEQ ID NO:2) for directing the CRP-CSPGs complex to lysosomes for degradation.

[0053] We have conducted experiments using behavioral assessments and histological studies (Figure 2) to investigate the efficacy of this scar-degrading peptide, CRP to reduce the gliosis in both brain (Figures 3, 4) and spinal cord (Figures 5, 6) and to improve functional recovery (Figure 7) in rats with chronic stroke induced by photo thrombotic ischemia.

[0054] CRP can be delivered by subcutaneous injection and distributes to the central nervous system (CNS) via its N-terminal blood-brain barrier- and cell membrane-penetrating motif, where it specifically binds to CSPGs via its CSPG-binding motif and is then degraded in the lysosome as directed by its lysosome-targeting motif to reduce CSPGs deposited in both intracellular and extracellular compartments (Figure 1).

[0055] Photothrombotic stroke procedure was first conducted in the forelimb sensorimotor cortex after craniotomy to result in ischemic stroke in the left hemisphere of cortex. The laser light was used to illuminate cortex following by the peritoneal injection of Rose Bengal to induce photothrombosis. Two weeks after photothrombotic stroke, the stroke animals were randomized and started to be subcutaneously injected (twice per day) with either CRP or scrambled peptide for an additional 6 weeks. The cylinder test (once every other week) was used to evaluate the motor function of the animals in response to photothrombotic stroke and the two- week delayed CRP treatment. The brain and spinal cord were harvested at 8 weeks after photothrombotic stroke for immunohistochemistry (IHC) of the levels of GFAP+ astrocytes and Chondroitin Sulfate A (CSA) (Figure 2).

[0056] As shown, the GFAP+ astrocytes (Figure 3) and CSA (Figure 4) were significantly increased at the sites surrounding the ischemic lesions in the cortex when animals were treated with scrambled peptide beginning 2 weeks after photothrombotic stroke. Encouragingly, the two-week delayed CRP treatment significantly decreased such increases in GFAP+ astrocytes (Figure 3) and CSA (Figure 4) at the peri-lesion site of the cortex, as compared to two-week delayed scrambled peptide treatment. Surprisingly, the GFAP+ astrocytes (Figure 5) and CSA (Figure 6) were also significantly increased at in the contralateral CST of the cervical spinal cord when animals were two-week delayed treated with scrambled peptide. These data suggest the gliosis occurs not only at the lesion site (gray matter) but also at the remote location in the CST (white matter) from chronic stroke. The chronic stroke-increased GFAP+ astrocytes (Figure 5) and CSA (Figure 6) were significantly decreased by the two-week delayed CRP treatment, as compared to two-week delayed scrambled peptide treatment. Of most importance, two-week delayed CRP treatment significantly improved motor function of the animals with chronic stroke, when compared to two-week delayed scrambled peptide treatment (Figure 7).

[0057] REFERENCES

[0058] 1. Donnan et al., Lancet 2008, 371, 1612-1623.

[0059] 2. Nowicka et al., Spatiotemporal dynamics of astroglial and microglial responses after photothrombotic stroke in the rat brain. Acta Neurobiol Exp (Wars) 2008, 68, 155-168.

[0060] 3. Lively et al., SCl / hevin and reactive gliosis after transient ischemic stroke in young and aged rats. J Neuropathol Exp Neurol 2011, 70, 913-929.

[0061] 4. Wojtasiewicz et al., De novo glioblastoma in the territory of a prior middle cerebral artery infarct. Case Rep Neurol Med 2013, 2013.

[0062] 5. Huang et al., Glial scar formation occurs in the human brain after ischemic stroke. Int J Med Sci 2014, 11, 344-348.

[0063] 6. Wang et al., White matter injury in ischemic stroke. Prog Neurobiol 2016, 141, 45-60.

[0064] 7. Paul et al., The role of corticospinal and extrapyramidal pathways in motor impairment after stroke. Brain Commun 2023, 5, fcac301.

[0065] 8. Rhodes, K.E.; Fawcett, J.W. Chondroitin sulphate proteoglycans: preventing plasticity or protecting the CNS? J Anat 2004, 204, 33-48.

[0066] 9. Deguchi et al. Expression of neurocan after transient middle cerebral artery occlusion in adult rat brain. Brain Res 2005, 1037, 194-199.

[0067] 10. Rolls et al., The bright side of the glial scar in CNS repair. Nat Rev Neurosci 2009, 10, 235-241.

[0068] 11. Fitch, M.T.; Silver, J. Activated macrophages and the blood-brain barrier: inflammation after CNS injury leads to increases in putative inhibitory molecules. Exp Neurol 1997, 148, 587-603. 12. Fawcett, J.W.; Asher, R.A. The glial scar and central nervous system repair. Brain

[0069] Res Bull 1999, 49, 377-391.

[0070] 13. Panickar, K.S.; Norenberg, M.D. Astrocytes in cerebral ischemic injury: morphological and general considerations. Glia 2005, 50, 287-298.

[0071] All publications and patents mentioned in the specification and / or listed below are herein incorporated hy reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope described herein.

Claims

CLAIMSWc Claim:

1. A method of treating glial scarring caused by injury to a central nervous system (CNS) site of a subject comprising: administering a composition comprising a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, to a subject with glial scarring caused by injury to a CNS site, wherein said CRP comprises: i) a cell membrane penetrating domain, ii) a lysosome targeting domain; and iii) a chondroitin sulfate proteoglycan (CSPG) binding domain.

2. The method of claim 1, wherein said administering at least partially causes reduction in the level of glial scarring at said CNS site.

3. The method of claims 1 or 2, wherein glial scarring reduction is measured by a computerized tomography (CT) scan or an MRI scan.

4. The method of any of claims 1-3, wherein said injury to said CNS site is caused by a stroke.

5. The method of any of claims 1-4, wherein said administering causes at least some cognitive ability gain in said subject, and wherein optionally said cognitive ability gain is measured by: Cognitive Assessment Scale for Stroke Patients (CASP); Montreal Cognitive Assessment (MoCA); IQCODE; or short-IQCODE.

6. The method of any of claims 1-5, wherein said CNS site is located in the spinal cord of said subject.

7. The method of any of claims 1-5, wherein said CNS site is located in the brain of said subject.

8. The method of any of claims 1 -5 or 7, wherein said subject is a human, wherein said CNS site was caused by a stroke, which is optionally a chronic ischemic stroke.

9. The method of any of claims 1-8, wherein administering is conducted within about 12 or 24 or 36 or 48 hours, or between 6 and 24 hours, or between 12 and 24 hours, of said injury to said CNS site.

10. The method of any of claims 1-8, wherein said administering is conducted after at least three days since said injury to said CNS site.

11. The method of any of claims 1-8, wherein said administering is conducted after a week or more, or a month or more, since said injury to said CNS site.

12. The method of any of claims 1-11, wherein said cell membrane penetrating domain is located at the N-terminal or C-terminal of said CRP.

13. The method of any of claims 1-12, wherein said lysosome targeting domain is located at the N-terminal or C-terminal of said CRP.

14. The method of any of claims 1-13, wherein said CSPG binding domain is located between said cell membrane penetrating domain and said lysosome targeting domain.

15. The method of any of claims 1-14, wherein said cell membrane penetrating domain comprises or consists of an amino acid sequence shown in any of SEQ ID NOS: 1 and 5-27 and 59-61, or a sequence with one or two amino acid additions, subtractions, or substitutions.

16. The method of any of claims 1-15, wherein said lysosome targeting domain comprises or consists of an amino acid sequence shown in any of SEQ ID NOS: 2 or 28-36, or a sequence with one or two amino acid additions, subtractions, or substitutions.

17. The method of any of claims 1-16, wherein said CSPG binding domain comprises or consists of an amino acid sequence shown in any of SEQ ID NO:3 or 37-53 or 62-71, or a sequence with one or two amino acid additions, subtractions, or substitutions.

18. The method of any of claims 1-17, wherein said administering is performed by a method selected from: subcutaneously, intravenously, intracranially, and intrathecal injection or infusion.

19. A kit or system comprising: a) composition comprising a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP comprises: i) a cell membrane penetrating domain, ii) a lysosome targeting domain; and iii) a chondroitin sulfate proteoglycan (CSPG) binding domain; and b) a device for administering said composition.

20. The kit or system or claim 19, wherein said composition is located inside said device.

21. The kit or system of claim 19, wherein said CRP is as recited in any of claims 1-17.

22. A composition comprising: a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP comprises: a) a cell membrane penetrating domain which comprises or consists of an amino acid sequence shown in any of SEQ ID NOs: 59-61, b) a lysosome targeting domain; and c) a chondroitin sulfate proteoglycan (CSPG) binding domain.

23. A composition comprising: a CSPG reduction peptide (CRP), or a nucleic acid sequence encoding said CRP, wherein said CRP comprises: a) a cell membrane penetrating domain, b) a lysosome targeting domain; and c) a chondroitin sulfate proteoglycan (CSPG) binding domain which comprises or consists of an amino acid sequence shown in any of SEQ ID NOs: 62-71.