WO2024200749A1

WO2024200749A1 - Treatment of spinal injury

Info

Publication number: WO2024200749A1
Application number: PCT/EP2024/058651
Authority: WO
Inventors: Leonor SAUDE; Isaura MARTINS; Dalila NEVES-SILVA
Original assignee: Instituto de Medicina Molecular João Lobo Antunes
Priority date: 2023-03-29
Filing date: 2024-03-28
Publication date: 2024-10-03

Abstract

This invention relates to methods of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof by reducing the expression or activity of CD9 and/or MYLIP at a site of spinal cord injury in the individual. Methods of treatment and medical uses are provided, along with pharmaceutical compositions and methods of screening for compounds useful in treating spinal cord injury or promoting spinal cord repair.

Description

Treatment of Spinal Injury Field The present invention relates to methods and compounds for the promotion of spinal cord repair and the treatment of spinal cord injury. Background After a mammalian traumatic spinal cord injury (SCI) there is an induced synaptic loss and neuronal cell death, which prompts a widespread deposition of cellular debris that triggers gliosis and neuroinflammation [1,2]. Reactive gliosis gives rise to a mature astrocytic border, within which resides a fibrotic scar, representing a major physical barrier to axonal regeneration [3,4]. Once activated microglia, neutrophils, macrophages, and lymphocytes initiate a secondary damage response, by releasing chemokines and cytokines, contributing to a strong inflammatory environment and constraining the regenerative capacity [5]. In addition, the actual mechanical force of the trauma disrupts the spinal cord vasculature, damaging all the components of the neurovascular unit (NVU) of the blood spinal cord barrier (BSCB), which can remain compromised [6]. The NVU is a crucial structure composed of endothelial cells (ECs), pericytes, astrocytic endfeet and neurons, that is responsible for the normal homoeostasis of the spinal parenchyma, protecting the cord during trauma and infection [7,8]. However, after injury, this system is compromised and BSCB is damaged. This scenario favours the increased circulation of immune cells and pro-inflammatory mediators, contributing to systemic inflammation [9], and induces ischemia, leading to greater tissue loss [10]. In fact, the degree of immune infiltrates into the cord has been associated with the status of BSCB in SCI [11]. In addition, pericytes from the NVU are able to detach from the vascular wall and contribute to the scar formation after injury [12], demonstrating another important role of the components of the BSCB in SCI. Although there is an angiogenic response to the injury, the new blood vessels that are formed in the lesion area are usually leaky with an impaired BSCB, further contributing to the secondary damage [13]. Abnormal permeability of BSCB is even more apparent during the initial process of angiogenesis, which proceeds during the sub-acute phase (3–7 days postinjury (dpi)) in the mouse model [14], further contributing to a hostile microenvironment that limits axonal regeneration and repair. Thus, acting during this period, when the BSCB is even more prone to impairment, might prevent irreversible damage and diminish the secondary injury. Promoting BSCB integrity and blood vessel vascularization/remodelling after SCI are therefore crucial for spinal cord repair [14–17]. Nevertheless, the numerous therapeutic interventions based on revascularization and targeting BSCB permeability have achieved limited therapeutic effects [16,17]. This demonstrates that there is still the need to better understand the instilled molecular changes on BSCB after injury, particularly at early stages, in order to identify potential players that could promote both vascularization and BSCB integrity, and therefore stimulate spinal cord regeneration Summary The present inventors have recognised that tetraspanin CD9 protein (CD9) and myosin regulatory light chain interacting protein (MYLIP) are independently associated with impaired function of the blood spinal cord barrier (BSCB) at sites of spinal cord injury. Reduction of CD9 and/or MYLIP expression or activity, for example using a therapeutic agent, may be useful in improving BSCB integrity after spinal cord injury and promoting the repair of spinal cord injury in patients. A first aspect of the invention provides a method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof comprising: reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury in the individual. A second aspect of the invention provides a method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof, the method comprising: reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury in the individual. A method of the first and second aspects may comprise (i) reducing CD9 expression at a site of spinal cord injury, for example by administering an agent that reduces CD9 expression (ii) reducing CD9 activity at a site of spinal cord injury, for example by administering an agent that reduces or inhibits CD9 activity (iii) reducing MYLIP expression at a site of spinal cord injury, for example by administering an agent that reduces MYLIP expression or (iv)reducing MYLIP activity at a site of spinal cord injury, for example by administering an agent that reduces MYLIP activity. A third aspect of the invention provides an agent that reduces CD9 or MYLIP expression or activity for use in a method according to the first or second aspect. A fourth aspect of the invention provides the use of an agent that reduces CD9 or MYLIP expression or activity in the manufacture of a medicament for use in a method according to the first or second aspect. A fifth aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of agent that reduces reducing CD9 or MYLIP expression or activity and a pharmaceutically acceptable excipient. The pharmaceutical composition of the fifth aspect of the invention may be useful in the first and second aspects of the invention. A sixth aspect of the invention provides a method of screening for a compound useful in (i) treating spinal cord injury in a patient or (ii) promoting spinal cord repair in a patient with a spinal cord injury, the method comprising; determining the activity of MYLIP or CD9 in the presence or absence of a test compound, wherein a decrease in MYLIP or CD9 activity in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound for use in treating spinal cord injury in a patient or promoting spinal cord repair in a patient with a spinal cord injury. A seventh aspect of the invention provide a method of screening for a compound useful in (i) treating spinal cord injury in a patient or (ii) promoting spinal cord repair in a patient with a spinal cord injury, the method comprising; determining the expression of MYLIP or CD9 in a mammalian cell in the presence or absence of a test compound, wherein a decrease in MYLIP /or CD9 expression in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound for use in treating spinal cord injury in a patient or promoting spinal cord repair in a patient with a spinal cord injury. Other aspects and embodiments of the invention are described in more detail below. Brief Description of the Figures Figure 1 shows the results of digital cytometry analysis to validate vascular cells as the overall most predominant cell type sorted by FACS at 3- and 7-days post-injury. Single cell gene expression data from spinal cord mouse tissue acquired from the Brain Mouse Atlas were used to build the cell signatures using 20 replicates from a pool of 50 cells classified with TaxonomyRank1 [19]. Next, estimation of cellular abundance was carried out with the bulk transcriptomic data using the subset of high confidence genes. Cell signatures and cell type abundance were determined using CIBERSORTx [18]. Figure 2 shows that spinal cord injury is associated with differences in the expression levels of a small subset of genes at 3- and 7-days post-injury. Volcano plots showing the effect size (in log2 fold-change; Xaxis) and the significance (in B statistic, i.e, the log-odds ratio of differential expression; Y-axis) of d differential gene expression specifically resulting from spinal cord injury (SCI) in (a) 3 and (b) 7 days post- injury (dpi) samples. Each point indicates one of the 1266 genes considered in the analysis. Figure 3 shows Gene Set Enrichment Analysis (GSEA) [20] of the 3 dpi SCI-specific transcriptomic alterations in vascular cells shows enrichment for immune- and leukocyte migration-related GO biological processes. The bar plot shows the significantly enriched terms ordered by normalized enrichment score (NES); blue bars for positive enrichment and red bars for negative enrichment (i.e., depletion). GSEA was run as described in detail in the respective Materials and Methods section. Briefly, we ran the GSEA pre- ranked method with empirical Bayes moderated t-statistic values against MsigDB’s gene ontology (GO) biological processes; terms were considered significantly enriched with an adjusted p-value < 0.05. Figure 4 shows that spinal cord injury induces an upregulation of Cd9 and Mylip mRNA levels. (a) Cd9 and (b) Mylip mRNA expression levels at 3 and 7 days post-injury (dpi) were evaluated by qPCR. For each cDNA sample, three technical replicates were included (n = 3). Cd9 and Mylip mRNA levels were significantly increased with the injury at 3 and 7 dpi. Data are expressed in fold change towards the housekeeping gene PPIA and bars represent the mean. *p<0.05 versus sham, Student’s t-test Figure 5 shows the CD9 and MYLIP protein expression increases after spinal cord injury. Representative Western Blot images of CD9 (a) and MYLIP (b) with GAPDH as protein loading control at 3 and 7 days post- injury (dpi), each lane corresponds to an independent biological replicate. CD9 (a) and MYLIP (b) protein levels were quantified in sham and injured animals with a significant increase at 7 dpi and only at 3 dpi for MYLIP (n=3-5). Data are expressed in relative intensity towards GADPH, and bars represent the mean. *p<0.05, ∗∗p < 0.01 versus sham, Student’s t-test. Figure 6 shows that CD9 and MYLIP expression is injury-induced at the caudal side of the lesion. Representative images of sham and injured spinal cords demonstrate that both CD9 and MYLIP (green) are only detected after injury with more prominent expression in the caudal region of the lesion in all animals analysed (n=3). White line: spinal cord delimitation; Circle line: core of the lesion; R: rostral; C: caudal. Scale bar: 500 µm; 20x amplification. Figure 7 shows that the injury-induced expression of CD9 is detected in close proximity of vascular and perivascular cells. Representative images of injured spinal cords demonstrate that when CD9 (green) is induced after injury it is detected in close proximity of both endothelial marker CD31 (magenta, arrows) and pericytic marker αSMA (cyan, arrowhead) at 7 dpi (n=3). DAPI in grey. Scale bar: 20 µm; 40x amplification Figure 8 shows that MYLIP expression is injury-induced in pericytes. Representative images of injured spinal cords identify MYLIP (green) expression in close proximity with the pericytic marker CD13 (cyan, arrows), surrounding the blood vessels (CD31, magenta) at 7 dpi (n=3). DAPI in grey. Scale bar: 20 µm; 40x amplification. Figure 9 shows that MYLIP is expressed in detached pericytes. Representative images of injured spinal cords demonstrate that MYLIP (green) is present within the pericytes (CD13, cyan, arrow) detached from blood vessels (CD31, magenta) at 7 dpi (n=3). DAPI in grey. Scale bar: 20 µm; 40x amplification Detailed Description This invention relates to the methods of treating spinal cord injury or promoting spinal cord repair in a patient and methods of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in a method. The methods may comprise reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury in the individual. Spinal cord injury is associated with BSCB damage and permeability. Traumatic spinal injury causes BSCB rupture and the death of cells within the BSCB. Repaired BSCB (following spinal injury) is associated with dissociation of pericytes from endothelial cells with the BSCB, a reduction in tight junction proteins between endothelial cells, and overexpression of adhesion molecules by endothelial cells, resulting in leakiness of the barrier. Damage to the BSCB can be readily determined by intravenous administration of a detectable dye. In an individual with an intact or functional BSCB the dye will not enter the spinal parenchyma and will only be detectable within the vasculature. Detection of the dye in the spinal parenchyma is indicative of BSCB damage. The methods provided herein may reduce the leakiness (or permeability) of a damaged BSCB at the site of spinal cord injury, improving BSCB integrity at the site. In the methods provided herein, these effects are achieved by reducing CD9 and/or MYLIP expression or activity at the injury site. A reduction in CD9 and/or MYLIP expression or activity may include (i) a reduction in CD9 expression (ii) a reduction in CD9 activity (iii) a reduction in MYLIP expression (iv) a reduction in MYLIP activity (v) a reduction in both CD9 and MYLIP expression or (vi) a reduction in both CD9 and MYLIP activity. A reduction in CD9 or MYLIP expression or activity as described herein may be a significant reduction. Significance may be measured, for example, using a t-test, such as Student’s t-test or Welch’s t-test with a significance level of p<0.001 indicating a significant increase or reduction. In other embodiments, a significance level of p<0.05, such as p<0.01 or p<0.005 may indicate a significant increase or reduction. In some embodiments, a reduction in CD9 or MYLIP expression or activity may be significant if the CD9 or MYLIP expression or activity is 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the CD9 or MYLIP expression, or activity at the site of injury before the start of the treatment or the CD9 or MYLIP expression, activity, level, amount or concentration in a reference or control sample. In some embodiments, reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity may be useful in the treatment of an impairment in the BSCB resulting from spinal cord injury. An impaired BSCB may be characterised by abnormal permeability. For example, the impaired BSCB may display increased permeability to immune cells relative to normal BSCB. Reducing CD9 and/or MYLIP expression or activity as described herein may for example prevent immune cell infiltration of the spinal cord at the injury site or lesion. Reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury as described herein may promote axonal regeneration and functional recovery of a patient, for example by promoting blood vessel revascularization and remodelling at the site of injury. Abnormal BSCB permeability can be diagnosed by any suitable method in the art, for example using a dye as set out above. Alternatively, BSCB damage, and associated abnormal permeability, may be assumed without formal diagnosis following a spinal injury. In some embodiments, a method described herein may reduce inflammatory responses and/or scarring at the site of injury and may maintain blood supply to the spinal cord and optimise the microenvironment for neuronal repair. By avoiding improper permeability of the BSCB, the method described herein may also prevent inflammatory mediators and markers from migrating from the site of the spinal injury into the body at large, and thereby prevent or reduce secondary inflammation. Secondary inflammation in spinal cord injury patients can cause chronic inflammation affecting other organs such as the liver, heart, bladder etc. Inflammatory conditions affecting these or other organs may be prevented, or their severity reduced in spinal cord injury patients by treatment according to the present invention. Without being bound by theory, reducing expression and/or activity of CD9 may directly reduce inflammation, whereas reducing expression and/or activity of MYLIP may impact scar formation. MYLIP is present in detached pericytes, which form the fibrotic tissue associated with scar formation, so targeting of MYLIP may impact on this process. CD9 and/or MYLIP expression or activity may be reduced in the sub-acute injury phase, for example 48 hours to 15 days after spinal cord injury in a human patient. For example CD9 and/or MYLIP expression or activity may be reduced 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 10 to 15, 5 to 12, 6 to 12, 7 to 12 or 8 to 12 days post injury (dpi). In some embodiments though CD9 and/or MYLIP expression or activity may be reduced after this period, i.e. more than 15 dpi. In other embodiments CD9 and/or MYLIP expression or activity may be reduced before this period, i.e. within the first 48 hours after injury, e.g. immediately after injury or upon initiation of medical treatment for the injury. In some embodiments, CD9 and/or MYLIP expression or activity may be reduced before the formation of scar at the site of injury. MYLIP expression and activity appears to be involved in scar formation, so reduction of MYLIP activity or expression before scar formation may be preferred. CD9 expression or activity on the other hand may usefully be reduced after scar formation also. In some embodiments, CD9 and/or MYLIP expression or activity may be reduced in the caudal region of the site of spinal cord injury, e.g. in the region at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm caudal to the injury site. In particular CD9 and/or MYLIP expression or activity may be reduced in the region 5 mm caudal to the injury site. In some embodiments, CD9 and/or MYLIP expression or activity may be reduced systemically. A reduction in CD9 and/or MYLIP expression or activity may include (i) a reduction in CD9 expression (ii) a reduction in CD9 activity (iii) a reduction in MYLIP expression (iv) a reduction in MYLIP activity (v) a reduction in both CD9 and MYLIP expression (vi) a reduction in both CD9 and MYLIP activity. A reduction in expression may comprise a reduction in transcription or translation of the CD9 or MYLIP gene. For example, reduced expression may include a decrease in the level or amount of CD9 or MYLIP encoding mRNA in a cell and/or a decrease in the level or amount of CD9 or MYLIP protein. A reduction in activity may comprise a reduction in the function or activity of the CD9 or MYLIP protein. For example, reduced activity may include a decrease in the level or amount of active CD9 or MYLIP protein and/or a decrease in activity of CD9 or MYLIP protein. CD9 and/or MYLIP expression or activity may be reduced in vascular cells, such as endothelial cells, and perivascular cells, such as pericytes, at the site of injury. CD9 expression may also or alternatively be reduced in immune cells which are associated with blood vessels around the injury site, e.g. macrophages. CD9 is a cell-surface glycoprotein of the tetraspanin family. The CD9 for which expression or activity are reduced herein may be human CD9 (Gene ID: 928) and may have the amino acid sequence of database accession number NP_001760.1 (SEQ ID NO: 1) or NP_00131724.1 (SEQ ID NO: 5), or a variant of either of these sequence which represent different isoforms of CD9. CD9 may be encoded by the nucleotide sequence of database accession number NM_001330312.2 (SEQ ID NO: 2), or a variant of this sequence, such as an allelic variant. MYLIP is an E4 ubiquitin-protein ligase that mediates the ubiquitination and proteasomal degradation of myosin regulatory light chain (MRLC) and LDL receptors LDLR and VLDLR. MYLIP may be human MYLIP (Gene ID: 29116) and may have the amino acid sequence of database accession number NP_037394.2 (SEQ ID NO: 3), or a variant of this sequence, such as an isoform variant. MYLIP may be encoded by the nucleotide sequence of database accession number NM_013262.4 (SEQ ID NO: 4), or a variant of this sequence, such as an allelic variant. A variant of a reference CD9 or MYLIP amino acid or nucleotide sequence may have a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the reference amino acid or nucleotide sequence. Sequence identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package^TM, Accelrys, San Diego CA). GAP uses the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol.215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol.147: 195-197), generally employing default parameters. Particular amino acid sequence variants may differ from a given sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20 or 20-30 amino acids. In some embodiments, a variant sequence may comprise the reference sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, up to 15, up to 20, up to 30, up to 40, up to 50 or up to 60 residues may be inserted, deleted or substituted. Particular nucleotide sequence variants may differ from a given sequence by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20 or 20-30 nucleotides. In some embodiments, a variant sequence may comprise the reference sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides inserted, deleted or substituted. For example, up to 15, up to 20, up to 30, up to 40, up to 50 or up to 60 nucleotides may be inserted, deleted or substituted. CD9 or MYLIP expression or activity may be reduced by administering an agent to the individual which reduces CD9 or MYLIP expression or activity, respectively, at the site of spinal cord injury of the individual. Suitable agents include CD9 antagonists. A CD9 antagonist is any agent capable of antagonising, inhibiting or blocking CD9. Suitable CD9 antagonists include an organic compound having a molecular weight of 900 Da or less; a protein or peptide that specifically binds CD9, for example, a receptor or antibody molecule that specifically binds CD9, or a peptide that binds CD9 and blocks its activity (a blocker peptide); and a nucleic acid that specifically binds CD9, for example, an aptamer that specifically binds CD9. For example, a CD9 blocker peptide with the sequence RSHRLRLH (SEQ ID NO: 6) has previously been reported (Suwatthanarak et al., Chemical Communications 57(40): 4906-4909, 2021), which inhibits CD9-mediated migration of cancer cells and may be suitable in the present invention. MYLIP activity may be reduced by administering an agent to the individual which reduces MYLIP activity and/or expression at the site of spinal cord injury of the individual. Suitable agents include MYLIP antagonists. In some embodiments, the MYLIP antagonist may be an organic compound having a molecular weight of 900 Da or less. In other embodiments, the MYLIP antagonist may be a protein that specifically binds MYLIP, for example, a receptor or antibody molecule that specifically binds MYLIP, or a peptide that binds CD9 and blocks its activity (a blocker peptide); or a nucleic acid that specifically binds MYLIP, for example, an aptamer that specifically binds MYLIP. MYLIP is a sterol-dependent inhibitor of cellular cholesterol uptake that has previously been found to be targeted by statins. In particular, statins have been found to reduce the level of MYLIP (also referred to as IDOL, or inducible degrader of low-density lipoprotein receptor) in serum and monocytes of human patients (Chan et al., Endocrine Connections 11: e220019, 2022). Atorvastatin has also previously been found to suppress inflammation in rats suffering from spinal cord injury (Bimbova et al., International Journal of Molecular Sciences 19: 1106, 2018). Statins may be used according to the present invention to reduce expression and/or activation of MYLIP. Statins which may be used for this purpose include atorvastatin, fluvastatin, pravastatin, rosuvastatin and simvastatin. In other embodiments, the agent which reduces MYLIP activity or expression is not a statin. The terms “antagonist” and “inhibitor” as used herein, cover pharmaceutically acceptable salts and solvates of these compounds. Techniques for the rational design of small molecule antagonists and inhibitors through structural analysis of target proteins are well-known in the art. Agents such as small molecules which may be useful in the invention may be identified by screening suitable cells to determine the impact on CD9 and/or MYLIP. For example, compounds of interest may be screened on co-cultures of endothelial cells and pericytes, or in vitro models of the BSCB such as an endothelial cell monolayer and measuring the impact of the compound on CD9 expression or activity. Expression may be monitored by standard techniques in the art such as qPCR or quantitative Western blot. Activity may be assayed by monitoring biological activities associated with CD9 and/or MYLIP, e.g. axonal growth. CD9 expression may be reduced by administering an agent to the individual which reduces CD9 expression at the site of spinal cord injury of the individual. Suitable agents include suppressor nucleic acids that reduces expression of active CD9 polypeptide. The use of nucleic acid suppression techniques such as anti- sense and RNAi suppression, to down-regulate expression of target genes is well-established in the art. In some embodiments, the suppressor nucleic acid may be a siRNA or shRNA. For example, the suppressor nucleic acid comprises a nucleotide sequence at least 95% identical to a contiguous sequence of 15 to 40 nucleotides of SEQ ID NO: 2. MYLIP expression may be reduced by administering an agent to the individual which reduces MYLIP expression at the site of spinal cord injury of the individual. Suitable agents include suppressor nucleic acids that reduces expression of active MYLIP polypeptide. In some embodiments, the suppressor nucleic acid may be a siRNA or shRNA. For example, the suppressor nucleic acid comprises a nucleotide sequence at least 95% identical to a contiguous sequence of 15 to 40 nucleotides of SEQ ID NO: 4. In some embodiments, the suppressor nucleic acid may be an antisense oligonucleotide. Cells at the site of injury may be transfected with a suppressor nucleic acid (i.e. a nucleic acid molecule which suppresses CD9 or MYLIP expression), such as an siRNA or shRNA, or a heterologous nucleic acid encoding the suppressor nucleic acid. The suppressor nucleic acid reduces the expression of active CD9 or MYLIP polypeptide by interfering with transcription and/or translation, thereby reducing CD9 or MYLIP activity in the cells. RNAi involves the expression or introduction into a cell of an RNA molecule which comprises a sequence which is identical or highly similar to the CD9 OR MYLIP coding sequence. The RNA molecule interacts with mRNA which is transcribed from the CD9 OR MYLIP gene, resulting in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of the mRNA. This reduces or suppresses expression of active CD9 OR MYLIP polypeptide (Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; Voinnet & Baulcombe (1997) Nature 389: pg 553). The RNA molecule is preferably double stranded RNA (dsRNA) (Fire A. et al Nature 391, (1998)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)). Suitable RNA molecules for use in RNAi suppression include short interfering RNA (siRNA). siRNA are double stranded RNA molecules of 15 to 40 nucleotides in length, preferably 15 to 28 nucleotides or 19 to 25 nucleotides in length, for example 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, two unmodified 21-mer oligonucleotides may be annealed together to form a siRNA. A siRNA molecule may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The overhang lengths of the strands are independent, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Other suitable RNA molecules for use in RNAi include small hairpin RNAs (shRNAs). shRNA are single- chain RNA molecules which comprise or consist of a short (e.g.19 to 25 nucleotides) antisense nucleotide sequence, followed by a nucleotide loop of 5 to 9 nucleotides, and the complementary sense nucleotide sequence (e.g.19 to 25 nucleotides). Alternatively, the sense sequence may precede the nucleotide loop structure and the antisense sequence may follow. The nucleotide loop forms a hairpin turn which allows the base pairing of the complementary sense and antisense sequences to form the shRNA. A suppressor nucleic acid, such as a siRNA or shRNA, may comprise or consist of a sequence which is identical or substantially identical (i.e. at least 90%, at least 95% or at least 98% identical) to all or part (for example, 15 to 40 nucleotides) of a reference CD9 or MYLIP nucleotide coding sequence, or its complement. Suitable reference sequences coding CD9 or MYLIP that may be used for the design of suppressor nucleic acids are publically available and include SEQ ID NO: 2. CD9 or MYLIP activity is suppressed in the immune cells by down-regulation of the production of active CD9 or MYLIP polypeptide by the suppressor nucleic acid. Suppressor nucleic acids, such as siRNAs and shRNAs, for reducing CD9 or MYLIP expression may be readily designed using reference CD9 or MYLIP coding sequences and software tools which are widely available in the art and may be produced using routine techniques. For example, a suppressor nucleic acid may be chemically synthesized; produced recombinantly in vitro or cells (Elbashir, S. M. et al., Nature 411:494-498 (2001); Elbashir, S. M., et al., Genes & Development 15:188-200 (2001)) or obtained from commercial sources (e.g. Cruachem (Glasgow, UK), Dharmacon Research (Lafayette, Colo., USA)). In some embodiments, two or more suppressor nucleic acids may be used to suppress the expression of CD9 or MYLIP. For example a pool of siRNAs may be employed. Other siRNAs and siRNA pools may be produced using standard technique. Nucleic acid suppression may also be carried out using anti-sense techniques. Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti- sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences. The construction of anti-sense sequences and their use is well known in the art (Peyman and Ulman, Chemical Reviews, 90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol.32:329-376, (1992)). Anti-sense oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within the immune cells in which down-regulation of CD9 or MYLIP is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. The complete sequence corresponding to the CD9 or MYLIP coding sequence in reverse orientation need not be used. For example, fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17. In other embodiments, the expression of active CD9 or MYLIP polypeptide is reduced at the site of injury by targeted mutagenesis. The use of targeted mutagenesis techniques such as gene editing, to knock out or abolish expression of target genes is well- established in the art (see for example Gaj et al (2013) Trends Biotechnol.31(7) 397-405). One or more mutations, such as insertions, substitutions, or deletions, may be introduced into the CD9 or MYLIP gene of a cell. Suitable mutations include deletions of all or part of the CD9 or MYLIP gene, for example, one, two or more exons, frameshift mutations, or nonsense mutations introducing premature stop codons. The mutations may prevent the expression of active CD9 or MYLIP polypeptide, for example by impairing transcription or translation of the CD9 or MYLIP gene or causing an inactive polypeptide to be expressed. Targeted mutagenesis to introduce one or more mutations may be performed by any convenient method. For example, cells may be transfected with a heterologous nucleic acid which encodes a targetable nuclease. The targetable nuclease may inactivate the CD9 or MYLIP gene encoding CD9 or MYLIP in one or more cells of the individual, for example, by introducing one or more mutations that prevent the expression of active CD9 or MYLIP polypeptide. The targetable nuclease may be site-specific (e.g. ZFN or TALEN) or may be expressed with one or more targeting sequences that target the nuclease to the CD9 or MYLIP gene (e.g. CRISPR/Cas). The heterologous nucleic acid may include an inducible promoter that promotes expression of the targetable nuclease and optional targeting sequence within a specific cell type, for example a vascular or perivascular cell. For example, the inducible promoter could be a promoter-enhancer cassette that selectively favours expression of the targetable nuclease and the optional targeting sequence within the vascular or perivascular cell over other types of host cells. Suitable targeting nucleases include, for example, site-specific nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases or RNA guided nucleases, such as clustered regularly interspaced short palindromic repeat (CRISPR) nucleases. Zinc-finger nucleases (ZFNs) comprise one or more Cys2-His2 zinc-finger DNA binding domains and a cleavage domain (i.e., nuclease). The DNA binding domain may be engineered to recognize and bind to any nucleic acid sequence using conventional techniques (see for example Qu et al. (2013) Nucl Ac Res 41(16):7771-7782). The use of ZFNs to introduce mutations into target genes is well-known in the art (see for example, Beerli et al Nat. Biotechnol.2002; 20:135–141; Maeder et al Mol. Cell.2008; 31:294–301; Gupta et al Nat. Methods.2012; 9:588–590) and engineered ZFNs are commercially available (Sigma-Aldrich (St. Louis, MO). Transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain comprising a series of modular TALE repeats linked together to recognise a contiguous nucleotide sequence. The use of TALEN targeting nucleases is well known in the art (e.g. Joung & Sander (2013) Nat Rev Mol Cell Bio 14:49-55; Kim et al Nat Biotechnol. (2013); 31:251–258. Miller JC, et al. Nat. Biotechnol. (2011) 29:143–148. Reyon D, et al. Nat. Biotechnol. (2012); 30:460–465). Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome (see for example Silva et al. (2011) Curr Gene Ther 11(1):11-27). CRISPR targeting nucleases (e.g. Cas9) complex with a guide RNA (gRNA) to cleave genomic DNA in a sequence-specific manner. The crRNA and tracrRNA of the guide RNA may be used separately or may be combined into a single RNA to enable site-specific mammalian genome cutting within the CD9 OR MYLIP gene or its regulatory elements. The use of CRISPR/Cas9 systems to introduce insertions or deletions into genes as a way of decreasing transcription is well known in the art (see for example Cader et al Nat Immunol 201617 (9) 1046-1056, Hwang et al. (2013) Nat. Biotechnol 31:227-229; Xiao et al., (2013) Nucl Acids Res 1-11; Horvath et al., Science (2010) 327:167–170; Jinek M et al. Science (2012) 337:816–821; Cong L et al. Science (2013) 339:819–823; Jinek M et al. (2013) eLife 2:e00471; Mali P et al. (2013) Science 339:823– 826; Qi LS et al. (2013) Cell 152:1173–1183; Gilbert LA et al. (2013) Cell 154:442–451; Yang H et al. (2013) Cell 154:1370–1379; and Wang H et al. (2013) Cell 153:910–918). In some preferred embodiments, the targetable nuclease is a Cas endonuclease, preferably Cas9, which is expressed in the immune cells in combination with a guide RNA targeting sequence that targets the Cas endonuclease to cleave genomic DNA within the CD9 OR MYLIP gene and generate insertions or deletions that prevent expression of active CD9 OR MYLIP polypeptide. Nucleic acid sequences encoding a suppressor nucleic acid or targetable nuclease and optionally a guide RNA may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the encoding nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in a range of expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts, such as E. coli and/or in eukaryotic cells, such as yeast, insect or mammalian cells. Vectors suitable for use in expressing a suppressor nucleic acid or targetable nuclease in mammalian cells include plasmids and viral vectors e.g. retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Suitable techniques for expressing a suppressor nucleic acid or targetable nuclease in mammalian cells are well known in the art (see for example; Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press or Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (Mar 1997) Humana Press Inc). Transfection with the vector or nucleic acid may be stable or transient. Suitable techniques for transfecting cells, such as vascular or perivascular cells, are well known in the art. A vector such nucleic acids may be delivered to a patient in the context of a cell. Particularly suitable for this purpose are mesenchymal stem cells (MSCs), which may be used as a delivery system for nucleic acids, particularly RNA molecules such as siRNA. MSCs may be autologous or allogeneic to the patient. In other embodiments, the expression of active CD9 or MYLIP polypeptide is reduced at the site of injury by cell therapy. The use of cell therapy techniques such as T-cell and CAR-T cells, to remove or deplete cells expressing target antigens is well- established in the art. For example, CD9 or MYLIP expression may be reduced by administering a T cell to the individual which comprises a heterologous antigen receptor that specifically binds CD9 or MYLIP. The T cell may reduce expression of active CD9 or MYLIP polypeptide at the site of injury by killing cells that express CD9 or MYLIP. Preferably, the heterologous antigen receptor is a chimeric antigen receptor (CAR). CARs are artificial receptors that are engineered to contain an immunoglobulin antigen binding domain, such as a single-chain variable fragment (scFv). A CAR may, for example, comprise an scFv fused to a TCR CD3 transmembrane region and endodomain. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, which may be connected with a short linker peptide of approximately 10 to 25 amino acids (Huston J.S. et al. Proc Natl Acad Sci USA 1988; 85(16):5879-5883). The linker may be glycine- rich for flexibility, and serine or threonine rich for solubility, and may connect the N-terminus of the V_H to the C-terminus of the VL, or vice versa. The scFv may be preceded by a signal peptide to direct the protein to the endoplasmic reticulum, and subsequently the T cell surface. In the CAR, the scFv may be fused to a TCR transmembrane and endodomain. A flexible spacer may be included between the scFv and the TCR transmembrane domain to allow for variable orientation and antigen binding. The endodomain is the functional signal-transmitting domain of the receptor. An endodomain of a CAR may comprise, for example, intracellular signalling domains from the CD3 ζ-chain, or from receptors such as CD28, 41BB, or ICOS. A CAR may comprise multiple signalling domains, for example, but not limited to, CD3z-CD28-41BB or CD3z- CD28-OX40. The CAR may bind specifically to a CD9 or MYLIP expressed by cells at the site of spinal injury. Techniques for generating CAR-T cells that specifically bind to target antigens are well-established in the art. An agent as described above may be administered alone or may be formulated into a pharmaceutical composition. A pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect. A suitable pharmaceutical composition for use as described herein may comprise an agent described above and a pharmaceutically acceptable excipient. For example, a pharmaceutical composition may comprise a therapeutic agent selected from (a) a CD9 or MYLIP antagonist or inhibitor (b) CD9 or MYLIP suppressor nucleic acid, (c) CD9 or MYLIP targetable nuclease, and (d) nucleic acid encoding a CD9 or MYLIP suppressor nucleic acid or targetable nuclease, as described herein, and a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH- adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003. The pharmaceutical compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the agent with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers. Formulations may for example be in the form of liquids or solutions. Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. The pharmaceutical compositions may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions. The precise nature of the carrier or other material will depend on the route of administration, which may be any convenient route, for example by injection, e.g. cutaneous, subcutaneous, or intravenous. Preferably, the agent is administered to the site of spinal injury, for example by intrathecal or intraspinal injection. Alternatively, the agent may be administered systemically, e.g. intravenously. The pharmaceutical compositions comprising the active compounds may be formulated in a dosage unit formulation that is appropriate for the intended route of administration. Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use. Methods of determining the most effective means and dosage of administration are well known in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician. Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Multiple doses of the agent may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered. The administration of the agent may continue for sustained periods of time. For example treatment with the agent may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the agent may be continued for as long as is necessary to reduce symptoms or improve functionality. An agent that reduces CD9 and/or MYLIP expression or activity or pharmaceutical composition comprising such an agent may be useful in treating a spinal cord injury (SCI), promoting spinal cord repair, reducing blood spinal cord barrier (BSCB) permeability and/or promoting BSCB integrity at a site of spinal cord injury in a patient. A spinal cord injury (SCI) may be a contusion or other injury that damages the spinal cord and, in particular the nerve fibres therein, and temporarily or permanently alters its function. The SCI may be in the cervical (C1 to C8), thoracic (T1 to T12), lumbar (L1 to L5) or sacral (S1 to S5) spine. An SCI may be traumatic or non-traumatic. An SCI may be a complete injury in which all functions mediated by nerves below the site of injury are lost, or an incomplete injury, in which some function, such as sensory or motor function, mediated by nerves below the site of injury is preserved. Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of spinal cord injury or post spinal cord injury complications. For example, one or more of locomotor function, sensory function; and autonomic function may be improved in the individual following spinal cord injury by reduction in CD9 or MYLIP expression or activity as described herein. An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human. In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or leporid) may be employed. An individual with a spinal cord injury may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of spinal cord injury in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine. It will be appreciated that appropriate dosages of an agent can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular agent, the route of administration, the time of administration, the rate of loss or inactivation of the agent, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The dosage of agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of injury which achieve the desired effect without causing substantial harmful or deleterious side-effects. In some embodiments, the agent may be administered at a dosage that is effective in reducing MYLIP or CD9 expression or activity at the injury site. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a agent described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. Treatment may comprise the administration of a therapeutically effective amount of the agent or pharmaceutical composition to the individual. “Therapeutically effective amount" relates to the amount of a agent or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio. For example, a suitable amount of a agent or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual. A therapeutic effect may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. In some embodiments, a treatment as described herein may have a duration of up to 3 weeks, up to 6 weeks, up to 3 months, up to 6 months or up to 12 months. The treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the agent, the route of administration and the nature of the condition being treated. Treatment may be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient. In other embodiments, an agent as described herein may be administered in combination with one or more other therapies, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated. Other therapies may include treatment with therapeutic agents that diminish neurological tissue destruction and ameliorate functional recovery, such as riluzole and minocycline. When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art. In other embodiments, CD9 and/or MYLIP may be useful in screening for compounds that may be useful in the development of therapeutics for treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord injury. A method of screening for a compound useful in treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord injury may comprise; determining the binding of a test compound to isolated CD9 and/or MYLIP, Binding of the test compound to CD9 and/or MYLIP may be indicative that the compound is useful in treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord. A method of screening for a compound useful in treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord injury comprising; determining the effect of a test compound on the expression of CD9 and/or MYLIP in a non- human mammal or determining the effect of a test compound on the activity of CD9 and/or MYLIP, for example in a non-human mammal. A decrease in expression or activity of CD9 and/or MYLIP may be indicative that the compound is useful in treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord. A reduction in expression of CD9 and/or MYLIP in the non-human mammal may be assessed by taking a sample from the animal and performing immunohistochemistry to assess expression levels of the protein of interest. Indirectly, a reduction in expression/activity of CD9 and/or MYLIP may be assessed by measuring levels of inflammation in the animals, e.g. levels of inflammatory markers etc. A reduction in CD9 and/or MYLIP activity is associated with a reduction in inflammation. The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments. A test compound may be an isolated molecule or may be comprised in a sample, mixture, or extract, for example, a biological sample. Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used. Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate CD9 or MYLIP activity. Such libraries and their use are known in the art, for all manner of natural products, small molecules, and peptides, among others. The use of peptide libraries may be preferred in certain circumstances. The amount of test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100μM, e.g. 0.1 to 50 μM, such as about 10 μM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development. A test compound identified in a screening method may be useful in the development of therapeutics for ^{treating spinal cord injury}; ^{promoting spinal cord repair}; ^{reducing blood spinal cord barrier (BSCB)} ^permeability; ^{or promoting BSCB integrity at a site of spinal cord injury. Test compounds which may be} screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Suitable compounds include CD9 and MYLIP antagonists and variants or derivatives thereof. A test compound identified using one or more initial screens as having ability to bind or neutralise CD9 and/or MYLIP may be assessed further using one or more secondary screens. A secondary screen may involve testing for a biological function or activity in vitro and/or in vivo, e.g. in an animal model. For example, the ability of a test compound to modulate endothelial barrier integrity may be determined. Following identification of a test compound which binds or neutralises CD9 and/or MYLIP, the compound may be isolated and/or purified or alternatively it may be synthesised using conventional techniques of chemical synthesis. The compound may be modified to optimise its pharmaceutical properties. This may be done using modelling techniques which are well-known in the art. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition. This may be useful as a CD9 or MYLIP antagonist in the development of therapies for treating spinal cord injury; promoting spinal cord repair; reducing blood spinal cord barrier (BSCB) permeability; or promoting BSCB integrity at a site of spinal cord injury. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term” consisting essentially of”. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes. “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

In this study, we explored the transcriptome of spinal cord vascular cells at 0, 3 and 7 dpi, a period in which the process of angiogenesis affects BSCB integrity, with the aim to characterize the molecular basis of the response to injury in a mouse contusion SCI model. We identified two genes, Cd9 and Mylip, whose expression is injury induced. Both CD9 and MYLIP proteins were found in association with vascular and/or perivascular cells (ECs and pericytes) and accumulate at the injury periphery, predominantly at the caudal region. Therapeutic strategies for spinal cord repair by targeting CD9 and MYLIP during the subacute phase of the injury hold promising results for BSCB control and functional recovery. Materials and Methods Animals All surgical and postoperative care procedures were performed in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) guidelines, were approved by Instituto de Medicina Molecular –João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Portugal (iMM) and followed the Portuguese Animal Ethics Committee (DGAV) regulations. Adult (8-9 weeks old) female C57BL/6J mice (Mus musculus) were purchased from Charles River Laboratory and housed in the animal facilities of iMM. Animals were housed under conventional conditions on a 12-hour light-dark cycle with ad libitum access to food and water. Spinal cord injury and post-operative care After two weeks of handling and acclimatization, body weight was assessed to ensure ideal weight (18-20 g) and animals were assigned to spinal cord injury. Mice (9–11-week-old) were anesthetized using a cocktail of ketamine (120 mg/kg) and xylazine (16 mg/kg) administered by intraperitoneal (ip) injection. For spinal contusion injuries, a laminectomy of the ninth thoracic vertebra (T9), identified based on anatomical landmarks, was first performed followed by a moderate (75 kdyne) contusion using the Infinite Horizons Impactor (Precision Systems and Instrumentation, LLC.) After SCI, the muscle and skin was closed with 4.0 polyglycolic absorbable sutures (Safil, G1048213). In control uninjured mice (sham), the wound was closed and sutured after the T9 laminectomy, and the spinal cord was not touched. Animals were injected with saline (0.5 ml) subcutaneously (sq) then placed into warmed cages until they recovered from anaesthesia and for the following recovery period (3 days). To prevent dehydration mice were supplemented with daily saline (0.5 ml, sq) for the first 5 dpi. Bladders were manually voided twice daily for the duration of experiments. Single-cell preparation for FACS Animals were sacrificed at 0, 3 and 7 dpi and the spinal cords harvested for Fluorescence Activated Cell Sorting (FACS). Approximately 6 mm spanning the injury/sham epicentre of the manipulated experimental and control spinal cords were collected and for each condition 3/4 biological replicates were used. The harvested spinal cord samples were homogenized according to a spinal cord specific and FACS-compatible protocol optimized in our laboratory. Briefly each spinal cord was dissected in DMEM and then transferred to a specific digestion mix (0.01% CaCl, 200 U/ml Collagenase I (Sigma 680U/mg), 0.000125% of 2% DNAseI in DMEM (GIBCO), for 30 minutes at 37°C to allow digestion of the spinal cord tissue.22% BSA was added in a 1:1 ratio to allow the separation between myelin and the vascular tubes followed by centrifugation at 1360 g for 10 minutes at 4°C. After the removal of myelin cold EC medium (DMEM + 10% FBS) was added and the suspension of cells was filtered through a 70 µm filter to remove undigested cell clumps and separate single cells. Additional steps were added to eliminate blood cells by adding ACK lysing buffer (GIBCO) followed by a wash with a FACSmax Cell Dissociation Solution (Amsbio). Cell suspension was incubated with a rat anti-mouse CD31-PE (BD Biosciences 1:50, 561073) on ice and in the dark for 45 minutes, followed by a wash of FACS solution and spin at 400 g for 5 minutes. Cells were resuspended in FACS medium, filtered and 7AAD (Miltenyi Biotec, 130-111-568) added. All samples were passed through a FACSAriaIII Cell Sorter to separate the specific vascular cells from non-endothelial and cell death fraction. The EC fractions were collected in RLT-plus buffer (Quiagen, 1053393) and stored at -80°C until RNA extraction was performed on the following day. Preparation of cDNA library and RNA-seq Cells in suspension were collected in 2.5 µL of Buffer RLT Plus (Qiagen, 1053393) and mRNA-library was prepared at IGC Genomics Unit using SMART-Seq [38]. Illumina libraries were generated with the Nextera based protocol and libraries quality were assessed in Fragment Analyzer before sequencing. Sequencing was carried out in NextSeq 500 Sequencer (Illumina) at the IGC Genomics facility using SE75bp and 30 million reads per library. Sequencing data were demultiplexed and converted to FASTQ format using bcl2fastq v2.19.1.403 (Illumina) Pre-processing of RNA-seq Provided FASTQ files with RNA-seq data from the six studied conditions (SCI and sham samples for each timepoint considered: 0-, 3- and 7-days post injury) were checked for the overall quality of sequencing reads using tool fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and information was compiled using MultiQC [39]. Despite differences in the total number of sequenced reads per sample (that were addressed in the following steps), all samples were considered to have enough quality. Vast-tools (Vertebrate Alternative Splicing and Transcription Tools) version 2 [40] was used for alignment and quantification of gene expression, considering the VastDB [41] gene annotation for mouse genome assembly mm9. The total number of aligned reads and the number of profiled genes per sample was inspected and only samples with more than 7 million read counts were considered for further analysis. Furthermore, sample 0dpisham3b_S_Run7_12 was not considered due to an abnormally low number of profiled genes compared to all other considered samples, suggesting a library of very low complexity. Thus, from the 24 original samples, only 15 were further considered. Gene expression quantification Using the vast-tools pipeline, gene expression quantification is performed by aligning RNA-seq reads against a reference transcriptome and counting those mapping to each given gene. Raw read counts for 22667 genes were obtained by vast-tools. In order to consider only the genes for which expression was detected in all samples, only genes with at least 1 read count for all samples and with read count variance different from 0 were considered (1266 genes). To prepare count data for differential gene expression analysis, normalisation factors to scale raw library sizes were obtained using the function calcNormfactors from package edgeR [42] after converting the filtered count matrix into a DGEList object. The voom method with quantile normalisation [43] was then applied on count data to obtain gene expression estimates in log2- counts per million (logCPM). Derivation of Gene Expression Signatures for the Major Spinal Cord Cell Types We employed CIBERSORTx [18] to infer gene signatures of major cell types present in the mouse spinal cord. The tutorials provided at the CIBERSORTx portal (https://cibersortx.stanford.edu/) were used as a basis for our analysis. The Zeisel dataset [19] was used to extract single-cell gene expression data from spinal cord mouse tissue (l5_all.loom file from mousebrain.org). Cells in the dataset are hierarchically classified according to Taxonomy levels, being the TaxonomyRank1 the top level and the one we used to create the signature matrix. A total of 50 cells of each taxon were sampled and renamed according to their taxon (expression_single_cell50_TaxonomyRank1.txt). Signatures were generated with the default parameter except for the following: Min. Expression: 1; Replicates: 20; Sampling: 0 Estimation of Cellular Composition on FACS Sorted Samples We used CIBERSORTx deconvolution to estimate the cellular composition of FACS sorted samples from bulk RNA sequencing. To build the mixture file containing bulk RNA-seq expression profile, we built a count matrix containing only the high confidence genes described above (mouse_per_sample_high_conf_genes.txt) at the analysed timepoints. We ran the CIBERSORTx deconvolution algorithm with default parameters except for the following: Batch correction mode: S-mode; Single cell reference matrix file used for S-mode batch correction: expression_single_cell50_TaxonomyRank1.txt; Permutations: 500 Differential gene expression analysis Differential gene expression analysis was performed with linear models using the limma R package [43]. The expression of each gene was fitted a model considering as baseline the average expression level of that gene across samples (centred design) and calculating the increment in expression for each of the following contrasts (coefficients in the model):

a) 3 dpi: GE increment from the 0 dpi average sample to the 3 dpi average sample; b) 7dpi: GE increment from the 0 dpi average sample to the 7 dpi average sample; c) Sci: GE increment in the average Sci sample compared to the average Sham sample; d) Interaction 3dpi with Sci: GE increment in 3dpi Sci samples that is not explained by a) and c) combined; e) Interaction 7dpi with Sci: GE increment in 7dpi Sci samples that is not explained by b) and c) combined. With such mathematical implementation, the isolated effects of SCI and time (3 or 7 dpi) can be assessed independently from the interaction coefficients β3SCI and β7SCI that in turn reflect the GE alterations that are specific of samples with the lesion (SCI) at the respective time-points With such mathematical implementation, the isolated effects of SCI and time (3 or 7 dpi) can be assessed independently from the interaction coefficients β3SCI and β7SCI that in turn reflect the GE alterations that are specific of samples with the lesion (SCI) at the respective time-points. Global empirical Bayes statistics are calculated for each gene and each coefficient in the model, allowing for the identification of genes significantly differentially expressed as those with an associated positive log-odds ratio (B statistic > 0) of the gene being differentially expressed and with magnitude of difference in expression measured in log2 fold-change between the two conditions. Using this approach, 13 genes (for the interaction effect between 3 dpi and Sci) and 5 genes (for the interaction effect between 7 dpi and Sci) were identified as significantly differentially expressed. Gene Set Enrichment Analysis We used the ‘clusterProfiler’R package [44] to perform gene set enrichment analysis (GSEA) on the t- statistics of differential gene expression of the high confidence genes at the analysed time-points. Gene symbols were checked for duplicates and, when found, the symbol with the highest absolute t-statistic value was kept. GO Biological Process ontology genes and terms for Mus musculus were retrieved with the package ‘msigdbr’. The GSEA algorithm was run with the default settings, except for the following: pvalueCutoff = Inf, eps = 0, seed = TRUE. We considered terms with adjusted p-value < 0.05 to be enriched in our analysis. Quantitative real-time PCR Total RNA was extracted from mouse spinal cord samples (6 mm of tissue spanning the lesion/sham site) using TRIzol (Invitrogen)/chloroform and purified using the RNA Clean & Concentrator-5 kit (Zymo Research), according to manufacturer’s instructions. RNA concentration was determined by NanoDrop (Thermo Scientific). cDNA synthesis was performed using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad), according to manufacturer’s instructions. qPCR was performed using 7500 Fast Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). For each cDNA sample, three technical replicates were included. Relative mRNA expression was normalized to PPIA mRNA expression using the ΔΔCt method. Western blot Protein extraction was performed from 6 mm of spinal cord tissue spanning the lesion/sham site of saline perfused animals. The samples were homogenized in lysis buffer (PBS/1% Triton X-100/protease and phosphatase inhibitors - cOmplete™, EDTAfree Protease Inhibitor Cocktail (Roche, 11873580001). Protein concentration was determined by DC Protein Assay (BioRad, 5000111). Spinal cord extracts were denatured and reduced in 4x Laemmli protein sample buffer (RioRab, 1610747) supplemented with 10% β- mercaptoethanol and boiled at 95°C for 10 min.50 μg of protein per sample was loaded and separated by 4 –15% SDS-PAGE gel (Bio-Rad, 4561084). Proteins were transferred to PVDF membranes (pore size 0.45 μm GE Healthcare, GE10600023) and blocked in 5% bovine albumin serum in TBS/0,1%Tween20 (TBS-T). Membranes were incubated overnight at 4°C with primary antibodies in blocking solution, and on the following day, the membranes were washed in TBS-T and incubated in the respective HRP-conjugated secondary antibody, in blocking solution, for 1 hour. GAPDH monoclonal antibody was used as loading control. Membranes were developed using the enhanced chemiluminescence kit Clarity Western ECL Subs (BioRad, 1705060) and visualized at Amersham 680 (GE Healthcare). The intensity of the specific bands was quantified using Image Studio Lite software. Unless described otherwise, all steps were performed at room temperature. Immunohistochemistry To perform immunostaining in sections, the frozen slides were thawed for 30 minutes. For MYLIP, the slides were washed 3 times with a pre-heated PBS in a water bath at 37°C for 5 minutes. For CD9 staining, the slides were subjected to antigen retrieval protocol. Afterwards, the slides were incubated for 10 minutes in a PBS/Triton X-100, and 3 hours in blocking. The slides were incubated overnight at 4°C in a humidified chamber with the specific primary antibodies’ combination in blocking solution. On the following day, a series of washes in PBS/Triton X-100 solution were performed for 15 minutes each. The cryosections were incubated with the appropriate secondary antibodies combination and 1 mg/ml DAPI (Sigma, D9564) for 2 hours, followed by 3 washes in PBS/Triton X-100 for 15 minutes, and 2 times in PBS. The slides were mounted in Mowiol mounting medium. All steps were performed at room temperature, unless described otherwise. Imaging Longitudinal images of spinal cords were acquired in a Zeiss Cell Observer Spinning Disk (SD) confocal microscope equipped with an Evolve 512 EMCCD camera. Images were acquired with a Plan-Apochromat 20x/0.80 Ph dry objective. DAPI fluorescence was detected using 405 nm for excitation (50 mW nominal output –20% transmission) and a BP 450/50 nm filter, with exposure time set to 150 ms. Alexa Fluor 488 fluorescence was detected using 488 nm for excitation (100 mW nominal output –10% transmission) and a BP 520/35 nm filter, with exposure time set to 100 ms. Alexa Fluor 561 fluorescence was detected using 561 nm for excitation (75 mW nominal output –12% transmission) and a BP 600/50 nm filter, with exposure time set to 100 ms. Alex Fluor 647 fluorescence was detected using 638 nm for excitation (75 mW nominal output –10% transmission) and a BP 690/50 nm filter, with exposure time set to 80 ms. EM Gain was set to 300 for all channels. Z-stacks of the four channels were acquired in tiled regions corresponding to the whole spinal cord tissue (typical acquisition volume ≈10 x 1.6 x 0.03 mm), with a Z interval of 0.49 µm and pixel size 0.67 µm. Stitching of the 3D dataset and maximum intensity projections were performed in Zeiss ZEN 3.2 (blue edition). Spinal cord closeup images were acquired in a Zeiss LSM 880 laser scanning confocal microscope, using a LD C-Apochromat 40x/1.10 water immersion objective. DAPI fluorescence was detected using 405 nm for excitation and a 415-485 nm detection window, with PMT gain set to 500 and offset to 2. Alexa Fluor 488 fluorescence was detected using the 488 nm laser line of an Ar laser for excitation and a 497-541 nm detection window, with GaAsP detector gain set to 500 and offset to 3. Alexa Fluor 561 fluorescence was detected using 561 nm for excitation and a 570-620 nm detection window, with GaAsP detector gain set to 500 and offset to 3. Alexa Fluor 647 fluorescence was detected using 633 nm for excitation and a 680-735 nm detection window, with GaAsP detector gain set to 550 and offset to 3. The pinhole size was set to 1 AU for Alexa Fluor 647, 1.31 AU for Alexa Fluor 561, 1.51 AU for Alexa Fluor 488 and 1.74 AU for DAPI to achieve the same optical slice thickness in all 4 channels. Z-stacks were acquired with Zoom set to 1 (212.55 x 212.55 µm area with 1024x1024 frame size –0.21 µm pixel size) with a line average of 2 and 1.02 µs pixel dwell time (unidirectional scan). Maximum intensity projections were performed in Zeiss ZEN 2.3 (black edition) and the image analysis software Fiji. Adobe Illustrator was used for assembly of figures. Statistical Analysis GraphPad Prism 6 was used for data visualization and statistical analysis. qPCR and Western blot data were analysed using an unpaired Student t-test. All data were expressed as mean, with statistical significance determined at p-values < 0.05. Details on statistical parameters, including sample sizes and precision measures (e.g., p-values), are described elsewhere herein. Results Validation of vascular identity of sorted cells from spinal cord samples To explore the vascular transcriptome, spinal cords from sham and injured animals (0, 3 and 7 dpi) were dissociated for single-cell analysis by Fluorescence Activated Cell Sorting (FACS) using the vascular marker CD31. Due to the complex microenvironment of the BSCB, rich in cell contacts and adhesion molecules, it can be hard to dissociate single cells. Therefore, we used CIBERSORTx [18], a ‚digital cytometry‛tool that estimates relative cell type abundances from bulk tissue transcriptomes, to assess the purity of our samples at 3 and 7 dpi, and their composition in different cell populations. For this, we resorted to gene expression signatures of the major cell types present in the mouse spinal cord, which are vascular, immune, glial and neuronal populations as defined by the Brain Mouse Atlas [19]. Considering these main cell populations, we estimated an average of 67% of mRNA in the analysed samples to be of vascular and perivascular origin, including ECs, pericytes and perivascular macrophages (Figure 1). This estimate is concordant with the method applied to sort vascular cells. The second most abundant cell type appeared to be of immune origin (average of 16%) (Figure 1). This is not surprising, given the close interaction of immune cells and the BSCB, which can be sorted following the incomplete dissociation of the vascular cells. With this validation we were confident that the studied transcriptomes were mostly of vascular origin. Identification of differentially expressed genes in vascular cells after spinal cord injury Differential gene expression was analysed for the subset of high-confidence genes considered, i.e., genes whose expression was detected and measured in all samples (see Materials & Methods), in order to identify GE alterations that are specific of injured samples at 3 and 7 dpi, respectively.13 genes were identified as significantly differentially expressed (B statistic > 0) at 3 dpi (when compared with 0 dpi) specifically in SCI samples (Figure 2a, Table 1).5 of these 13 genes were identified as also significantly differentially expressed at 7 dpi (when compared with 0 dpi) specifically in SCI samples (Figure 2b, Table 1). From those 13 genes specifically altered in injury at 3 dpi, 8 were upregulated (Mylip, Cd9, Selplg, Map3k1, Slc3a2, Rpr16, Ssr1, H2-DMb2) and 5 downregulated (Nek7, Trak2, Rock1, Ppk4, S100β). At 7 dpi, from those 5 differentially expressed genes, 3 showed upregulation (Cd9, Mylip, Scl3a2) and 2 were downregulated (Nek7 and Rock1). Given that they were consistently the two injury-specifically most over-expressed genes at 3 and 7 dpi (Table 1), we selected Cd9 and Mylip as candidate SCI-associated players at the perivascular microenvironment at 3 and 7 dpi. Spinal cord injury induces an upregulation of immune-related biological processes in vascular cells To understand whether the modulation of the 13 genes we identified at 3 dpi was part of a larger transcriptional program orchestrated by the vascular cells upon injury, we performed Gene Set Enrichment Analysis (GSEA) [20] to look for potentially enriched biological processes. Interestingly, we found that most of the terms positively enriched were immune-related, including those associated with immune regulation, immune effector function, immune response regulating cell surface receptor signalling, leukocyte migration and activation processes (Figure 3). CD9 and MYLIP mRNA and protein levels increase after spinal cord injury Given that the differential gene expression data obtained from spinal cord vascular cells showed Cd9 and Mylip as two genes being overexpressed after injury in both timepoints, we focused our analysis in validating their expression in total spinal cord tissue at 3 and 7 dpi. First, we evaluated their mRNA levels by qPCR and both Cd9 (Figure 4a) and Mylip (Figure 4b) were significantly increased after injury when compared with sham controls at 3 and 7 dpi, as detected in the transcriptome analysis. To assess if the increase of mRNA levels of Cd9 and Mylip was translated into increased protein levels, we performed a Western blot using 6 mm of spinal cord tissue spanning from the injury or sham epicentre. No changes were found between sham and SCI samples at 3 dpi for CD9 (Figure 5a) although we observed increased protein levels for MYLIP (Figure 5b). However, at 7 dpi, both CD9 and MYLIP protein levels were significantly increased when compared to controls (Figure 5a, b). Taken together, these results are consistent with the vascular transcriptome data and suggest increase of protein translation of CD9 and MYLIP at 7 dpi. CD9 and MYLIP expression is injury-induced predominantly at the caudal lesion site We determined the spatial localization of CD9 and MYLIP expression at 7 dpi in longitudinal cryosections of injured and sham spinal cords, a time-point in which we observed an increase in protein levels for both proteins. In sham injured animals no expression of both CD9 and MYLIP (Figure 6) was observed in all animals analysed (n=3). However, after injury, CD9 and MYLIP were detected at the injury site with more prominent expression in the caudal region of the lesion in all animals analysed (n=3) (Figure 6). CD9 expression is detected in close proximity with endothelial and pericytic markers To explore CD9 expression after injury in situ, we further investigated its expression in association with endothelial and pericytic markers. Using CD31 as a marker for ECs, and αSMA for pericytes, we were able to identify that, when observed, CD9 is in close proximity with both vascular and perivascular markers (both ECs and pericytes) (Figure 7). MYLIP expression is detected in association with pericytic markers, including in pericytes detached from blood vessels To investigate the injury-induced expression of MYLIP in the spinal cord, CD31 and CD13 were used (see Materials & Methods, Table S4), to assess both endothelial and pericytic populations, respectively. We observed that, in contrast to CD9, MYLIP expression was only observed in close proximity with the marker CD13 (Figure 8). In addition, MYLIP is also detected in pericytes that are not attached with blood vessels (Figure 9). In mammals, SCI leads to irreversible tissue loss, characterized by a chronic state of neuroinflammation. The complexity of the disorder involves two waves of injury: the first associated with the physical damage to the cord caused by the trauma; and a second injury, produced by a series of molecular and cellular events that perpetuate tissue dysfunction, inhibiting axonal regeneration and preventing functional recovery [21]. One of the main structures that are destroyed with the mechanical force of the trauma is the vasculature [15], which in turn is one of the key players in disseminating the second injury and propagating neuroinflammation [9,10,13]. Once broken down, the blood vessels become dysfunctional and the cellular complex that they are part of –the BSCB –becomes leaky, allowing the infiltration of immune cells and inhibiting blood supply to the spinal cord. Therefore, several studies in the past years have focused on therapeutic strategies to promote regeneration of the vasculature and restore BSCB integrity, as a means to create an optimal microenvironment for neuronal repair [14,17]. Little to no recovery has been attained, demonstrating the complexity of the vascular response after trauma and the importance of other unknown molecular players. In this study, we used the contusion mouse model of SCI, to explore the vasculature response after a traumatic injury. We focused our attention during the subacute injury phase, between 3 to 7 dpi, a time when the abnormal permeability of BSCB is even more apparent and that precedes the settlement of scar formation. We hypothesized that, by resorting to RNA sequencing analysis of the vascular population at spinal cord injury epicentre, we might have a sight of key vascular players that could be involved in BSCB integrity and could be targeted in future studies. To understand vascular response, we used CD31, a specific marker of vascular differentiation, to isolate spinal cord vascular cells from the injury or sham epicentre at 0, 3 and 7 dpi. The FACS-sorted cells were sequenced and CIBERSORTx deconvolution [18] was used to estimate their cellular composition using inferred gene expression signatures for each of the major cellular populations (vascular, immune, glial and neuronal) present in the spinal cord. This analysis allowed us to estimate the purity of the isolated cells. We observed an average vascular purity of 67% in the sorted samples (Figure 1). Knowing the complexity of the vasculature, with high cell to cell contacts and adhesion molecules, we cannot exclude that single cell dissociation might have not been completely accomplished and some cell duplets might have been sorted, in particular of cells that are part of the BSCB, such as pericytes and perivascular macrophages. In fact, our CIBERSORTx analysis identifies the vascular population being constituted of ECs, pericytes and perivascular macrophages. Our second main cellular population were immune cells (Figure 1), particularly high in injured samples (SCI 3 dpi: 35,4%; SCI 7 dpi: 17,0%) when compared with controls (sham 3 dpi: 3,3%; sham 7 dpi: 5,9%) (Figure 1). As we know, particularly in the case of injured spinal cords, immune cell infiltrates can considerably affect sorting of vascular cells [22]. Furthermore, although CD31 is fairly specific for vascular cells, some reactivity may also be seen in macrophages and other immune cells [23], therefore we must consider the 15% contribution of immune cells in our analysis and not exclude the input of immune cells in BSCB integrity and dysfunction. With a purity of approximately 70% in our FACS-based isolation, we next analysed the expression dynamics for the subset of high confidence genes (i.e., genes with detectable expression in all samples). We identified 13 genes specifically differentially expressed in SCI samples at 3 dpi (Figure 2a), and 5 genes within these 13 were identified as also specifically differentially expressed in SCI samples at 7 dpi (Figure 2b). To further investigate the changes in the transcriptome after SCI in the vasculature, we used GSEA at 3 dpi, the time- point in which we see more differentially expressed genes (Figure 3). GSEA revealed that after a SCI there is an upregulation of immune-related gene ontologies in our FACS-isolated vascular cells, concomitant with the subacute phase in which the cells were sorted and with our ‘digital cytometry‛ analysis (16% of immune population). At this time-point we observe an activation of processes associated with regulation of immune effector, cell surface receptor signalling, myeloid leukocyte migration and lymphocyte activation (Figure 3). As known, all the cellular components of the NVU play an important role in the immune response during homeostasis and injury. Therefore, it is expected that particularly after SCI, these cells activate these same pathways, in a first instance to respond to the damage, but later as a chronic and disruptive manner, that contributes to neuroinflammation. In contrast, we observed a downregulation of processes associated with synaptic and cell to cell signalling, and axonal transport, correlated with the aggressive microenvironment of the spinal tissue as the result of the injury (Figure 3). We focused our attention on the 2 top genes identified as overexpressed in both timepoints with the highest B statistic: Cd9, that encodes an endothelial tetraspanin protein that although expressed in a variety of cell types, is particularly high in ECs [24,25], where it plays a crucial role in the transendothelial migration of leukocytes [24,26,27]; and Mylip, which encodes a novel ERM-like protein that interacts with myosin regulatory light chain, that inhibits neurite outgrowth [28] and was shown to be overexpressed in pericytes during tumour progression [29]. To further validate these results, CD9 and MYLIP mRNA and protein levels were assessed at 3 and 7 dpi. mRNA levels were significantly increased for both Cd9 and Mylip (Figure 4), however, this increase was only translated into augmented protein levels at 7 dpi for both proteins (Figure 5). This proposes that, for CD9, although there is a significant increase in gene expression at 3 dpi, the effector protein may possibly only have an important role at 7 dpi after injury. However, MYLIP protein levels follow gene expression already at 3 dpi. Taken that for both CD9 and MYLIP proteins, 7 dpi appears as an important time-point where protein levels were significantly increased, we next pursue the in situ validation at 7 dpi. Our histological analysis revealed that both CD9 and MYLIP are injury-induced, as no expression was observed in sham animals (Figure 6). CD9 and MYLIP expression was detected at the injury site with a caudal predominance (Figure 6). As expected, when detected, both CD9 and MYLIP exhibited perivascular expression. CD9 showed to be associated with the endothelial marker CD31 and the pericytic marker αSMA (Figure 7). As pericytes and ECs can share the same membrane in specific vessel locations, denominated as peg–socket pockets [30], it is not surprising that CD9 expression might be shared between ECs and pericytes at different levels in the vasculature. In fact, CD9 expression has already been described as a crosstalk signalling peptide between ECs and pericytes, in particular in pocket regions where both cells share the intramembrane space [31,32], reinforcing the importance of our results. In addition, other studies have already associated CD9 with SCI, both in proteomic data of contusion rat models at later time-points (8 weeks) [33] and in mouse cervical injury [34]. Nevertheless, although this supports CD9 as being injury- induced, this is the first time that CD9 expression is associated with vascular and perivascular populations after SCI. On the other hand, MYLIP expression revealed to be only associated with pericytes, due to its close proximity with the pericytic marker CD13 (Figure 8, 9) to be also present in a small population of pericytes dissociated from blood vessels (Figure 9) and were in close proximity to the lesion epicentre, suggesting that these pericytes might contribute to the scar formation. MYLIP expression has already been identified in pericytes in other contexts, such as in transcriptomic data of tumours [29] and in single cell profiling of lung tissue [35]. However, to our knowledge no study has ever shown MYLIP expression associated with pericytes in the context of a SCI. Pericytes regulate the capillary tone and blood flow in the spinal cord below the site of lesion [36], suggesting that there are pericyte-mediated constriction mechanisms that decrease spinal blood flow below the lesion that, if identified, can contribute to a decrease in hypoxia and improve myelination and regrowth. We hypothesise that both CD9 and MYLIP might play a role in such mechanisms but also have a crucial part in the inflammatory response, although further research should be done in near future. CD9 in particular has already a described role in the immune control [24,26,27], but can also dynamically interact with other transmembrane and cytoplasmatic proteins and therefore have a multitude of biological functions, such as affecting the activity of metalloproteinases, cytokines and chemokines, among others [25]. MYLIP could regulate the integrity of the BSCB as it plays a key role in the maintenance of cellular morphology, modulation of cell motility, remodelling of cytoskeletal proteins, and adhesion of cells with extracellular matrix (ECM) and other cells, through ICAM-1 and integrins [37]. Here, we identify for the first time CD9 and MYLIP as new vascular players in SCI. Forthcoming strategies targeting C9 and/or MYLIP may hold promising results for BSCB integrity, by decreasing inflammatory response and promoting a more permissive regenerative spinal microenvironment.

Table 1 Injury specific differentially expressed genes at 3 and 7 days post-injury 5

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SEQ ID NO: 1 (NP_001317241.1) MPVKGGTKCIKYLLFGFNFIFWLAGIAVLAIGLWLRFDSQTKSIFEQETNNNNSSFYTGVYILIGAGALMMLVG FLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHY ALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILCCAIRRN REMV SEQ ID NO: 2 (NM_001330312.2) 1 actctggcag caggccttgg ccaaggggcc tttagccctg acgacccggg gaagagtctc 61 ccaaagcaga acgcccggtc cggcgcccag accaaacgcg ggggaaccgg aagggcgagg 121 cctccacctt gccgggattg ctgtccttgc cattggacta tggctccgat tcgactctca 181 gaccaagagc atcttcgagc aagaaactaa taataataat tccagcttct acacaggagt 241 ctatattctg atcggagccg gcgccctcat gatgctggtg ggcttcctgg gctgctgcgg 301 ggctgtgcag gagtcccagt gcatgctggg actgttcttc ggcttcctct tggtgatatt 361 cgccattgaa atagctgcgg ccatctgggg atattcccac aaggatgagg tgattaagga 421 agtccaggag ttttacaagg acacctacaa caagctgaaa accaaggatg agccccagcg 481 ggaaacgctg aaagccatcc actatgcgtt gaactgctgt ggtttggctg ggggcgtgga 541 acagtttatc tcagacatct gccccaagaa ggacgtactc gaaaccttca ccgtgaagtc 601 ctgtcctgat gccatcaaag aggtcttcga caataaattc cacatcatcg gcgcagtggg 661 catcggcatt gccgtggtca tgatatttgg catgatcttc agtatgatct tgtgctgtgc 721 tatccgcagg aaccgcgaga tggtctagag tcagcttaca tccctgagca ggaaagttta 781 cccatgaaga ttggtgggat tttttgtttg tttgttttgt tttgtttgtt gtttgttgtt 841 tgtttttttg ccactaattt tagtattcat tctgcattgc tagataaaag ctgaagttac 901 tttatgtttg tcttttaatg cttcattcaa tattgacatt tgtagttgag cggggggttt 961 ggtttgcttt ggtttatatt ttttcagttg tttgtttttg cttgttatat taagcagaaa 1021 tcctgcaatg aaaggtacta tatttgctag actctagaca agatattgta cataaaagaa 1081 tttttttgtc tttaaataga tacaaatgtc tatcaacttt aatcaagttg taacttatat 1141 tgaagacaat ttgatacata ataaaaaatt atgacaatgt cctgga SEQ ID NO: 3 (NP_037394.2) MLCYVTRPDAVLMEVEVEAKANGEDCLNQVCRRLGIIEVDYFGLQFTGSKGESLWLNLRNRISQQMDGLAP YRLKLRVKFFVEPHLILQEQTRHIFFLHIKEALLAGHLLCSPEQAVELSALLAQTKFGDYNQNTAKYNYEELCA KELSSATLNSIVAKHKELEGTSQASAEYQVLQIVSAMENYGIEWHSVRDSEGQKLLIGVGPEGISICKDDFSPI NRIAYPVVQMATQSGKNVYLTVTKESGNSIVLLFKMISTRAASGLYRAITETHAFYRCDTVTSAVMMQYSRDL KGHLASLFLNENINLGKKYVFDIKRTSKEVYDHARRALYNAGVVDLVSRNNQSPSHSPLKSSESSMNCSSCE GLSCQQTRVLQEKLRKLKEAMLCMVCCEEEINSTFCPCGHTVCCESCAAQLQSCPVCRSRVEHVQHVYLPT HTSLLNLTVI SEQ ID NO: 4 (NM_013262.4) 1 agttgggctg ctggagtgcg gcgccaccgc ggaggacagg ggcagctggc gggcagcggg 61 tgagggggtg gcggggacgc gagtggcggc cgcggggccc cggacaaggg tccgcagagc 121 tgcagccttc gagggccagc cctctccgag tccggggctg ggtcccacca gtgacaaggc 181 ggcagccccg cgcacaccaa agagaaggcg gctgtggcgg cagcggcagc cccagccatg 241 ctgtgttatg tgacgaggcc ggacgcggtg ctgatggagg tggaggtgga ggcgaaagcc 301 aacggcgagg actgcctcaa ccaggtgtgc aggcgactgg gaatcataga agttgactat 361 tttggactgc agtttacggg tagcaaaggt gaaagtttat ggctaaacct gagaaaccgg 421 atctcccagc agatggatgg gctagcccct tacaggctta aacttagagt caagttcttc 481 gtggagcctc atctcatctt acaggagcag actaggcata tctttttctt gcacatcaag 541 gaggccctct tggcaggcca cctcttgtgt tccccagagc aggcagtgga actcagtgcc 601 ctcctggccc agaccaagtt tggagactac aaccagaaca ctgccaagta taactatgag 661 gagctctgtg ccaaggagct ctcctctgcc accttgaaca gcattgttgc aaaacataag 721 gagttggagg ggaccagcca ggcttcagct gaataccaag ttttgcagat tgtgtcggca 781 atggaaaact atggcataga atggcattct gtgcgggata gcgaagggca gaaactgctc 841 attggggttg gacctgaagg aatctcaatt tgtaaagatg actttagccc aattaatagg 901 atagcttatc ctgtggtgca gatggccacc cagtcaggaa agaatgtata tttgacggtc 961 accaaggaat ctgggaacag catcgtgctc ttgtttaaaa tgatcagcac cagggcggcc 1021 agcgggctct accgagcgat aacagagacg cacgcattct acaggtgtga cacagtgacc 1081 agcgccgtga tgatgcagta tagccgtgac ttgaagggcc acttggcatc tctgtttctg 1141 aatgaaaaca ttaaccttgg caagaaatat gtctttgata ttaaaagaac atcaaaggag 1201 gtgtatgacc atgccaggag ggctctgtac aatgctggcg ttgtggacct cgtttcaaga 1261 aacaaccaga gcccttcaca ctcgcctctg aagtcctcag aaagcagcat gaactgcagc 1321 agctgcgagg gcctcagctg ccagcagacc cgggtgctgc aggagaagct acgcaagctg 1381 aaggaagcca tgctgtgcat ggtgtgctgc gaggaggaga tcaactccac cttctgtccc 1441 tgtggccaca ctgtgtgctg tgagagctgc gccgcccagc tacagtcatg tcccgtctgc 1501 aggtcgcgtg tggagcatgt ccagcacgtc tatctgccaa cgcacaccag tcttctcaat 1561 ctgactgtaa tctaatctgt tgtgcttttg ttggacttgg catgtttcca tgaactgcac 1621 tattataaac tattaaaatg atagattgtg gagaaagtaa ttattccaac acccatctgc 1681 catgcgatgt taaaaaaaaa aaaaaggaag aaaaataaca cagctactcc tcactgcaaa 1741 aacatatcca tgcgtagaat caacaactcc agtcatggga ccaggaggag ctctgggacg 1801 cagacacatt ccttggatgt tgattttttt tatgatctag taaaggaata ggtaaagtct 1861 ttgatgtcag tgaagtggca acatagccaa aaagttgggt accttttagg aaatgatgtt 1921 gtaagtctcc ttaatgtatc ctgaggtaag tttcctactg gcagcagatt ttgtaagaat 1981 tacttttaag aatttcattc tttttgtatg gtcatggagc tccaaccatt tttaatagga 2041 aagtcttttg taaattgttg tcgttttaat gtcatttctg tctttataac ttgatcaaga 2101 atgattggaa ggcaaacagg tttacaaatc aattctgtga cttttaaaaa gttgacaatg 2161 ttgtcagatt taaaccagtg tggctagtaa aaagcagctc actcaatgtg ggtggctccc 2221 tattccttta cgctccccct atccctaccc cacaagcctt tcgattataa aatactacca 2281 atcttgttat aagattactg tggagtagtc aagtactccc cgggccttct gagctggtgg 2341 aatattttat ttcagactga aaacagagag cactctcctt gggaagggaa agcggagctt 2401 gctgagtgag agatggagcc tcatggtgta caactgaggg tagttaactc atcacttctc 2461 ccaagcactc gatcccagct tcacccactg gtgttgcttt gcttgaactg ttcaagcctt 2521 ttatagcctt accataagta tttagatatg gtgtcctttt ctgtttttgg ggggggagtt 2581 ttgttgtgtt tttttaaagt aagtgcttaa gtattaactt tgggttgtcc cctctgtatg 2641 tttcgaaggg gttttggttc tttttgcttc tgttttctta aacatgtttt ccactcccac 2701 ttgggcattt tggaagctgg tcagctagca ggttttctgg gatgtcggga gacctagatg 2761 accttatcgg gtgcaatact agctaaggta aagctagaaa cctacactgt cactttactg 2821 agatttctga gtatactttt catattgcct taatgtagca gtaatgtgtt tatgcatttg 2881 tttctttgca cagacatttt gtcaaatatt aaaactctac ttttttatgg cacatattag 2941 catataagcc tttattccaa gaggtattta ttttttcact tgtaaaaaaa taatgtttcc 3001 acgtaaagaa ctctgttata tcctagagga ctctgtcttt tatattcggg ataataaaga 3061 ctttaaagca aa SEQ ID NO: 5 (NP_00131724.1) MMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRET LKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMIFGMIFSMILC CAIRRNREMV SEQ ID NO: 6 (CD9 blocking

RSHRLRLH

Claims

Claims 1. A method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof comprising; reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury in the individual.

2. A method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof, the method comprising; reducing CD9 and/or MYLIP expression or activity at a site of spinal cord injury in the individual.

3. A method according to claim 1 or claim 2 wherein CD9 and/or MYLIP expression or activity is reduced in the sub-acute injury phase following a spinal cord injury.

4. A method according to any one of the preceding claims wherein CD9 and/or MYLIP expression or activity is reduced by administering a therapeutically effective amount of an agent that reduces CD9 expression or activity to the individual.

5. A method according to claim 4 wherein the agent is a CD9 antagonist.

6. A method according to claim 5 wherein the CD9 antagonist is an organic compound having a molecular weight of 900 Da or less.

7. A method according to claim 5 wherein the CD9 antagonist is a protein that specifically binds CD9.

8. A method according to claim 7 wherein the CD9 antagonist is an antibody molecule that specifically binds CD9.

9. A method according to claim 4 wherein the agent is a suppressor nucleic acid that reduces expression of active CD9 polypeptide.

10. A method according to claim 9 wherein the suppressor nucleic acid is a siRNA or shRNA.

11. A method according to claim 10 wherein the suppressor nucleic acid comprises a nucleotide sequence at least 95% identical to a contiguous sequence of 15 to 40 nucleotides of SEQ ID NO: 2.

12. A method according to claim 9 wherein the suppressor nucleic acid is an antisense oligonucleotide (ASO).

13. A method according to claim 4 wherein the agent is a targeted nuclease that reduces expression of active CD9 polypeptide at the site of injury.

14. A method according to claim 13 wherein the targeted nuclease is a ZFN, TALEN or meganuclease that recognises a target sequence within the CD9 gene.

15. A method according to claim 13 wherein the targeted nuclease is a CRISPR associated nuclease, said CRISPR associated nuclease being administered in combination with a guide RNA that recognises a target sequence within the CD9 gene.

16. A method according to any one of claims 13 to 15 wherein the targeted nuclease cleaves genomic DNA at the target sequence of the CD9 gene, thereby causing a deletion or insertion which reduces expression of active CD9 polypeptide.

17. A method according to claim 4 wherein the agent is a T cell comprising a heterologous antigen receptor that specifically binds to CD9.

18. A method according to claim 17 wherein the heterologous antigen receptor is a chimeric antigen receptor (CAR).

19. A method according to any one of claims 1 to 3 wherein CD9 and/or MYLIP expression or activity is reduced by administering a therapeutically effective amount of an agent that reduces MYLIP expression or activity to the individual.

20. A method according to claim 19 wherein the agent is a MYLIP antagonist.

21. A method according to claim 20 wherein the MYLIP antagonist is an organic compound having a molecular weight of 900 Da or less.

22. A method according to claim 20 wherein the MYLIP antagonist is a protein that specifically binds MYLIP.

23. A method according to claim 20 wherein the MYLIP antagonist is an antibody molecule that specifically binds MYLIP.

24. A method according to claim 19 wherein the agent is a suppressor nucleic acid that reduces expression of active MYLIP polypeptide.

25. A method according to claim 24 wherein the suppressor nucleic acid is a siRNA or shRNA.

26. A method according to claim 24 wherein the suppressor nucleic acid comprises a nucleotide sequence at least 95% identical to a contiguous sequence of 15 to 40 nucleotides of SEQ ID NO: 4.

27. A method according to claim 24 wherein the suppressor nucleic acid is an antisense oligonucleotide (ASO).

28. A method according to claim 19 wherein the agent is a targeted nuclease that reduces expression of active MYLIP polypeptide at the site of injury.

29. A method according to claim 28 wherein the targeted nuclease is a ZFN, TALEN or meganuclease that recognises a target sequence within the MYLIP gene.

30. A method according to claim 28 wherein the targeted nuclease is a CRISPR associated nuclease, said CRISPR associated nuclease being administered in combination with a guide RNA that recognises a target sequence within the MYLIP gene.

31. A method according to any one of claims 28-30 wherein the targeted nuclease cleaves genomic DNA at the target sequence of the MYLIP gene, thereby causing a deletion or insertion which reduces expression of active MYLIP polypeptide.

32. A method according to claim 19 wherein the agent is a T cell comprising a heterologous antigen receptor that specifically binds to MYLIP.

33. A method according to claim 32 wherein the heterologous antigen receptor is a chimeric antigen receptor (CAR).

34. An agent that reduces CD9 expression or activity for use in a method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof.

35. An agent that reduces CD9 expression or activity for use in a method of method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof.

36. An agent that reduces MYLIP expression or activity for use in a method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof.

37. An agent that reduces MYLIP expression or activity for use in a method of method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof.

38. An agent for use according to any one of claims 34 to 37 wherein the method is a method according to any one of claims 1 to 33.

39. Use of an agent that reduces CD9 expression or activity in the manufacture of a medicament for use in a method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof.

40. Use of an agent that reduces CD9 expression or activity in the manufacture of a medicament for use in a method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof.

41. Use of an agent that reduces MYLIP expression or activity in the manufacture of a medicament for use in a method of treating spinal cord injury or promoting spinal cord repair in an individual in need thereof.

42. Use of an agent that reduces MYLIP expression or activity in the manufacture of a medicament for use in a method of method of reducing blood spinal cord barrier (BSCB) permeability or promoting BSCB integrity at a site of spinal cord injury in an individual in need thereof.

43. Use according to any one of claims 40 to 42 wherein the method is a method according to any one of claims 1 to 33.

44. A pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces CD9 expression or activity or an agent that reduces MYLIP expression or activity and a pharmaceutically acceptable excipient.

45. A pharmaceutical composition according to claim 44 for use in a method according to any one of claims 1 to 33.

46. A method of screening for a compound useful in (i) treating spinal cord injury in a patient or (ii) promoting spinal cord repair in a patient with a spinal cord injury, the method comprising; determining the activity of MYLIP or CD9 in the presence or absence of a test compound, wherein a decrease in MYLIP or CD9 activity in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound for use in treating spinal cord injury in a patient or promoting spinal cord repair in a patient with a spinal cord injury.

47. A method of screening for a compound useful in (i) treating spinal cord injury in a patient or (ii) promoting spinal cord repair in a patient with a spinal cord injury, the method comprising; determining the expression of MYLIP or CD9 in a mammalian cell in the presence or absence of a test compound, wherein a decrease in MYLIP /or CD9 expression in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound for use in treating spinal cord injury in a patient or promoting spinal cord repair in a patient with a spinal cord injury.