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WO2024200749A1 - Traitement d'une lésion rachidienne - Google Patents

Traitement d'une lésion rachidienne Download PDF

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Publication number
WO2024200749A1
WO2024200749A1 PCT/EP2024/058651 EP2024058651W WO2024200749A1 WO 2024200749 A1 WO2024200749 A1 WO 2024200749A1 EP 2024058651 W EP2024058651 W EP 2024058651W WO 2024200749 A1 WO2024200749 A1 WO 2024200749A1
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Prior art keywords
mylip
spinal cord
expression
activity
agent
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Inventor
Leonor SAUDE
Isaura MARTINS
Dalila NEVES-SILVA
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Instituto de Medicina Molecular Joao Lobo Antunes
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Instituto de Medicina Molecular Joao Lobo Antunes
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides

Definitions

  • 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].
  • NVU neurovascular unit
  • BSCB blood spinal cord barrier
  • 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.
  • BSCB blood spinal cord barrier
  • 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.
  • 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].
  • 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.
  • 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.
  • Figure 8 shows that MYLIP expression is injury-induced in pericytes.
  • Figure 9 shows that MYLIP is expressed in detached pericytes.
  • 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.
  • 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.
  • a significance level of p ⁇ 0.05 such as p ⁇ 0.01 or p ⁇ 0.005 may indicate a significant increase or reduction.
  • 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.
  • 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.
  • 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.
  • BSCB damage, and associated abnormal permeability may be assumed without formal diagnosis following a spinal injury.
  • 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.
  • 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.
  • CD9 and/or MYLIP expression or activity 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.
  • 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).
  • 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.
  • 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.
  • CD9 and/or MYLIP expression or activity may be reduced in the region 5 mm caudal to the injury site.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the MYLIP antagonist may be an organic compound having a molecular weight of 900 Da or less.
  • 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.
  • 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.
  • the agent which reduces MYLIP activity or expression is not a statin.
  • antagonists and inhibitors 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.
  • the suppressor nucleic acid may be a siRNA or shRNA.
  • 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.
  • the suppressor nucleic acid may be a siRNA or shRNA.
  • 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.
  • 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.
  • PTGS post-transcriptional gene silencing
  • the RNA molecule is preferably double stranded RNA (dsRNA) (Fire A.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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)).
  • two or more suppressor nucleic acids may be used to suppress the expression of CD9 or MYLIP.
  • 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.
  • anti- sense techniques may be used to target control sequences of a gene, e.g.
  • 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.
  • 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.
  • a suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.
  • the expression of active CD9 or MYLIP polypeptide is reduced at the site of injury by targeted mutagenesis.
  • 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.
  • 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.
  • 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.
  • 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.
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Zinc-finger nucleases ZFNs
  • 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.
  • TALENs 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • MSCs mesenchymal stem cells
  • RNA molecules such as siRNA
  • MSCs may be autologous or allogeneic to the patient.
  • the expression of active CD9 or MYLIP polypeptide is reduced at the site of injury by cell therapy.
  • 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.
  • 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.
  • 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).
  • scFv single-chain variable fragment
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • pharmaceutically acceptable refers 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.
  • a subject e.g., human
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the individual is a human.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic agents 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.
  • 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.
  • 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 100 ⁇ M, e.g. 0.1 to 50 ⁇ M, such as about 10 ⁇ M. 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.
  • BSCB blood spinal cord barrier
  • 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.
  • 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.
  • 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.
  • BSCB blood spinal cord barrier
  • 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.
  • 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.
  • a laminectomy of the ninth thoracic vertebra (T9) 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).
  • saline 0.5 ml
  • sq subcutaneously
  • 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.
  • FACS Fluorescence Activated Cell Sorting
  • 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 ⁇ m filter to remove undigested cell clumps and separate single cells.
  • DMEM + 10% FBS myelin cold EC medium
  • 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 ⁇ L 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.
  • 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.
  • 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 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).
  • 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.
  • 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.
  • the isolated effects of SCI and time (3 or 7 dpi) can be assessed independently from the interaction coefficients ⁇ 3SCI and ⁇ 7SCI that in turn reflect the GE alterations that are specific of samples with the lesion (SCI) at the respective time-points
  • the isolated effects of SCI and time (3 or 7 dpi) can be assessed independently from the interaction coefficients ⁇ 3SCI and ⁇ 7SCI that in turn reflect the GE alterations that are specific of samples with the lesion (SCI) at the respective time-points.
  • 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 ⁇ Ct 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 - cOmpleteTM, 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% ⁇ - mercaptoethanol and boiled at 95°C for 10 min.50 ⁇ g of protein per sample was loaded and separated by 4 –15% SDS-PAGE gel (Bio-Rad, 4561084).
  • lysis buffer PBS/1% Triton X-100/protease and phosphatase inhibitors - cOmpleteTM, EDTAfree Protease Inhibitor Cocktail (Roche, 11873580001). Protein concentration was determined by DC Protein Assay (BioRad, 5000111). Spin
  • Proteins were transferred to PVDF membranes (pore size 0.45 ⁇ m 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.
  • 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.
  • SD Zeiss Cell Observer Spinning Disk
  • 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 ⁇ m and pixel size 0.67 ⁇ m. Stitching of the 3D dataset and maximum intensity projections were performed in Zeiss ZEN 3.2 (blue edition).
  • 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 ⁇ m area with 1024x1024 frame size –0.21 ⁇ m pixel size) with a line average of 2 and 1.02 ⁇ s pixel dwell time (unidirectional scan).
  • GSEA Gene Set Enrichment Analysis
  • CD9 and MYLIP mRNA and protein levels increase after spinal cord injury
  • 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
  • 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.
  • MYLIP expression is detected in association with pericytic markers, including in pericytes detached from blood vessels
  • CD31 and CD13 were used (see Materials & Methods, Table S4), to assess both endothelial and pericytic populations, respectively.
  • CD9 MYLIP expression was only observed in close proximity with the marker CD13 ( Figure 8).
  • MYLIP is also detected in pericytes that are not attached with blood vessels ( Figure 9).
  • 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].
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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].
  • ECM extracellular matrix

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Abstract

La présente invention concerne des méthodes de traitement d'une lésion de la moelle épinière ou de promotion de la réparation de la moelle épinière chez un individu en ayant besoin par réduction de l'expression ou de l'activité de CD9 et/ou de MYLIP au niveau d'un site de lésion de la moelle épinière chez l'individu. L'invention concerne également des méthodes de traitement et des utilisations médicales, ainsi que des compositions pharmaceutiques et des méthodes de criblage de composés utiles pour traiter une lésion de la moelle épinière ou pour favoriser la réparation de la moelle épinière.
PCT/EP2024/058651 2023-03-29 2024-03-28 Traitement d'une lésion rachidienne Pending WO2024200749A1 (fr)

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