Title: Inhibitors of lysine methyltransferase for treatment of pain

The invention is in the field of medical treatment methods and therapeutic compositions for use in such methods. In particular, the invention relates to therapeutic compounds for treatment of pain, especially persistent inflammatory and/or neuropathic pain.

INTRODUCTION

Chronic pain is a major clinical problem and affects approximately 20% of the population (Breivik et al., 2006. Eur J Pain 10, 287-333; Breivik et al., 2013. BMC. Public Health 13, 1229; Steglitz et al., 2012. Transl Behav Med. 2, 6-8).

Inflammation, tissue and nerve damage induce long-lasting changes in the nociceptive circuitry causing pain and exaggerated responses to noxious and innocuous stimuli (Basbaum et al., 2009. Cell 139, 267-284;Hucho and Levine, 2007. Neuron 55, 365-376). Although many efforts have been undertaken to elucidate the molecular pathways driving chronic pain, a complete understanding of the mechanisms leading to chronic pain is missing, hampering the development of highly needed novel therapeutic approaches to treat debilitating pain conditions.

At the mechanistic level, the activation of spinal cord glial cells is thought to drive persistent pain. In various rodent models of chronic pain, including neuropathic and persistent inflammatory pain, spinal cord microglia have an activated phenotype and produce inflammatory mediators that trigger or maintain the long-lasting changes in nociceptive circuitry, thereby contributing to persistent pain (Clark and Malcangio, 2014. Front Cell Neurosci 8, 121; Graeber and Christie, 2012. Exp. Neurol. 234, 255-261; Milhgan and Watkins, 2009. Nat Rev Neurosci 10, 23-36; Old et al., 2015. Handb Exp Pharmacol 227, 145-170; Ren and Dubner, 2010. Nat Med 16, 1267-1276). Many efforts have been undertaken to elucidate how peripheral sensory neurons drive the engagement of these glial cells in chronic pain conditions. Sensory neurons engage spinal glial cells through the release of soluble factors (Clark and Malcangio, 2014. Front Cell Neurosci. 8, 121; Grace et al., 2014. Nat Rev Immunol 14, 217-231; Guan et al., 2016. Nat Neurosci 19, 94-101). However, the intracellular pathways in sensory neurons upstream of the release of glia- activating factors are still unknown.

Another driving force of pathological pain is the formation of reactive oxygen species (ROS) (Flatters, 2015. Prog Mol Biol Transl Sci 131, 119- 146). ROS are derived from electrons leaking from the mitochondrial electron transport chain (Flatters, 2015. Prog Mol Biol Transl Sci 131, 119- 146). ROS production can initiate pro-inflammatory cascades and activate microglia in the central nervous system (Kallenborn-Gerhardt et al., 2013. Pharmacol Ther 137, 309-317).

Importantly, increased ROS levels in the dorsal root ganglia (DRG) and/or spinal cord contribute to chronic pain development in different rodent models (Fidanboylu et al., 2011. PLoS One 6, e25212; Flatters, 2015. Prog Mol Biol Transl Sci 131, 119- 146; Kim et al, 2008. Neurosci Lett 447, 87-91; Schwartz et al., 2009. J. Neurosci 29, 159- 168) and altered ROS levels are associated with chronic pain development in humans (Meeus et al., 2013. Expert Opin Ther Targets 17, 1081-1089; Sanchez- Dominguez et al., 2015. Mitochondrion 21, 69-75; Tan et al., 2011. Eur J Pain 15, 708-715).

The identification of novel 'pain genes' that lie at the root of the transition from acute to persistent pain, possibly through glial cell engagement and ROS formation, aids in the understanding of the mechanism driving pathological pain and could identify highly needed novel targets for therapeutic pain interventions. Several genome-wide association studies (GWAS) in humans have offered a glimpse of the genetic contributions to pain syndromes. Nevertheless, very few have pinpointed to new pain genes that provided novel insights in pain

neurobiology. Recently, specific single nucleotide polymorphisms (SNPs) have been identified in patients with chronic widespread pain (CWP) in a large-scale GWAS (Peters et al., 2013. Ann Rheum Dis 72, 427-436). Two top intronic SNPs on chromosome 5pl5.2 were shown to be associated with a 30% higher risk of developing CWP. This genomic region encodes Chaperonin Containing TCP1 Subunit 5 (CCT5) and the hitherto functionally uncharacterized FAM173B protein, indicating potential novel pain genes. However, the molecular function of

FAM173B and its potential role in the neurobiology of chronic pain have not been revealed. SUMMARY OF THE INVENTION

FAM173B was identified as a lysine-specific protein methyltransferase that controls the development of chronic pain. Protein FAM173B resides in

mitochondria. Chronic pain was found to be mediated by increased expression of neuronal FAM173B methyltransferase activity, and involves reactive oxygen species-dependent pathway and/or activation of glial cells.

The invention therefore provides a specific inhibitor of FAM173B

mitochondrial lysine methyltransferase for use as a medicament, especially for use in a method of treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain.

The invention further provides a use of a specific inhibitor of FAM173B mitochondrial lysine methyltransferase in the manufacture of a medicament, especially a medicament for the treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain. Said inhibitor preferably is a specific inhibitor of the family of seven-6-strand lysine methyltransferase, more preferably a specific inhibitor of FAM173B lysine methyltransferase.

Said inhibitor preferably is a peptide or peptide analogue. Said peptide or peptide analogue preferably comprises a cell-penetrating domain.

Said inhibitor preferably is a specific inhibitor of mitochondrial FAM173B lysine methyltransferase such as mitochondria-targeted chaetocin or a functional analogue thereof.

Said inhibitor may be provided to primary sensory neurons of an individual suffering from chronic pain, preferably by systemic or local administration, including percutaneous injection and/or infusion, such as intrathecal,

paravertebral, intraforaminal and transforaminal epidural injection or infusion.

The provision of a specific inhibitor of FAM173B mitochondrial lysine methyltransferase preferably is combined with a ROS inhibitor compound and/or a mitochondrial inhibitor. Said ROS inhibitor compound preferably is selected from ascorbic acid, L-galactonic acid-g-galactone, pyruvate, mannitol, Trolox, a- tocopherol, Ebselen, uric acid and/or imidazole. Said mitochondrial inhibitor preferably is metformin.

The provision of a specific inhibitor of FAM173B mitochondrial lysine methyltransferase, with or without a ROS inhibitor compound and/or a mitochondrial inhibitor, may further be combined with the provision of microglia cells of the individual with a glial cell activation modulator compound.

The invention further provides a pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyl transferase and a pharmaceutically acceptable excipient.

Said pharmaceutical composition may further comprise an inhibitor of the generation of reactive oxygen species (ROS), a mitochondrial inhibitor and/or an inhibitor of glial cell activation.

Said pharmaceutical composition preferably is for use in a method of treatment of chronic pain, preferably of persistent inflammatory and neuropathic pain.

The invention further provides a method of typing an individual suffering from pain, the method comprising providing a sample of said individual;

determining a level of expression and/or activity of FAM173B in said sample; and typing said individual as suffering from chronic pain, based on the determined level of expression and/or activity of FAM173B.

The invention further provides a method of treating an individual suffering rom pain, the method comprising typing an individual according to the method of the invention; and treating an individual with a specific inhibitor of FAM173B, optionally combined with a ROS inhibitor and/or glial cell activation modulator compound, if the level of expression of FAM173B is enhanced in said individual when compared to a reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Sensory neuron FAM173B promotes chronic pain

a, mFaml73b mRNA expression 24 hours after the last intrathecal mFaml73b asODN (mFaml73b-AS) injections at day 5, 6, 7, 9 and 10 after intraplantar injection of complete Freund's adjuvant (CFA) (n=6). Statistical analysis was performed by Student's t test, b-d, Time course of (b) thermal and (c,d) mechanical hyperalgesia following (b,c) intraplantar injection of CFA (n=8), vehicle (veh; n=8), or (d) after spared nerve injury (n=8, ipsi = ispilateral, contra = contralateral). Mice received intrathecal injections of mFaml73b-AS or mismatch (MM) ODN (MM- ODN) at days 5, 6, 7, 9 and 10 during inflammatory pain or day 1-9 after SNI. e, Mouse Faml73b is expressed in sensory neurons and intraplantar injections with HSV-amplicons (day 1 and 3) encoding either for hFAM173B or nothing (EV) induced hFAM173B expression selectively in sensory neurons at day 4. Scale bar is 100 μηι. f-g, Intraplantar HSV-hFAM173B injection at day 5 and 7 rescued mFaml73b-AS-mediated attenuation of (1) thermal and (g) mechanical

hypersensitivity (n=8) in the CFA-model of persistent inflammatory pain. Mice received intrathecal asODN at day 5, 6, 7, 9 and 10 after CFA. h-i, Intraplantar HSV-hFAM173B injections at 3 and 1 day prior to intraplantar carrageenan injection prolonged transient inflammatory (h) thermal and (i) mechanical hypersensitivity (carrageenan: n=10, vehicle: n=6). j-k, Intraplantar HSV- liFAM173B injections at 3 and 1 day prior to an unilateral intraplantar

carrageenan injection induced (j) persistent reduced weight bearing of the affected paw as measured with dynamic weight bearing apparatus (n=6) and (k) ongoing spontaneous pain measured by the gabapentin-induced place preference in HSV- hFAM173B but not HSV-EV treated mice 1 month after intraplantar carrageenan (n=7). Statistical analysis was performed by Student's t test. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, statistical analyses were performed by 2-way ANOVA with Holm-Sidak multiple comparison test unless otherwise indicated.

Figure 2: FAM173B characterization

a, Topology diagram of archetypical sevens-strand methyltransferase with alpha- helices (boxes) and beta-strands (arrows), b, Protein sequence alignment of FAM173A/B from Homo sapiens (h), Mus musculus (m) and the aKMT homolog of FAM173B (FAM173hom) from Sulfolobus islandicus (Si). Predicted secondary structure of mFaml73b above alignment, coded as in a. Bars with white stripe indicate predicted N- and C-terminus of mFaml73b. Motif I and Post I (boxed) are involved in binding of SAM. Asp94 (*) was mutated to generate an enzymatically inactive protein. The first residue (Thr56) in recombinant truncated hFAM173B (FAM173BA55) is also indicated (vertical arrow), c, Fluorography of HEK293- extracts incubated with [3H]-SAM and recombinant hFAM173BA55. d,e, wt FAM173BA55 (d,e), but not FAM173BA55-D94A (e) methylated lysine- homopolymers (n=3-6). Statistical analysis was performed by Holm-Sidak multiple comparison test (d) or Student's t test (e). f, hFAM173B-GFP co-localized with the mitochondrial dye MitoTracker, but not with endoplasmic reticulum (PDI) or Golgi (PGM130). Scale bar 10 μπι g, Electron microscopy GFP-tagged hFAM173B- expressing HEK293. N: Nucleus, M: mitochondrion, dotted line: boundary between non-transfected (left) and transfected cell (right). Scale bar 500 nm. h, Cultured primary sensory neurons were stained for mFaml73b and the mitochondrial marker COX IV. Right panel is the co-localization profile at the white line shown in panel 3 of the double immunostaining for mFaml73b and COX IV. Scale bar 10 μιη. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3: FAM173B and mitochondrial function

Mitotracker fluorescence, as measure of mitochondrial potential, 48 hours after (a) mFaml73b knockdown (n=14- 16) or (b) hFAM173B overexpression (n=10-l l) in N2A cells. Statistical analyses were performed by Student's t test, c-d, HSV- mediated hFAM173B expression in cultured primary sensory neurons increased (c) TMRM fluorescence (n=100- 150 cells, 7 cultures) and (d) ROS production (DHE) after vehicle or 6 hours stimulation with 100 ng/nl TNFoc (n= 100- 130 cells, 9 cultures), e-f, In vivo expression of hFAM173B in sensory neurons with HSV- hFAM173B prior to intraplantar carrageenan increased (e) DHE fluorescence intensity at day 5 (n=7) and (1) MitoTrackerRedCMH2-ROS fluorescence intensity at day 3 (n =8) and day 6 (EV: HSV-EV, n=4; HSV-hFAM173b: hFAM173B, n=6) in small diameter neurons after intraplantar carrageenan injection. Statistical analysis was performed by 2-way ANOVA with Holm-Sidak multiple comparison test, (g) Intraperitoneal injection of the ROS scavenger PBN attenuated the hFAM173B-mediated prolongation of carrageenan-induced mechanical

hypersensitivity (n=5-6, veh: vehicle) Statistical analyses were performed by 2-way ANOVA with Holm-Sidak multiple comparison test. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, statistical analyses were performed by 1-way ANOVA with Holm-Sidak multiple comparison test unless otherwise indicated. For exemplar picture of a-c and f see supplementary figure 3. Figure 4: FAM173B-induced microglia activation

a, Spinal microglia ILGDrelease after stimulation for 24 hours with supernatants of hFAM173B-expressing sensory neurons that were previously stimulated with 100 ng/ml TNFot with or without the ROS scavenger PBN (2mM) for 6 hours, washed, and cultured for subsequent 15 h to capture sensory derived factors (n=7- ll; 100% = 59.4 pg/ml based on 3 independent experiments ). Statistical analysis was performed by 1-way A OVA with Holm-Sidak multiple comparison test, b,

Intrathecal anti-TNFa (100 μg/mouse) or (c) minocycline (30 μg/mouse) injection 7 days after intraplantar carrageenan attenuates hFAM173B-mediated prolongation of carrageenan-induced hyperalgesia (anti-TNFa: n=6 vehicle: n=3). d-e,

Intraplantar HSV-hFAM173B injection prior to induction of paw inflammation increased Ibal+ area in (d) spinal cord and (e) DRG at day 5 (n=4) and day 10 (n=6) after intraplantar carrageenan injection, f, Examplar images of quantified Ibal staining in d and e. (g-n) Mice received intraplantar CFA to induce persistent hyperalgesia and received intrathecal mFaml73b-AS at day 5, 6, 7, 9, and 10. At day 11 after CFA injection microglia activation in (g-i) DRG and (j-1) spinal cord was assessed by analysis of (d,j) fluorescent Ibal+ area (n=4) and (h,k) Ibal+ mRNA (n=8) in the dorsal horn of spinal cord or DRG. Statistical analyses were performed by Student's t test. i,l, Examplar images of Iba l staining of (i) DRG and (1) spinal cord as quantified in D an J. The specific area quantified in the spinal cord is shown in supplementary figure 4d. m-n, Inflammatory mediator mRNA expression 24 hours after the last intrathecal injection of niFaml73b-AS (day 11 after CFA) in (m) spinal cord and (n) DRG (n=8). Scale bars 50 Dm. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, statistical analyses were performed by 2-way ANOVA with Holm-Sidak multiple comparison test unless otherwise indicated.

Figure 5: Methyltransferase activity requirement

Wild-type hFAM173B but not hFAM173B-D94A expression in sensory neurons prolonged carrageenan-induced (a) thermal and (b) mechanical hypersensitivity (n=6). (c-d) Compared to wt hFAM173B, the methyltransferase inactive mutant hFAM173B-D94A did not increase (c) Ibal+ area in DRG and (d) dorsal horn of the spinal cord (n=5) at day 5 after intraplantar carrageenan injection, or enhance (e) TMRM fluorescence in N2A in vitro (n=10-15) and (1) ROS production in small diameter neurons in vivo (n=5-7) at 5 days after intraplantar carrageenan.

Statistical analyses were performed by 1-way ANOVA (C-F) with Holm-Sidak multiple comparison test, g, Supernatants of 15h cultured sensory neurons expressing hFAM173B-D94A that were stimulated with 100 ng/ml TNFa for 6 hours and subsequently washed did not enhance IL6 release in spinal microglia in vitro (n=4-8). Statistical analysis was performed by Student's t test. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, statistical analyses were performed by 2-way ANOVA with Holm-Sidak multiple comparison test unless otherwise indicated.

DETAILED DESCRIPTION OF THE INVENTION A Definitions

The term "FAM173R', as used herein, refers to a gene on human chromosome 5 in the 5pl5.2 region. Said gene encodes a protein with Uniprot accession number Q6P4H8. The protein sequence was found to have homology to the family of 7 beta strand S-adenosyl-L-methionine-dependent, lysine-specific methyltransferases. This family of methyltransferases structurally differs from the family of SET domain comprising methyltransferases. Seven-6-strand methyltransferases methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dotl/DOTIL was the only identified eukaryotic seven-6-strand methyltransferase (Falnes et al., 2016.

Biochem J 473: 1995-2009). As is shown in the examples, the encoded protein FAM173B is a mitochondrial lysine methyltransferase.

The term "Farnl 73b", as used herein, refers to the homologous gene of mouse.

The term "chronic pain", as used herein, refers to a pain in humans without apparent biologic value that has persisted beyond the normal tissue healing time (usually taken to be three months) that lasts more than 12 weeks. Chronic pain, also termed persistent pain, also refers to pain in rodent observed in model of persistent pain, for example persistent neuropathic or inflammatory pain models, that persists beyond normal tissue healing time or that persists during ongoing damage (at least several days). Chronic pain may arise from an initial injury, damage or inflammation, such as a back sprain, or from an ongoing cause, such as illness. However, often there is no clear cause. Chronic pain often results in disability and despair.

The term "peptide", as used herein, refers to a molecule with an amino acid chain of between 5 and 100 amino acid residues, preferably between 10 and 50 amino acid residues. The term peptide includes a peptide in which one or more of the amino acid monomers have been modified, for example by acetylation, amidation and/or glycosylation.

The term "peptide analogue", as used herein, refers to peptidomimetics which are or which comprise small peptide-like chains such as peptoids and 6-peptides designed to mimic a peptide. The altered chemical structure is preferably designed to adjust one or more properties such as, for example, stability, of a peptide, cell-penetrating domain.

The term "chaetocin", as used herein, refers to a natural product from

Chaetomium species, (3S,3'S,6R,6'R, 14R, 14'R, 16S, 16'S)-3,3'-bis(hydroxymethyl)- 2,2'-dimethyl-2,2',3,3',6,6',7,7'-octahydro- lH, rH-[14, 14'-bi(3, lla- epidithiopyrazino[1^2': l,5]pyrrolo[2,3-b]indole)]- l, l',4,4X15H, 15'H)-tetraone.

Chaetocin is known as a broad spectrum, non-specific lysine methyltransferase inhibitor, especially of SET domain comprising methyltransferases.

The term "functional analogue of chaetocin", as is used herein, refers to other lysine methyltransferase inhibitors, including lysine methyltransferase inhibitor of the epidithiodiketopiperazine class, BIX01294 (N-(l-benzylpiperidin-4-yl)-6,7- dimethoxy-2-(4-methyl-l,4-diazepan- l-yl)quinazolin-4-amine;trihydrochloride), UNC0224 (7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl- l,4-diazepan-l- Yl)-N-(l-methylpiperidin-4-Yl)quinazohn-4-amine), UNC0321 (7- [2- [2- (dimethylamino)ethoxy]ethoxy]-6-methoxy-2-(4-methyl-l,4-diaz epan-l-yl)-N-(l- methylpiperidin-4-yl)quinazolin-4-amine), DZNep (lS,2R,5R)-5-(4-amino- lh- imidazo[4,5-c]pyridin- l-yl)-3-(hydroxymethyl)cyclopent-3-ene- l,2-diol), Neplanocin A (lS,2R,5R)-5-(6-aminopurin-9-yl)-3-(hydroxymethyl)cyclopent- 3-ene-l,2-diol), CHEMBL61824 (2-(9H-xanthen-9-ylsulfanyl)-N-[2-[2-(9H-xanthen-9- ylsulfanyl)propanoylamino]ethyl]propanamide), CHEMBL468927 (2S)-2-amino-N- [[3-[5-[5-(l,3-benzothiazol-7-yl)- l,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyrazol- l- yl]phenyl]methyl]propanamide), Sinefungin ((2S,5S)-2,5-diamino-6-[(2R,3S,4R,5R)- 5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]hexanoic acid ), S-adenosyl-L- homocysteine (SAH), UNC0638 (2-cyclohexyl-N-(l-isopropylpiperidm-4-yl)-6- methoxy-7-(3-(pyrrolidin- l-yl)propoxy) quinazolin-4-amine), UNC0642 (2-(4,4- difluoropiperidin-l-yl)-6-methoxy-N 1-(propan-2-yl)piperidin-4-yl]-7 3-(pyrrolidin- l-yl)propoxy]quinazolin-4-amine) and BRD4770 (methyl 2-(benzoylamino)-l-(3- phenylpropyl)- lH-benzimidazole-5-carboxylate).

Said functional analogue preferably is a 7 beta strand lysine

methyltranferase inhibitor, preferably a specific inhibitor of a 7 beta strand lysine methyltranferase, of which class FAM173B is a member. Said functional analogue preferably is specific inhibitor of FAM173B.

The term "specific inhibitor of FAM173B", as is used herein, refers to an methyltransferase-inhibiting compound that has a binding affinity Kd for a seven- 6-strand methyltransferase such as FAM173B that is at least 10 fold lower than the Kd for a SET domain containing methyltransferase. A specific inhibitor of FAM173B preferably has a Kd of less than 10 ⁷ M, preferably less than 1( ⁸ M. Said specific inhibitor preferably is selected from the group consisting of adenosine-2',3'- dialdehyde (Adox) and, more preferably, specific inhibitors of DOT1L such as 7-[5- deoxy-5- [[3- [[[[4-(l, l-dimethylethyl)phenyl] amino]carbonyl] amino]propyl] time thLylethyl)amino]-6-I)-ribofuranosyl]^^

(EPZ004777), l-[3-[[[(2R,3S,4R,5R)-5-(4-amino-5-bromo-7H-pyrrolo[2,3- d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2- yl]methyl](isopropyl)amino]propyl]-3-[4-(2,2-dimethylethyl)p henyl]urea (SGC0946) and (2R ₎ 3R,4S,5R)-2 6-ammo-9H-purin )-yl)-5 (((lr,3S)-3-(2-(5-(tert-butyl)- lH- benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)meth yl)tetrahydrofuran-

A more general methyltransferase inhibitor such as, for example, chaetocin may be rendered into a specific inhibitor of FAM173B by targeting the inhibitor to mitochondria. As is known to a person skilled in the art, mitochondria have a strong negative internal potential of about -180mV. Cationic molecules are attracted to negatively charged mitochondria. Therefore, a molecule such as a general methyltransferase inhibitor can be targeted to mitochondria by conjugating this molecule to one or more cell-penetrating, lipophilic peptides such as

rhodamine-based and cyanine-based fluorophores (Zielonka et al., 2017. Chemical Reviews 117: 10043-10120), oligoguanidinium, or triphenylphosphonium moieties. As an example, 2,2,6, 6-tetramethyl-4-[[5-(triphenylpliospliomo)pentyl]oxy]- l- piperidinyloxy, monobromide (mitoTEMPOL) is a mitochondria-targeting superoxide dismutase mimetic that combines an antioxidant moiety (TEMPO L, also known as 4-hydroxy- TEMPO) with a lipophilic cation triphenylphosphonium, which allows it to pass through lipid bilayers and accumulate in mitochondria. Similarly, chaetocin, or a functional analogue of chaetocin, may rendered into a specific inhibitor of FAM173B by targeting the inhibitor to mitochondria, for example by conjugating this molecule to one or more cell-penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores,

oligoguanidinium, or triphenylphosphonium moieties.

A further preferred specific inhibitor of FAM173B is based on the nucleic acid sequence of the FAM173B-encoding gene, or based on the amino acid sequence of FAM173B. Said sequence -based specific inhibitor of FAM173B preferably is provided to a person in need thereof by intraplantar and/or intrathecal

administration, preferably by lumbar intrathecal injection.

Said sequence-based specific inhibitors of FAM173B include antisense oligodeoxynucleotides (asODN) and small interfering RNA (siRNA) molecules mediating RNA interference, which are 18-to 23-nucleotide dsRNA molecules with 2 nucleotide-long 3' overhangs. Said asODN and/or siRNA molecules preferably target human FAM173B. Said asODN and/or siRNA molecules may be provided to relevant cells as in vitro synthesized oligonucleotide. Said oligonucleotide may be modified, for example by phosphorothioate (PS) bonds, to prevent degradation of the oligonucleotide. As an alternative, or in addition, said oligonucleotide is targeted to mitochondria by conjugating the oligonucleotide to one or more cell- penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores (Zielonka et al., 2017. Chemical Reviews 117: 10043-10120), oligoguanidinium, or triphenylphosphonium moieties.

For expression of asODN and/or siRNA molecules may be provided to relevant cells by generating an expression cassette encoding the asODN and/or siRNA molecules. Said expression cassette preferably comprises a polymerase III enhancer/promoter. A preferred polymerase III enhancer/promoter is selected from the U6 and HI promoter. Said expression cassette preferably is provided in a vector, preferably a viral vector that is able to transduce neural cells, preferably sensory neurons. Said viral vector preferably is a recombinant adeno-associated viral vector, a herpes simplex virus-based vector, or a lentivirus-based vector such as a human immunodeficiency virus-based vector. Said viral vector most preferably is a herpes simplex virus-based vector.

A specific inhibitor of FAM173B that is based on the amino acid sequence of

FAM173B, for example, is an antibody or antibody mimetic that specifically binds and inhibits FAM173B. The term antibody includes a single heavy chain variable domain antibody such as a camelid VHH, a shark immunoglobulin-derived variable new antigen receptor, a scFv, a tandem scFv, a scFab, and an improved scFab (Koerber et al., 2015. J Mol Biol 427: 576-86).

The term "antibody mimetic", as is used herein, refers to a molecule that can specifically bind FAM173B, but that is not structurally related to an antibody. Antibody mime tics include a designed ankyrin repeat protein, a binding protein that is based on a Z domain of protein A, a binding protein that is based on a fibronectin type III domain, engineered lipocalin, and a binding protein that is based on a human Fyn SH3 domain (Skerra, 2007. Current Opinion Biotechnol 18: 295-304; Skrlec et al., 2015. Trends Biotechnol 33: 408-418).

The term "cell-penetrating domain", as is used herein, refers to peptides that facilitate cellular intake/uptake of various molecules. Examples of suitable cell- penetrating domains are penetratin or Antenapedia (N-terminus

RQIKWFQNRRMKWKK), TAT (N-termmus YGRKKRRQRRR), SynBl (N- terminus RGGRLSYSRRRFSTSTGR), SynB3 (N-termmus RRLSYSRRRF), PTD-4 (N-terminus PIRRRKKLRRLK), PTD-5 (N-terminus RRQRRTSKLMKR), FHV Coat-(35-49) (N-terminus RRRRNRTRRNRRR VR) , BMV Gag-(7-25) (N-terminus KMTRAQRRAAARRNRWTAR) , HTLV-II Rex-(4-16) (N-terminus

TRRQRTRRARRNR), D-Tat (N-termmus GRKKRRQRRRPPQ), R9-Tat (N- terminus GRRRRRRRRRPPQ), transportan (N-termmus

GWTLNSAGYLLGKINLKALAALAKKIL), MAP ((N-terminus

KLALKLALKLALALKLA) , SBP (N-termmus

MGLGLHLLVLAAALQGAWSQPKKKRKV) and FBP (N-terminus

GALFLGWLGAAGSTMGAWSQPKKKRKV) .

The term "mitochondrial targeting peptide", as is used herein, refers to a peptide of 10-70 amino acid residues that directs a protein to mitochondria. It is often present at the N- terminus and may consist of an alternating pattern of hydrophobic and positively charged amino acids. Examples of suitable

mitochondrial targeting peptides are MLSLRQSIRFFKPATRTLCSSRYLL, amino acid residues 289- 18 of the C terminus of APE 1

(HSLLPALCDSKIRSKALGSDHC PITLYLAL), and a 24 amino acid residues sequence from manganese-superoxide dismutase

(MLSRAVCGTSRQLAPALGYLGSRQ).

The term "inhibitor of the generation of ROS", as is used herein, refers to a molecule that may quench free radicals and/or inhibits production of reactive oxygen species (ROS). Examples of suitable ROS inhibitors include Trolox (6- hydroxy-2,5,7,8-tetramethylchiOman-2-carboxylic acid), a combination of Trolox and ascorbate, dihydroethidium (DHE), TEMPOL (2,2,6, 6-tetramethyl-4- piperidinol-N-oxyl), Tiron (disodium;4,5-dihydroxybenzene- 1,3-disulfonate), phenyl-N-t-butyl nitrone (PBN) and ebselen (1.2-phenyl- l,2-benzoisoselenazol- 3(2H)-one). Said term includes specific mitochondrial ROS inhibitors such as MitoQ (l()-(4,5-dimethoxy-2-methyl-3,6-dioxo-l,4-cyclohexadien- l- yl)decyl](triphenyl)phosphonium methanesulfonate), a mixture of [10-(4,5- dimethoxy-2-methyl-3,6-dioxocyclohexa-l,4-dienyl)decyl] triphenylphosphonium bromide and [l()-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)decyl]triph enyl phosphonium bromide}, MitoVitE (vitamin E attached to a triphenylphosphonium cation), mitoTEMPO (2-(2,2,6,6-tetramethylpiperidin- l-oxyl-4-ylamino)-2- oxoethyl)triphenylphosphonium chloride) and SKQ1 (10-(4,5-dimethyl-3,6- dioxocyclohexa- 1,4-dien- l-yl)decyl)triphenylphosphonium bromide.

The term "IC50", as is used herein, refers to a concentration of a compound that inhibits an enzymatic activity to 50% of the maximal activity. An IC50 value typically is expressed as a molar concentration.

B Treatment of patients

The present invention is based on the finding that FAM173B is a

mitochondrial lysine methyltransferase which is involved in mediating chronic pain. Chronic pain may occur after an injury or after a disease, or it may occur without any known physical cause. In some cases, it is accompanied by a tissue pathology such as chronic inflammation in some types of arthritis. Chronic pain is a very general concept and there are several varieties of chronic pain related to the musculoskeletal system, visceral organs, skin, and nervous system.

Examples of known diseases that may cause chronic pain include

fibromyalgia, irritable bowel syndrome, chronic arthropathy, post herpetic neuralgia, trigeminal neuralgia, neuropathy including mononeuropathy and polyneuropathy, small fiber neuropathy, metabolic neuropathy, diabetes-induced neuropathy, post-surgical pain syndrome, spinal nerve compression syndrome, migraine, chemotherapy-induced neuropathy, cancer-induced neuropathy, kidney and/or thyroid-induced neuropathy and rheumatoid and osteoarthritis.

In embodiments of this invention, the term chronic pain especially chronic widespread pain, chronic inflammatory and/or chronic neuropathic pain.

As is shown in the examples, down-regulation of Faml73b expression in a mouse model in vivo by lumbar intrathecal injections of antisense

oligodeoxynucleotides (asODN), resulted in a reduction of Faml73b mRNA expression and, consequently, also in a reduction of the steady state level of the encoded protein in lumbar dorsal root ganglia, without affecting spinal cord Fam l73b mRNA expression. This intrathecal administration abrogated thermal and mechanical hyperalgesia. Intrathecal injections of different Faml 73b asODN, but not of control ODNs, consistently abrogated thermal and mechanical hyperalgesia after inflammation and/or nerve ligation, indicating that

downregulation of Faml73b attenuates persistent neuropathic and inflammatory pain.

Rescue of Faml73b in primary sensory neurons by intraplantar and/or intrathecal administration of a viral vector transducing human FAM173B prevented rnFaml 73b as ODN-mediated attenuation of persistent thermal and mechanical hypersensitivity indicating that sensory neuron FAM173B is required for persistent inflammatory pain.

The knowledge that a mitochondrial lysine methyltransferase is required for development and/or sustainment of chronic pain provides an inhibitor, preferably a specific inhibitor, of said a mitochondrial lysine methyltransferase for use in a method of treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain. Said inhibitor of mitochondrial lysine methyltransferase, preferably specific inhibitor of FAM173B, may be an organic or inorganic compound, a peptide, a polynucleotide, a lipid, or a hormone or hormone analog.

Said organic or inorganic compound preferably is characterized by a relatively low molecular weight. A low molecular weight compound, i.e. with a molecular weight of 500 Dalton or less, is likely to have good absorption and permeation in biological systems and is consequently more likely to be a successful drug candidate than a compound with a molecular weight above 500 Dalton (Lipinski et al., 1997. Advanced Drug Delivery Reviews 23: 3-25). Synthetic compound libraries (e.g. LOP AC™, Sigma Aldrich) or natural compound libraries (Specs, TimTec) may be screened to identify said organic or inorganic compound.

The invention further relates to a method for identifying a compound that inhibits FAM173B mitochondrial lysine methyltransferase, comprising (a) contacting a compound with a FAM173B mitochondrial lysine methyltransferase polypeptide, (b) determining a binding affinity of the compound to the polypeptide,

(c) contacting a population of mammalian cells, preferably human neural cells, expressing said FAM173B mitochondrial lysine methyltransferase polypeptide with the compound that exhibits a binding affinity of at most 10 micromolar, and

(d) identifying a compound that inhibits the mitochondrial lysine

methyltransferase activity of FAM173B in the cells.

In one embodiment, said inhibitor is a peptide or peptide analogue, preferably a peptide or peptide analogue that inhibits the lysine methyltransferase of

FAM173B. Said inhibition of the lysine methyltransferase of FAM17B preferably is selective over inhibition of other lysine methyl transferase, whereby the term selective indicates that the IC50 for inhibition of FAM173B is more than 2x lower than the IC50 for inhibition of another lysine methyltransferase, preferably said IC50 for inhibition of FAM173B is more than 4x lower than the IC50 for inhibition of another lysine methyltransferase, more preferably more than lOx lower.

Inhibition might be achieved by competition with a peptide substrate and/or by competition with a co-factor, S-adenosyl methionine. Said inhibition preferably is achieved by competition with a peptide substrate or a peptide analogue of a substrate. To enable entrance into a cell, preferably a primary sensory neuron, said peptide substrate or peptide analogue of a substrate preferably comprises a cell- penetrating domain. Said cell-penetrating domain preferably is present at the N- terminus of the peptide or peptide analogue. Said cell-penetrating domain preferably is selected from penetratin, TAT, SynBl and SynB3.

As an alternative, said peptide substrate or peptide analogue of a substrate may be expressed by a viral vector, preferably a viral vector that is able to transduce neural cells, preferably sensory neurons. Said viral vector preferably is a recombinant adeno-associated viral vector, a herpes simplex virus -based vector, or a lentivirus-based vector such as a human immunodeficiency virus-based vector. Said viral vector most preferably is a herpes simplex virus-based vector.

Said peptide substrate or a peptide analogue of a substrate preferably additionally comprises a mitochondrial targeting moiety, preferably a

mitochondrial targeting peptide to direct said peptide or peptide analogue to the mitochondria. A preferred mitochondrial targeting moiety is a cell-penetrating, lipophilic peptide such as rhodamine-based and cyanine-based fluorophore, an oligoguanidinium, or a triphenylphosphonium moiety. A preferred mitochondrial targeting peptide comprises amino acid residues 289-318 of the C terminus of APEl (HSLLPALCDSKIRSKALGSDHC PITLYLAL). Said mitochondrial targeting peptide preferably is present at the C-terminus of the peptide substrate or a peptide analogue of a substrate.

Said inhibitor may also be a known inhibitor of a lysine methyltransferase, preferably a broad spectrum inhibitor of lysine methyltransferases. The term broad spectrum, as is used herein, refers to an inhibitor that is able to inhibit multiple different lysine methyltransferases with a similar IC50 concentration. Said broad spectrum inhibitor of lysine methyltransferases, such as chaetocin, is preferably targeted to mitochondria, for example by conjugating the inhibitor to one or more cell-penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores, oligoguanidinium, or triphenylphosphonium moieties. A preferred specific inhibitor of FAM173B lysine methyltransferase is a mitochondria targeted chaetocin or functional analogue thereof.

An inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173P), preferably is provided to primary sensory neurons of an individual suffering from chronic pain. Said primary sensory neurons preferably include thermoreceptor neurons, somatosensory neurons visceral neurons and/or mechanoreceptor neurons. The cell bodies of these neurons are located in ganglia throughout the spine. Methods of providing a compound such as a peptide or peptide analogue, or a small molecule, to primary sensory neurons, preferably to cell bodies of primary sensory neurons, are known in the art. Said methods include systemic administration, and local administration by percutaneous injection such as intrathecal, paravertebral, intraforaminal and transforaminal administration, as will be specified herein below.

Inhibition might further be achieved by alteration of the gene encoding

FAM173B in vivo, preferably in primary sensory neurons. Said alteration includes silencing of the gene, for example by expression of ZFP transcription factors attached to a "gene repression" domain in order to down-regulate (repress) the expression of FAM173B in vivo, preferably in primary sensory neurons; introducing inactivating alterations in the gene encoding FAM173B in vivo, preferably by alteration, including deletion of part or all, of the active domain of the lysine methyl transferase, especially of amino acid aspartic acid (D) at position 94 of the human FAM173B amino acid sequence within Motif 1, as depicted in Figure 2B. Said alteration may be accomplished, for example, by CRISPR-CAS (Sander and Joung, 2014. Nature Biotech 32, 347-355) and exon skipping (McNally et al., 2016. J Clin Invest 126: 1236-1238).

An inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173B, preferably is combined with a ROS inhibitor compound. As is shown herein, activation of FAM173B by overexpression of FAM173B in sensory neurons results in increased production of ROS. Inhibition of the production of ROS, or at least a decrease of the production of ROS, was shown to attenuate chronic pain such as persistent neuropathic pain. A combination of an inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173B, with a ROS inhibitor compound is likely to result in both immediate and sustained treatment of said chronic pain. A ROS inhibitor compound likely provides immediate, transient attenuation of chronic pain, while an inhibitor of lysine

methyltransferase, preferably a specific inhibitor of FAM173B, likely provides longer term, more persistent attenuation of chronic pain. A preferred ROS inhibitor compound is selected from ascorbic acid, DHE, L- galactonic acid-g-galactone, pyruvate, mannitol, Trolox, TEMPOL, mitoTEMPO, PBN, a-tocopherol, Ebselen, uric acid and/or imidazole.

The provision to primary sensory neurons of an inhibitor of lysine

methyltransferase, preferably a specific inhibitor of FAM173B, optionally in combination with a ROS inhibitor, may additionally be combined with the provision of a mitochondrial inhibitor. Said inhibitor preferably is a biguanide, such as metformin (3-(diaminomethylidene)- l, l-dimethylguanidine) and

phenformin (l-(diaminomethylidene)-2-(2-phenylethyl)guanidine).

Besides the biguanide family, other compounds and pathways have recently been identified in preclinical models as possible therapeutic approaches that ultimately seem to impair mitochondrial bioenergetic capacity, including gamitrinib, a resorcinolic small molecule comprising a benzoquinone ansamycin backbone, a linker region, and a mitochondrial targeting moiety, either provided by 1 to 4 repeats of cyclic guanidinium, or by a triphenylphosphonium moiety (Kang et al, 2009. J Clin Invest 119: 454-464), tigecycline ((4S,4aS,5aR, 12aR)-9-[[2-(tert- butylamino)acetyl] amino] -4, 7-bis(dimethylamino)- 1, 10, 11, 12a-tetrahydroxy-3, 12- dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide), and VLX600 (l-(2- pyridinyl)-ethanone, 2-(6-methyl-5H- l,2,4-triazino[5,6-b]indol-3-yl)hydrazine). Although these compounds have not undergone clinical trials for safety and therapeutic efficacy, they highlight the progress in targeting mitochondrial ATP production in cancer cells as a therapeutic strategy (Weinberg and Chandel, 2015. Nat Chem Biol 11: 9-15).

The provision to primary sensory neurons of an inhibitor of lysine

methyltransferase, preferably a specific inhibitor of FAM173B, optionally in combination with a ROS inhibitor and/or a mitochondrial inhibitor, may

additionally be combined with the provision of microglia cells of the individual suffering from chronic pain, with a glial cell activation modulator. Said glial cell activation modulator compound preferably is selected from minocycline

((4S,4aS,5aR, 12aR)-4, 7-bis(dimethylamino)- 1, 10, 11, 12a-tetrahydroxy-3, 12-dioxo- 4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide), ibudilast (2-methyl- 1-(2- propan-2-ylpyrazolo[l,5-a]pyridin-3-yl)propan- l-one), ATL313 (methyl 4-(3-{6- amino-9-[(2R,3R,4S,5S)-5-(cyclopropylcarbamoyl)-3,4-dihydrox ytetrahydro-2- furanyl]-9H-purin-2-yl}-2-propyn- l-yl)-l-piperidinecarboxylate), synerkine, a chimeric IL4-IL10 molecule (WO2013/070076) and/or XT101, a non-viral

interleukin- 10 delivery product. Studies have shown that one aspect of chronic pain is that activated glia in the spinal cord release proinflammatory products that drive pain and weaken the effects of opioid analgesia. Administration of

minocycline, ibudilast, ATL313, synerkine and/or XT101 will reverse glial cell activation and may further provide attenuation of chronic pain.

Said inhibitor may further find use, alone or in combination with a ROS inhibitor compound, a mitochondrial inhibitor, and/or a glial cell activation modulator compound, in the treatment of other neuroimmune-related diseases such as Alzheimer, Parkinson and multiple sclerose, in which microglia and activation of reactive oxygen species also play a role.

Said inhibitor of lysine methyltransferase, preferably a specific inhibitor of

FAM173B, alone or in combination with a ROS inhibitor compound, a

mitochondrial inhibitor, and/or a glial cell activation modulator compound, can be administered by a number of routes.

Said inhibitor of lysine methyltransferase, preferably a specific inhibitor of

FAM173B, alone or in combination with a ROS inhibitor compound, a

mitochondrial inhibitor, and/or a glial cell activation modulator compound preferably is provided to a patient in need thereof by systemic, or local

administration, including intrathecal, paravertebral, transforaminal and intraforaminal administration.

The term "systemic administration" as is used herein, refers to oral, intravenous, intramuscular, intra-articular, intra- arterial, intramedullary, intrathecal, epidural, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, inhalational, intraocular, intra-aural or rectal injection or infusion, preferably intravenous or intramuscular infection or infusion.

The term "intraforaminal administration" also termed intradiscal

administration, as is used herein, refers to administration in or through the foramen intervertebrale, preferably such that the inhibitor, optionally in

combination with one or more compounds, is provided to the spinal nerve, preferably the spinal ganglion of the spinal nerve. The term "intrathecal administration" as is used herein, refers to injection or infusion of said inhibitor, optionally in combination with one or more compounds, into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid. Said injection or infusion may be accomplished by use of external pump or of a fully implantable device. Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter. An implantable drug delivery system with a constant flow may be beneficial for long-term delivery of said inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173B, alone or in combination with a ROS inhibitor compound and/or a glial cell activation modulator compound.

The term "paravertebral administration" as is used herein, refers to injection or infusion into a space immediately lateral to where the spinal nerves emerge from the intervertebral foramina.

The term "transforaminal administration" or "intraforaminal

administration", as is used herein, refers to injection or infusion into the opening at the side of the spine where a nerve root exits. This opening is known as a foramen. There is a small sleeve of the epidural space that extends out over the nerve root for a short distance.

C Compositions

The invention further provides a pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyltransferase as defined herein above and a pharmaceutically acceptable excipient.

Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as a-lactose monohydrate, anhydrous a-lactose, anhydrous β-lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof.

Said pharmaceutical composition for intrathecal administration preferably is a sterile isotonic solution. Said buffer preferably is citrate-based buffer, preferably lithium-, sodium-, potassium-, or calcium- citrate monohydrate, citrate trihydrate, citrate tetrahydrate, citrate pentahydrate, or citrate heptahydrate; lithium, sodium, potassium, or calcium lactate; lithium, sodium, potassium, or calcium phosphate; lithium, sodium, potassium, or calcium male ate; lithium, sodium, potassium, or calcium tartarate; lithium, sodium, potassium, or calcium succinate; or lithium, sodium, potassium, or calcium acetate, or a combination of two or more of the above. The pH of said buffer may be adjusted, preferably to a pH of 7.27 - 7.37 by hydrochloric acid, sodium hydroxide, citric acid, phosphoric acid, lactic acid, tartaric acid, succinic acid, or a combination of two or more of the above. The volume of may range from 0.5 ml to 5 ml. Said excipient preferably is selected from, but not limited to, urea, L-histidine, L-threonine, L-asparagine, L-serine, L- glutamine, polysorbate, polyethylene glycol, propylene glycol, polypropylene glycol, or a combination of two or more of the above.

A pharmaceutical composition as defined herein above may further comprise an inhibitor of the generation of reactive oxygen species (ROS), a mitochondrial inhibitor, and/or an inhibitor of glial cell activation. A preferred ROS inhibitor compound is selected from ascorbic acid, DHE, TEMPOL, mitoTEMPO, PBN, L- galactonic acid-g-galactone, pyruvate, mannitol, Trolox, a- tocopherol, Ebselen, uric acid and/or imidazole. Said mitochondrial inhibitor preferably is metformin. Said glial cell activation modulator compound preferably is selected from minocycline, ibudilast, ATL313, synerkme and/or XT 101.

Said pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyltransferase and, optionally, an inhibitor of reactive oxygen species (ROS) generation, a mitochondrial inhibitor, and/or an inhibitor of glial cell activation may be provided as a kit of parts, comprising two or more receptacles comprising said inhibitor of FAM173B mitochondrial lysine

methyltransferase, in combination with said inhibitor of reactive oxygen species (ROS) generation, said mitochondrial inhibitor, and/or said inhibitor of glial cell activation.

A pharmaceutical composition as defined herein above preferably is for use in a method of treatment of chronic pain, preferably of persistent inflammatory and neuropathic pain. Said composition preferably is administered to a person in need thereof by systemic or local administration to the spinal canal, or to the subarachnoid space by injection or by infusion. Said injection or infusion may be accomplished by use of external pump or of a fully implantable device. Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter. An implantable drug delivery system with a constant flow may be beneficial for long-term delivery of said composition.

D Diagnosis and treatment

The invention further provides a method of typing an individual suffering from pain, the method comprising providing a sample of said individual;

determining a level of expression, and/or a level of activity, of FAM173B in said sample; and typing said individual as suffering from chronic pain, based on the determined level of expression and/or activity of FAM173B. Said sample preferably is a liquid biopsy. The term "liquid biopsy", as is used herein, refers to a liquid sample that is obtained from a subject. Said liquid biopsy is preferably selected from blood, urine, milk, cerebrospinal fluid, interstitial fluid, lymph, amniotic fluid, bile, cerumen, feces, female ejaculate, gastric juice, mucus pericardial fluid, pleural fluid, pus, saliva, semen, smegma, sputum, synovial fluid, sweat, tears, vaginal secretion, and vomit. A preferred liquid biopsy is a cerebrospinal fluid.

Methods for determining a level of expression and/or activity of FAM173B are known in the art. At first, a proteinaceous sample may be prepared from the liquid sample, as is known to a person skilled in the art. Said proteinaceous sample may be fractionated used standard techniques such as chromatography methods including ion exchange chromatography and/or size-exclusion chromatography, as is known to the skilled person. For direct determination of a level of FAM173B in said sample, FAM173B may be concentrated by affinity chromatography, for example by employing affinity partners such as antibodies or functional parts thereof that bind specifically to FAM173B. Said concentration step preferably removes proteins and/or peptides that interfere with the subsequent detection of FAM173B. A preferred method for determining a level of FAM173B comprises high performance liquid chromatography (HPLC), preferably coupled to tandem mass spectrometry (LC-MS MS). The LC-MS/MS analysis may be performed, for example by using a HPLC chromatographic system coupled to a triple-quadrupole mass- spectrometer.

Methods for determining lysine methyl transferase activity of FAM173B are exemplified herein below.

The invention further provides a method of treating an individual suffering from pain, the method comprising typing an individual according to a method of the invention, and treating an individual with a specific inhibitor of FAM173B, whether or not combined with a ROS inhibitor, a mitochondrial inhibitor, and/or a glial cell activation modulator compound, if the level of expression and/or activity of FAM173B is enhanced in said individual when compared to a reference. Said reference is a liquid biopsy, preferably a cerebrospinal fluid, from one or more individuals, preferably at least 5, more preferably at least 10 individuals, not suffering from chronic pain. A similarity score may be provided, which similarity score may vary between +1, indicating a prefect similarity, and - 1, indicating a reverse similarity, with a reference. Preferably, an arbitrary threshold is used to type a sample as likely from an individual not suffering from chronic pain. A similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.

It will be clear to a person skilled in the art that said reference may also be a liquid biopsy, preferably a cerebrospinal fluid, from one or more individuals, preferably at least 5, more preferably at least 10 individuals, that are suffering from chronic pain. In this case, an individual will be treated with a specific inhibitor of FAM173B, whether or not combined with a ROS inhibitor, a

mitochondrial inhibitor, and/or a glial cell activation modulator compound, if the level of expression of FAM173B is similar in said individual when compared to the reference.

E Dosage

The exact dosage of a specific inhibitor of FAM173B, whether or not combined with a ROS inhibitor, a mitochondrial inhibitor, and/or a glial cell activation modulator compound will be determined by the individual to which the dose is administered, in light of factors related to the individual's requiring treatment. Said dosage preferably is between 1 microgram and 10 milligram per kg per hour. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired therapeutic effect. Factors that can be taken into account include the severity of the pain and other factors, including the general health of the subject, age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy, as is known to a person skilled in the art.

F Protein production

To screen for inhibitors of lysine methyl transferase activity of FAM173B, including organic molecules and peptide inhibitors, the FAM173B protein preferably is expressed in a host cell. Commonly used expression systems for heterologous protein production include E. coli, baculovirus, yeast, Chinese

Hamster Ovary cells (CHO), human embryonic kidney (HEK) cells and derivatives thereof including HEK293 cells including HEK293T, HEK293E, HEK-293F and HEK-293FT (Creative Biolabs, NY, USA), PER.C6® cells (Thermo Fisher

Scientific, MA, USA) and plants. The efficiency of expression of recombinant proteins in heterologous systems depends on many factors, both on the

transcriptional level and the translational level.

FAM173B protein, or at least a part of said protein that encompasses the catalytic domain, may be produced using prokaryotic cells or eukaryotic cells, preferably E. coli, or fungi, most preferably filamentous fungi or yeasts such as

Saccharomyces cerevisiae and Pichia pastoris.

Production of FAM173B protein in filamentous fungi is preferably performed as described by Joosten et al., 2005. J Biotechnol 120:347-359, which is included herein by reference. A preferred method for producing FAM173B protein in S. cerevisiae is according to the method as described by v. d. Laar et al., 2007. Biotech

Bioeng 96, 483-494; or Frenken et al., 2000. J Biotechnol 78: 11-21, which are all included herein by reference.

Another preferred method of FAM173B protein production is by expression in Pichia pastoris as described by Rahbarizadeh et al., 2006. J Mol Immunol 43:426-

435, which is included herein by reference.

Said FAM173B protein is preferably produced by the provision of a nucleic acid encoding said the FAM173B protein to a cell of interest. Said nucleic acid, preferably DNA, is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, as is known to a skilled person. Alternatively, said nucleic acid is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person. Said nucleic acid is preferably codon-optimised to enhance expression of FAM173B protein in the selected cell or cell line. Further

optimization preferably includes removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that lead to unfavourable folding of the mRNA. The presence of an intron flanked by splice sites may encourage export from the nucleus. In addition, the nucleic acid preferably encodes a protein export signal for secretion of the FAM173B protein out of the cell into the periplasm of prokaryotes or into the growth medium, allowing efficient purification of the FAM173B protein. The single transmembrane domain, which is located between ammo acids 38 - 58 of the human protein (UniProt Q6P4H8 (F173B_HUMAN)), may be amended or deleted, for example to prevent incorporation of FAM173B protein in inclusion bodies. A preferred FAM173B protein comprises a protein export signal and lacks an intact transmembrane domain, for example by deletion of amino acid residues 1-55.

Methods for purification of FAM173B protein are known in the art and are generally based on chromatography, such as protein A affinity for antibody purification, and ion exchange, to remove contaminants. In addition to

contaminants, it may also be necessary to remove undesirable derivatives of the product itself such as degradation products and aggregates. Suitable purification process steps are provided in Berthold and Walter, 1994. Biologicals 22: 135- 150.

As an alternative, or in addition, recombinant FAM173B protein may be tagged with a specific tag by genetic engineering to allow the protein attach to a column specific to the tag and therefore be isolated from impurities. The purified protein is then exchanged from the affinity column with a decoupling reagent. The method has been increasingly applied for purifying recombinant protein.

Conventional tags for proteins, such as histidine tag, is used with an affinity column that specifically captures the tag ( eg., a Ni-IDA column for Histidine tag) to isolate the protein from other impurities. The protein is then exchanged from the column using a decoupling reagent according to the specific tag (eg., immidazole for histidine tag). This method is more specific, when compared with traditional purification methods. Suitable further tags include c-myc domain (EQKLISEEDL), hemagglutinin tag (YPYDVPDYA), and maltose-binding protein, glutathione-S- transferase, maltose-binding protein, FLAG tag peptide, biotin acceptor peptide, streptavidin-binding peptide and calmodulin-binding peptide, as presented in Chatterjee, 2006. Cur Opin Biotech 17, 353-358). Methods for employing these tags are known in the art and may be used for purifying FAM173B protein. G Assay

Methods for determining lysine methyl transferase activity of FAM173B are known in the art and include methods for detecting products of methyl transferase activity, methods for detecting residual substrate peptide, and methods for detecting a decrease in concentration of the methyl donor, S-adenosyl-L-methionine (SAM). The methods available for detection and assay of methyl transferase activity vary in their simplicity, rapidity, range of detection and sensitivity. Said methods comprise qualitative assays such as protein agar plate assay, radial diffusion and thin layer enzyme assay and, preferred, quantitative assays which provide a measure of the methyl transferase activity of the enzyme. The commonly used methods employ natural or synthetic substrates using techniques such as enzyme-linked immunosorbent assay-based assays (ELISA), spectrophotometry, fluorimetry, and radiometry. Said methods may be used for identification of an individual suffering from chronic pain who may benefit from treatment aimed at reducing expression and/or activity of FAM173B in relevant cells of the individual, and for screening and identifying inhibitors, preferably specific inhibitors, of FAM173B.

A peptide or analog preferably is labeled, for example with a chromogenic group, a (chemo)luminescent group, a radiolabel and/or, most preferred, a fluorescent group. A labeled peptide is preferably used in spectrophotometry, fluorimetry, and radiometry. Said label is preferably present at a terminus of the peptide. Said terminus is either the amino-terminus (N-terminus) or the carboxy- terminus (C-terminus). The term "terminus" indicates that the label preferably is present on one or more of the first five amino acid monomers from the N-terminus, and/or on one or more of the last five amino acid monomers at the C-terminus. Said label is preferably present at the N-terminal amino acid monomer, and/or at the C- terminal acid monomer. The skilled person will understand that said label can be indirectly coupled to the N- or C-terminus, for example through a linker that is attached to N-terminus and/or C-terminus. Said linker preferably is an amino acid residue, for example a glutamic acid residue at the N-terminus, and/or an aspartic acid or a cysteine residue at the C-terminus.

A preferred chromogenic group is 2'-azino-bis-(3-ethylbenzothiazoline-6- sulfonic acid (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), p-nitroanilide, paranitrophenol and/or 5-bromo-4-chloro-3-hydroxyindole. A preferred (chemo)luminescent group is a dioxetane derivative such as 1,2- dioxetanedione (C204), 3,3,4,4-tetramethyl- l,2-dioxetane and 3,3,4-trimethyl-l,2- dioxetane, 3-(4-methoxyspiro[l,2-dioxetane-3,2'-tricyclo[3,3, l, 13,7]decan]-4-yl- )- l- aniline and luminol. A preferred radiolabel is 35S and/or 14C. The labeled methylated products are preferably separated from the unmethylated substrate, for example by employing magnetic beads.

A peptide or analog preferably is labeled with one or more fluorescent group. Fluorescent groups are known and have been described, for example, in U.S. Pat. No. 7,256,012 and U.S. Pat. No. 7,410,769. Some non-limiting examples of fluorescent groups include coumarin derivatives such as 7-amino-4- methylcoumarin (AMC), 7-acetoxy-4-methylcoumarin (7-AC-4-MC) and 7- hydroxycoumarin, fluorescein, tetramethylrhodamine, rhodamine B, lissamine, rhodamine X, Texas Red, cyanine dyes, Dabcyl, BODIPY dyes, alexa dyes, QSY 7 and QSY 9 dyes, and other fluorescent dyes commonly available from, for example, Invitrogen Corp (Carlsbad, Calif.). Other dyes known to those skilled in the art may also be used.

A preferred fluorescence-based protease assay is simple, inexpensive and sensitive. Said protease assay preferably comprises a soluble fluorescein

isothiocyanate (FITC)-labeled peptide, or a soluble Alexa 488-labeled peptide.

Existing universal methyltransierase assays, based on the determination of any S-adenosyl-L-methionine (AdoMet/SAM) dependent methyltransierase activity may also be used for determining lysine methyl transferase activity of FAM173B, including end-point and kinetic read options. Preferably, such assays are amendable to miniaturization and high throughput screening. Examples of such kits are Universal Methyltranslerase Activity Assay Kit (Abeam; Cambridge, MA), MTase-Glo™ Methyltransferase Assay (Promega; Madison, WI ), and SAM

Methyltransferase Assay (Merck/Millipore; Darmstadt, Germany).

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be

appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

Examples

Example 1

Materials and methods

Animals

Experiments were conducted using both male and female (aged 8- 12 weeks)

C57BL/6 mice (Harlan Laboratories), since we did not overt sex differences during pain behaviour measurements. Mice received an intraplantar injection unilateral or in both hind paws of 5 μΐ λ-carrageenan (1% w/v, Sigma-Aldrich) to induce transient inflammatory pain (Wang et al., 2013a. J Clin Invest 123, 5023-5034) or 20 μΐ Complete Freund's Adjuvant (CFA, Sigma-Aldrich) to induce persistent inflammatory pain (Ren and Dubner, 1999. Nat Med 16, 1267-1276). Spared nerve injury (SNI) was performed as described previously (Decosterd and Woolf, 2000. . Pain 87, 149-158; Willemen et al., 2012. Nat Med. 16 1267- 1276). Heat withdrawal latency times were determined using the Hargreaves test (IITC Life Science) (Eijkelkamp et al., 2010. J Neurosci 30, 2138-2149; Hargreaves et al., 1988. Pain 32, 77-88). Mechanical thresholds were determined using the von Frey test

(Stoelting) with the up-and-down method as we described (Chaplan et al., 1994. . J Neurosci Methods 53, 55-63; Eijkelkamp et al., 2010. J Neurosci 30, 2138-2149). Changes in weight bearing were evaluated using a dynamic weight bearing (DWB) apparatus (Bioseb, France) as described (Robinson et al., 2012. Neurosci Lett 524, 107-110). The weight bearing of the affected paw was calculated as ratio of the weight between the affected paw and total weight and expressed relative to baseline. The conditioned place preference test was used as described previously (Park et al, 2013. Anesth Analg 116, 224-231). In short, conditioned place preference (Stoelting) was calculated by subtracting the mean time spent in the white room during pre-conditioning (days 1 and 2) with the time spent in the white room (day 5) after 2 days of conditioning (day 3-4) with intraperitoneal injections of gabapentin (100 mg/kg, Sigma-Aldrich).

In experiments were mice received intraplantar injections latency times and 50% thresholds of left and right paws were considered as an independent measure whilst experiments with intrathecal or intraperitoneal drug administration the average of the left and right paw were considered as an independent measure. All experiments were performed by experimenters blinded to treatment. Mice were maintained in the animal facility of the University of Utrecht. All experiments were performed in accordance with international guidelines and approved by the University Medical Center Utrecht experimental animal committee.

DNA and viral constructs

Full-length mFaml73b (NM_ 026546.1) and hFAM 173B (NM_199133.3) were cloned into several vectors including pAcGFP-Nl, pIRES2-AcGFPl, bacterial expression vector pET28a and pCMV6 containing a myc-tag at the C-terminal of human or mouse FAM173B (Origene). pIRES2-Ac,GFP vectors were used for functional experiments and GFP expression was used to verify successful transfection. The pCMV6 and pAcGFP-Nl vectors were used for identification of cellular and subcellular localization of FAM173B and pET28a was used for the production of recombinant FAM173B in Escherichia coli.

We generated a bicistronic herpes simplex virus (HSV) construct by cloning hFAM173B or hFAM173B-O94A in which residue (Asp94) is mutated to alanine in order to generate enzymatically inactive protein (Malecki et al., 2015. J Biol Chem 290, 423-434), under control of the a4 promotor and with GFP under control of the a22 promoter (Wang et al., 2013. J Clin Invest 123, 5023-5034). Control empty HSV (HSV-EV) only expresses GFP. HSV was produced as previously described (Roy et al., 2002. Gene Ther 9, 214-219). Mice were inoculated twice (day -3 and day -1 prior to carrageenan or at day 5 and 7 after CFA) with 2.5 μΐ of 1.4 x 107 pfu ml (intraplantar) or 5 μΐ 5 x 106 pfu/ml (intrathecal). Drug administration

For behavioural analysis, mice received an intraperitoneal injection (day 5 after carrageenan) with 100 μΐ phenyl-N-t-butyl nitrone (PBN, 100 mg/kg, Sigma- Aldrich). For spinal cord analysis mice received two PBN injections (2 hours apart) at one month after carrageenan. Spinal cords were collected 2 hours after the last PBN administration. Intrathecal injections (5μ1) with minocycline (6 g l, Sigma- Aldrich), neutralising TNF antibody (20 mg/ml, Enbrel®) and (Cy3-labeled, setl) asODN (3 μg l day 5,6,7,9 and 10, Sigma Aldrich), were performed under light isoflurane anaesthesia as described (Eijkelkamp et al., 2010. J Neurosci 30, 2138- 2149; Hylden and Wilcox, 1980. Eur J Pharmacol 67, 313-316.). The following phosphorothioated asODN sequences were used that specifically target niFam ,173b and not hFAM173B:

Set l Faml 73b: CCCGCCTGTCTTTC TTCC TC MM : CGCCTC CGTTCC TT TCTCCT Set 2 Farn l 73b: GGGTCCTCTTCTGT GTCGCA MM : GTGCTC GTCTTGCC GACGCT

Cell lines and primary cell cultures, and transfections

HEK293 and mouse neuroblastoma Neuro2a (N2a) cells were kept in Dulbecco's Modified Eagle medium (DMEM) with Glutamax-1 containing 4,5g/L D-Glucose, pyruvate and 10% fetal calf serum. FAAI173B expression was downregulated (100 μΜ asODN) or overexpressed with plasmids as described above using Lipofectamin 2000 (Life Technology) according to manufactures instructions. For measuring mitochondrial membrane potential, the cells were incubated for 20-30 minutes with 50 nM Mitotracker Red FM (Life technology) or 50 nM TMRM (Sigma-Aldrich) 2 days after transfection and following manufacturer's instructions. Cells were fixed with 4% PFA (paraformaldehyde) after Mitotracker or directly imaged without fixation (TMRM experiments). Fluorescence was captured using AxioCAM MRm from Zeiss Axio Observer microscope and analyzed with Imaged software.

DRGs were collected and primary sensory neurons were cultured as described (Eijkelkamp et al., 2013. Nat Commun 4, 1682). Twenty-four hours after plating, sensory neuron cultures were inoculated with HSV (10,000 pfu) for 3 days. The anti-mitotic fluoro-deoxyuridine (FDU 13.3 microg/ml, Sigma-Aldrich) was added to inhibit satellite glia cell growth in the neuronal cultures. Sensory neurons were stimulated with 100 ng recombinant TNFa (Peprotech) with or without phenyl-N-t- butyl nitrone (PBN 2 niM, Sigma-Aldrich). Six hours after neuronal TNFa stimulation (+/- PBN) the cultures were washed for three times with media

(DMEM) and new media was added and after 15 hours supernatants were collected. The collected supernatants were diluted 1: 1 with DMEM and added to spinal microglia cultures for 24 hours. Spinal microglia were cultured as described previously (Yip et al., 2009). After collection of the supernatants IL6 and TNFa contents were determined by ELISA according to manufacturer's protocol (R&D systems). The detection limit of IL-6 was 15 pg/ml and of TNFa 31 pg/ml. Electron microscopy

HEK293 and N2A cells were grown in 6-well plates and transfected with pCMV6- FAM173Bmyc as described above. The cells were treated as described previously (Slot and Geuze, 2007. Nat Protoc 2, 2480-2491). Briefly, cells were chemically fixed using 2% formaldehyde (FA), 0.2% glutaraldehyde in 0.1M phosphate buffer pH 7.4 (Pi) for 2 hours and stored overnight in 1% FA in Pi. After rinsing with PBS (3x) and PBS-0.15 M glycine 1 ml, a 1 % gelatin solution was put on the cells and using a cell scraper the cells were removed from the Petri dish, transferred to an Eppendorf vial and spun down. The 1% gelatin was removed and the cells were suspended in 1 ml 12 % gelatin 37°C. After 10 min the cells were spun down and the gelatin is allowed to solidify at 0°C. Small (0.5x0.5x0.5 mm) blocks were prepared and transferred to 2.3 M sucrose. After overnight infiltration of sucrose in a rotator, the blocks were mounted on specimen holders and frozen in liquid nitrogen. Ultrathin sections (70 nm) were prepared on a Leica UC7/FC7 (Leica, Vienna, Austria) at - 120°C. Immunolabeling was performed with Rabbit anti-GFP (Acris antibodies) and protein A-Gold (CMC, The Netherlands). The immunogold labeled sections were examined with a Tecnai 12 or 20 (FEI, The Netherlands).

ROS and superoxide detection

In vivo, dihydroethidium (DHE) (50 μΜ, 5 μΐ, Life technology) or

MitoTrackerRedCM-H2XROS (10 μΐ of 100 μΜ, Life technology) (Flatters, 2015.

Prog Mol Biol Transl Sci 131, 119- 146) was injected intrathecal respectively at day four or day three/six after intraplantar carrageenan administration. Twenty four hours later mice were perfused with PBS and 4% PFA as described below and DRGs were collected (Gwak et al., 2013. Pain 154, 1699-1708). DHE and

MitoTrackerRedCM-H2XROS fluorescence was analyzed in small diameter neurons < 25 μιη and large diameter neurons > 25 μηι.

For ROS or mitochondrial superoxide production measurements in vitro, primary sensory neurons or N2A were incubated with 10 μΜ DHE or 5 μΜ MitoSoX (Life, technology) in HBSS for 20 minutes. After HBSS washes cells were fixed with 4% PFA after DHE incubations or directly imaged without fixation (MitoSox experiments). Fluorescence was captured using AxioCAM MRm from Zeiss Axio Observer microscope and analyzed with Imaged software. Immunohistochemistry

Mice were deeply anesthetized with an overdose of sodium pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde and spinal cords and DRGs were collected. Tissues were postfixed, cryoprotected in sucrose, embedded in OCT compound (Sakura), and frozen at -80°C. Cryosections (10 pm) of lumbar DRG and of lumbar spinal cord segments L3-L5 were stained with anti- Ibal (1:500, Wako). DRGs were stained with rabbit anti-NF200 (1:200, Millipore), biotinylated anti-IB4 (1:25, Vector laboratories ) and rabbit anti-GFAP (1:2000, Dako). N2A cells were stained with anti-PDl (1: 100, Enzo life sciences) and anti- pGM130 (1: 100, BD Transduction laboratories). For the DRG's, sciatic nerves and hind paws staining's for FAM173B (1:500, Biorbyt), biotinylated anti-IB4 (1:25, Vector laboratories ), GFP (1:3000, Abeam) and periplierin (1: 100, Sigma Aldrich), tissues were fresh frozen, cut and post-fixed in PFA prior for staining. Staining's were visualized by using Alexafluor 488-(streptavidin) or 594-conjugated secondary antibody's. Nuclei were stained with DAPI. Photographs were captured with a confocal laser scanning microscope LSM700 (co-localization experiments) or with a Zeiss Axio Observer microscope (Zeiss) using identical exposure times for all slides within one experiment. Fluorescence intensity was analyzed with ImageJ software. Bioinformatics

Homo sapiens FAM173A (NP_076422.1) and FAM173B (NP_954584.2), Mus musculus Faml73a (NP_663385.2) and Faml73b (NP_()80822.1) and the homolog of FAM173 proteins (FAM173hom) from the Archaea Sulfolobus islandicus (Si) (gi I 227827841) were used for the alignment. The alignment was generated using the MUSCLE algorithm embedded in Jalview (Edgar, 2004. Nucleic Acids Res 32, 1792- 1797; Waterliouse et al., 2009. Bioinformatics 25, 1189-1191) and prediction of protein secondary structure was performed with Jpred 3 (Cole et al., 2008.

Nucleic Acids Res 36, W197-W201). Expression and purification of recombinant FAM173B

Human full length FAM173B, wt FAM173BA55 (without the putative

transmembrane domain to avoid the formation of inclusion bodies) and

FAM173BA55-D94A (enzymatically inactive), were cloned into pET28a and expressed as N-terminally hexahistidine tagged proteins in Escherichia coli BL21- CodonPlus(DE3)-RIPL cells (Agilent) and purified using nickel-nitrilotriacetic acid- agarose (Qiagen), according to manufacturer's instructions and as described (Malecki et al, 2015. J Biol Chem 290, 423-434). Eluted protems were buffer- exchanged (Malecki et al., 2015. J Biol Chem 290, 423-434) and protein purity was asses by SDS-PAGE and Coomassie staining. Protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific).

Methyltr ansfer ase ass ay

Methyltransferase reactions contained 10 μg of homopolymers or equivalent amounts of cell extracts from adenosine dialdehyde (AdOx)- treated HEK293 cells (Davydova et al., 2014. J Biol Chem 289, 30499-30510), [3H]-SAM (2 μ(¾ and recombinant hFAM173B (100 pmol) in 50 μΐ reactions and were incubated for 1 hour at 37°C, as described (Jakobsson et al., 2015. PLoS One 10, e0131426;

Kernstock et al., 2012. Nat Commun 3, 1038). Radioactivity in 10% trichloroacetic acid (TCA) precipitated material was measured by scintillation counting or proteins were resolved by SDS-PAGE and subjected to fluorography (Kernstock et al, 2012. Nat Commun 3, 1038). Western Blot

Isolation of mitochondria from N2A cells was performed with the Mitochondria Isolation Kit for Cultured Cells (ThermoFisher Scientific) according to

manufacturer's protocol. Protein concentrations of the total cell lysates or mitochondrial/cytosol fractions were determined using a Bradford assay (Bio-Rad). Protein samples (20 μg) were separated by 12% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). Membrane was stained with 1: 1000 goat anti-FAM173B, 1: 1000 mouse anti-COXIV (Invitrogen) or 1: 1000 goat ant- β-actin, followed by incubation with 1:5000 donkey anti goat-HRP (others all Santa Cruz Biotechnology). Specific bands were visualised by chemiluminescence (ECL, Advansta) and imaging system Proxima (Isogen life sciences) Real-time RT-PCR

Total RNA from freshly isolated DRGs and spinal cords was isolated using TRizol and RNeasy mini kit (Qiagen). cDNA was synthesized using Reverse Transcriptase (Biorad)(Peters et al., 2013). Quantitative real-time PCR reaction was performed with an I-cycler iQ5 (Bio-Rad) as described. The following primers were used: mFaml73b forward: TggTgTgCCCCAgATgAT

reverse : TgCCCTCTCCAgTggTgT

TNFa forward: gCggTgCCTATgTCTCAg

reverse: gCCATTTgggAACTTCTCATC

ΪΙΐβ forward: CAACCAACAAgTgATATTCT

reverse : gATCCACACTCTCCAgCTgCA

GFAP forward: ACAgACTTTCTCCAACCTCCAg

reverse : CCTTCTgACACggATTTggT

Ibal forward: ggATTTgCA.gggAggAA.AAg

reverse: TgggATCATCgAggAATTg

BDNF forward: CACATTACCTTCCAgCATCT

reverse: ACCATAgTAAggAAAAggATgg

CCL2 forward: ggTCCCTgTCATgCTTCTg

reverse : CATCTTgCTggTgAATgAgTAg

GAPDH forward: TgAAgCAggCATCTgAggg

reverse: CgAAggTggAAgAgTgggAg,

HPRT forward: TCCTCCTCAgACCgCTTTT

reverse : CCTggTTCATCATCgCTAATC

Data were normalized for GAPDH and HPRT expression.

Conventional PCR

cDNA was synthesized from 1 μg total RNA (Clontech) and PGR was performed using Phusion polymerase (ThermoFisher Scientific) following manufacturing instructions. Human and mouse FAM173B was detected in a tissue panel

(Clontech) using the following primers;

hFAMl 73B forward: gTAgCCACgCCgTTTgTAAC

reverse: CATCATCTgAggCACACCgA

β-actin forward: CCTggCACCCAgCACAAT

reverse: GggCCggACTCgTCATACT

mFaml73b forward: TggTgTgCCCCAgATgAT reverse: TgCCCTCTCCAgTggTgT

HPRT forward: TCCTCCTCAgACCgCTTTT

reverse: CCTggTTCATCATCgCTAATC Statistical analysis

All data are presented as mean ± SEM and were analyzed using Student's t-test, one-way or two-way ANOVA when appropriate followed by post-hoc Holm-Sidak multiple comparison tests. A p value less than 0.05 was considered statistically significant and each significance is indicated with *p<0.05, **p<0.01, ***p<0.001.

Results

Role of FAM173B in chronic pain

To determine whether FAM173B is involved in chronic pain we down-regulated Faml 73b expression in vivo by lumbar intrathecal injections of a nuclease resistant antisense oligodeoxynucleotide (asODN), a method that has been shown to reduce mRNA expression and protein translation (Dias and Stein, 2002. Mol Cancer Ther 1, 347-355). We injected mouse Fam l 73b (rnFaml 73b) asODN intrathecal into the lumbar enlargement because through this application route asODNs mainly target the lumbar DRGs (Alessandri-Haber et al., 2009. J Neurosci 29, 6217-6228; Eijkelkamp et al., 2013. Nat Commun 4, 1682; Ferrari et al., 2012. Neuroscience 222, 392-403; Wang et al., 2013. J Clin Invest 123, 5023-5034). Five daily intrathecal injections of mFaml 73b asODN reduced mFaml 73b mRNA expression in vivo in lumbar dorsal root ganglia (DRG) in the complete Freund's adjuvant (CFA) model of persistent inflammatory pain (Ren and Dubner, 1999. ILAR J 40, 111- 118) without affecting spinal cord mFaml 73b mRNA expression (Fig. la). Intrathecal injection of a fluorescent labeled Faml 73b asODN targeted almost all sensory neurons in the DRG and some other cells including Ibal and GFAP positive cells in the DRG (data not shown). Intrathecal administration of Fain 173b asODN at day 5 till 10 in the CFA model of persistent inflammatory pain abrogated thermal and mechanical hyperalgesia (Fig. Vole). These result were confirmed by using another Faml 73b asODN targeted to a different region of mFaml 73b mRNA (data not shown), indicating the asODN-induced effects are likely not due to off-target effects. Intrathecal injections of Fam l 73b asODN from day 1 till 9 also attenuated the development of neuropathic pain in the spared nerve injury model (Decosterd and Woolf, 2000. Pain 87, 149- 158) (Fig. Id).

Mechanical thresholds in the contralateral paw were not affected by Faml 73b asODN treatment (Fig. Id).

To test if sensory neuron FAM173B is central to the inhibitory effect of intrathecal mFaml73b asODN on chronic inflammatory pain, we performed a rescue experiment. We expressed human FAM173B (hFAM173B) specifically in primary sensory neurons in vivo using Herpes simplex virus (HSV)-mediated gene transfer in mice treated intrathecally with mFaml73b asODN that does not recognize human FAM173B mRNA. HSV selectively infects primary sensory neurons and intraplantar HSV amplicons can be used to selectively express proteins in primary sensory neurons without inducing expression in cells in the spinal cord or non- neuronal cells locally at the intraplantar injection site (Singhmar et al., 2016.

PNAS 113, 3036-3041; Wang et al., 2013. J Clin Invest 123, 5023-5034; Wolfe et al., 2012. Neurosci Lett 527, 85-89). Intraplantar administration of HSV amplicons encodmg for hFAM173B and GFP selectively transferred hFAM173B (Fig. le) and GFP (data not shown) into sensory neurons in the DRG, sciatic nerve fibers, and nerve endings in the skin of the injected hind paw (data not shown). In addition, intraplantar HSY-hFAM173B injections induced expression of hFAM173B in the DRG but not in spinal cord (data not shown). Intraplantar (to only target sensory neurons innervating the hind paw) (Fig. lf/g) or intrathecal (data not shown) administration of HSY-hFAMl 73B completely prevented the mFarnl73b asODN- mediated attenuation of persistent thermal and mechanical hypersensitivity in the CFA-model, indicating sensory neuron FAM173B is required for persistent inflammatory pain.

Next, we tested whether increasing sensory neuron hFAM173B is sufficient to promote the transition of transient inflammatory pain into persistent pain.

Intraplantar injection of 5 μΐ 1% carrageenan induced transient hyperalgesia (Aley et al, 2000. J Neurosci 20, 4680-4685; Wang et al., 2013. J Clin Invest 123, 5023- 5034) that resolved within 4-6 days (Fig. lh/i). Intraplantar (Fig. lh/i/j) or intrathecal (data not shown) administration of HSY-hFAMl 73B prior to the induction of transient inflammatory pain markedly prolonged carrageenan-induced evoked thermal and mechanical hyperalgesia after intraplantar carrageenan as compared to mice treated with control HSV-empty vector (EV). Importantly, expression of hFAM173B in sensory neurons induced ongoing carrageenan-induced non-evoked inflammatory hyperalgesia (Fig. lj) and spontaneous pain that was present one month after carrageenan injection (Fig. Ik). Overall, the results indicate that FAM173B in sensory neurons promotes development of chronic pain. We have previously evaluated mFaml73b expression levels during acute inflammatory pain (Peters et al., 2013. Ann Rheum Dis 72, 427-436). Here we evaluated whether endogenous niFaml 73b mRNA expression levels are increased in the DRG during the persistent phase of CFA-induced inflammatory pain. At 1 week after intraplantar CFA injection mFaml73b mRNA expression was increased in the DRG compared to naive animals. In contrast, during acute inflammation at day 1 and 3 after induction of paw inflammation mFaml73b expression levels were indistinguishable from controls (data not shown), consistent with our previous findings (Peters et al., 2013. Ann. Rheum. Dis. 72, 427-436).

FAM173B characterization

Bioinformatics analysis of FAM173B protein sequences shows that FAM173B harbors characteristic motifs involved in binding of the methyl donor S adenosyl-L- methionine (SAM). Moreover it shows similarities for a subclass of

methyltransferases characterized by a topology of seven β-strands (7BS) (Fig. 2a/b) (Petrossian and Clarke, 2011. Mol Cell Proteomics 10, MHO). Human and mouse FAM173B are ubiquitously expressed (data not shown). An Archaeal lysine-specific methyltransferase shows some homology with human FAM173B (Chu et al., 2012. J Bacteriol 194, 6917-6926), therefore, we explored whether hFAM173B specifically methylated lysine residues. To this end, we incubated a radioactive methyl donor, [3H]-SAM with protein extracts of HEK293 cells together with purified

recombinant hFAM173BA55 (without putative transmembrane domain) and detected methyltransferase activity by fluorography. These experiments revealed hFAM173B-mediated methylation of high-molecular weight proteins (Fig. 2c). To assess the product specificity of the enzyme we evaluated homopolymers of lysine and arginine, the two most commonly methylated amino acid residues in proteins, as artificial substrates. When incubating recombinant hFAM173BA55 (Fig. 2d) or full-length hFAM173B (data not shown) with [3H]-SAM and lysine or arginine homopolymers, hFAM173B displayed significant methyltransferase activity on poly-L-lysine but not on poly-L-arginine (Fig. 2d). Importantly, a putative ly enzymatically inactive mutant of hFAM173B (hFAM173B-D94A), generated by mutating a key conserved residue (Asp94) in the SAM-binding Motif I of

FAM173B (Fig. 2b) (Malecki et al., 2015. J Biol Chem 290, 423-434), did not show significant methyltransferase activity (Fig. 2e). The D94A mutation did not affect expression (data not shown).

To determine the subcellular localization of FAM173B, we expressed C-terminally GFP-tagged hFAM173B and mFaml73b in Neuro2a (N2A), a sensory neuron cell line. Confocal imaging of GFP-tagged hFAM173B indicated that FAM173B co- localized with the mitochondrial dye Mitotracker Red but not with the endoplasmic reticulum marker protein disulfide -isomer ase (PDI) or the Golgi scaffolding protein PGM130 (Fig. 2f). Mouse Faml73b-GFP and hFAM173B-D94A also co-localized with Mitotracker Red (data not shown). The localization of FAM173B and the methyltransferase death mutant FAM173B-D94A in mitochondria was further confirmed by western blot analysis of mitochondrial and cytosol fractions of N2A cells (data not shown). Electron microscopy of immuno-gold labeling of GFP-tagged hFAM173B showed that hFAM173B was predominantly present in the cristae of mitochondria when expressed in HEK293 (Fig. 2g) or N2A cells (data not shown).

FAM173B and mitochondrial function

To determine whether FAM173B modulates mitochondrial function, we assessed mitochondrial membrane potential (ΔΨηι) (Perry et al., 2011. Biotechniques 50, 98- 115). Knockdown of mFaml73b in N2A cells with mFaml73b asODN (data not shown) reduced accumulation of the ΔΨηι- sensitive dye Mitotracker Red compared to cells treated with control mismatch (MM) asODN (Fig. 3a and data not shown), whilst overexpression of liFAM173B in N2A cells (data not shown) increased accumulation of Mitotracker Red (Fig. 3b). These data indicate that FAM173B promotes mitochondrial hyperpolarization. Similarly, overexpression of hFAM173B using HSV-M^AMlf.JB-amplicons ) in N2A cells (data not shown) or in primary sensory neurons (Fig. 3c) in vitro increased fluorescence of tetramethylrhodamine methyl ester (TMRM), a dye sequestered by active mitochondria in a ΔΨηι- dependent manner (Perry et al., 2011. Biotechniques 50, 98-115). The sequestration of the dye was completely blocked by the respiratory uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) that abolishes ΔΨηι without affecting cell membrane potential (Perry et al., 2011. Biotechniques 50, 98- 115), indicating hFAM173B expression in sensory neurons hyperpolarizes mitochondria (Fig. 3c). Mitochondrial hyperpolarization has been reported to cause increased reactive oxygen species (ROS) formation (Brookes et al., 2004. Am J. Physiol Cell Physiol 287, C817-C833; Tirosh et al., 2003. Biochem Pharmacol 66, 1331- 1334). Therefore, overexpression of hFAM173B may increase ROS formation in sensory neurons. Human FAM173B overexpression in N2A and HEK293 cells significantly increased fluorescence of the ROS sensitive dye dihydroethidium (DHE) (Wang et al., 2013. J Mol Med 91, 917-927), indicating that FAM173B promotes ROS formation in these cells (data not shown). Similarly, overexpression of hFAM173B in primary sensory neurons in vitro significantly increased DHE fluorescence (Fig. 3d). Stimulation of sensory neurons with the prototypic inflammatory mediator TNFa for 6 hours, known to promote ROS formation (Valko et al., 2007. Int J Biochem Cell Biol 39, 44-84.), enhanced DHE fluorescence which was further increased when hFAM173B was expressed in sensory neurons with HSY-FAM173B (Fig. 3d). Next, we addressed if increased sensory neuron ROS formation also occurs during the hFAM173B-mediated switch from transient to persistent inflammatory pain in vivo. Expression of hFAM173B in sensory neurons increased DHE fluorescence in small (<25 μηι) diameter neurons that are central in inflammatory pain (Abrahamsen et al., 2008. Science 321, 702-705), but not in large diameter neurons (>25 μηι) 5 days after intraplantar carrageenan (Fig. 3e). Next we assessed whether FAM173B promotes mitochondrial superoxide production in vitro and in vivo. Overexpression of hFAM173B significantly increased fluorescence of the mitochondrial superoxide sensor MitoSox in N2A cells (data not shown). In vivo, HSV-mediated expression of hFAM173B in sensory neurons increased MitoTrackerRedCM-H2XROS fluorescence in small (<25 μπι) diameter 3 and 6 days after intraplantar carrageenan (Fig. 31), indicating liFAM173B expression in sensory neurons promotes ongoing mitochondrial superoxide production in vivo. To assess if the increased ROS production in sensory neurons contributes to hFAM173B-mediated prolongation of inflammatory pain, we administered the ROS scavenger phenyl-N-t-butylnitrone (PBN) during hFAM173B-induced persistent inflammatory hyperalgesia. PBN administration at day 5 completely reversed the persistent carrageen-induced mechanical hyperalgesia (Fig. 3g) in mice expressing hFAM173B in sensory neurons. PBN administration did not affect mechanical thresholds in mice treated with control HSV (Fig. 3g). These data indicate that sensory neuron FAM173B-mediated prolongation of inflammation-induced hypersensitivity is maintained through a ROS-dependent pathway.

FAM173B-induced microglia activation

Microglia/macrophage activation and the production of pro-inflammatory mediators in the spinal cord/DRG play a key role during persistent pain, including persistent inflammatory pain (Eijkelkamp et al., 2016. J Neurosci 36, 7353-7363; Grace et al., 2014. Nat Rev Immunol 14, 217-231; Ji et al., 2014. Nat Rev Drug Discov 13, 533-548; Milhgan and Watkms, 2009. Nat Rev Neurosci 10, 23-36; Old et al., 2015. Handb Exp Pharmacol 227, 145- 170). ROS formation can initiate proinflammatory cascades and activate microglia in the central nervous system (Kallenborn-Gerhardt et al., 2013. Pharmacol Ther 137, 309-317). As a next step, we evaluated if FAM173B expression in primary sensory neurons promotes the ability of sensory neurons to activate glial cells in vitro in an ROS-dependent manner. Sensory neuron cultures were stimulated with 100 ng/ml TNFa (Czeschik et al., 2008. Neurosci Lett 434, 293-298) for 6 hours with or without the ROS scavenger PBN, extensively washed to remove TNFa, and then further cultured overnight for 15 hours in medium to capture inflammatory mediator-induced sensory neuron- derived factors that could drive glial cell activation. Culture of primary spinal microglia with the supernatant of these TNFa- stimulated sensory neurons for 24h promoted microglia to release IL6, and this IL6 release by microglia was strongly enhanced by overexpression of hFAM173B in sensory neurons and completely abolished by incubating sensory neurons with the ROS scavenger PBN during TNFa stimulation (Fig. 4a). Overexpression of hFAM173B in sensory neurons also increased TNFa release by microglia (data not shown). IL6 and TNFa were not detectable in the conditioned medium or in supernatants of unstimulated microglia, indicating that IL6 and TNFa were released by microglia and not already present in sensory neuron cultures. The supernatants of unstimulated sensory neurons overexpressing hFAM173B did not trigger microglia to release detectable levels of IL6 and TNFa. These in vitro data indicate that hFAM173B expression in TNFcc-stimulated sensory neurons promotes the release of glial cell activating factors in a ROS-dependent manner. To test whether in vivo sensory neuron FAM173B promotes the engagement of microglia and subsequent TNFa release to drive ongoing inflammatory pain, we inhibited TNFa signaling in the spinal cord and DRG by intrathecal injection of a neutralizing anti-TNFa antibody. Intrathecal injection of the neutralizing anti-TNFa antibody at day 7 inhibited the sensory neuron-specific hFAM173B mediated persistent

inflammatory pain (Fig. 4b). Subsequently, we tested if activation of

microglia/macrophages contributes to the FAM173B-mediated prolongation of inflammatory pain. Intrathecal injection of the glial cell inhibitor minocycline at day 7 completely inhibited hFAM173B-induced persistent inflammatory hyperalgesia (Fig. 4c). To further validate the contribution of microglia to

FAM173B-mediated prolongation of inflammatory pain, we asked whether in vivo overexpression of hFAM173B would engage glial cells after induction of inflammatory pain. Expression of hFAM173B specifically in sensory neurons using HSV amplicons significantly increased the Ibal+ immunofluorescence in DRG and spinal cord at 5 and 10 days after carrageenan-treatment compared to mice treated with empty HSV amplicons (Fig. 4d-f). This neuronal FAM173B-mediated spinal microglia activation in vivo was attenuated after inhibition of ROS with the ROS scavenger PBN (data not shown). Conversely, asODN-mediated knockdown of FAM173B during CFA- induced persistent pain prevented activation of glial cells in the DRG and spinal cord as shown by the reversion of the CFA induced increase in Ibal+ area and mRNA expression in DRG (Fig. 4g-i) and spinal cord (Fig. 4j-l). niFarn l 73b asODN treatment during CFA-induced persistent hyperalgesia did not affect GFAP mRNA expression in the spinal cord and DRG (data not shown). The reduced signs of glia activation were associated with reduced expression of several inflammatory mediators in the spinal cord and DRG known to play a role in persistent pain statesmen and Dubner, 2010. Nat Med 16, 1267-1276). Knockdown of mFaml73b at day 5-11, prevented the CFA-induced increase in both TNFa and ILi mRNA expression in the spinal cord (Fig. 4m). In the DRG, mFaml73b knockdown prevented the CFA-induced expression of the chemokine CCL2 but not of the growth factor BDNF (Fig. 4n). Overall these data indicate that neuronal FAM173B drives the persistence of inflammatory hyperalgesia through a ROS- dependent engagement of glial cells. FAM173B methyltransferase activity & persistent pain

We next determined whether the methyltransferase activity of FAM173B in sensory neurons is required to regulate chronic inflammatory pain through a ROS and glial cell dependent mechanisms. Intraplantar (Fig. 5a b) or intrathecal administration of the methyltransferase deficient mutant hFAM173B-D94A (data not shown) did not prolong carrageenan-induced thermal and mechanical hyperalgesia, whilst expression of wt hFAM173B prolonged transient

inflammatory hyperalgesia (Fig. 5a b). In vivo overexpression of hFAM173B-D94A in sensory neurons prior to induction of inflammatory hyperalgesia did not increase Ibal+ area in the DRG and spinal cord at day 5 after carrageenan injection (Fig. 5c/d), indicating the requirement of FAM173B methyltransferase activity in sensory neurons to promote chronic pain and glial cell activity. In vitro,

overexpression of hFAM173B-D94A did not affect mitochondrial membrane potential, in contrast to wt hFAM173B which increased ΔΨηι (Fig. 5e). In addition, expression of wt hFAM173B but not hFAM173B-D94A increased the fluorescence of the ROS-sensitive dye DHE in small (< 25 μηι) diameter neurons at day 5 during carrageenan-induced inflammatory hyperalgesia, indicating FAM173B-mediated increase in ΔΨηι and ROS production is also methyltransferase dependent (Fig. 5f). Finally, culturing spinal microglia with supernatants of TNFa- stimulated sensory neurons expressing wt FAM173B increased IL6 (Fig. 5g) and TNFa (data not shown) release by microglia whilst overexpressing FAM173B-D94A had no such effect. Overall these results indicate that the methyltransferase activity of FAM173B, and not the protein per se, is important to control the development of chronic pain through a ROS-dependent mechanism involving the activation of glial