WO2022136344A1 - Modulation of prdm12 for use in treatment of pain conditions - Google Patents

Modulation of prdm12 for use in treatment of pain conditions Download PDF

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WO2022136344A1
WO2022136344A1 PCT/EP2021/086946 EP2021086946W WO2022136344A1 WO 2022136344 A1 WO2022136344 A1 WO 2022136344A1 EP 2021086946 W EP2021086946 W EP 2021086946W WO 2022136344 A1 WO2022136344 A1 WO 2022136344A1
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prdml2
pain
modulator
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prdm12
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PCT/EP2021/086946
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French (fr)
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Eric BELLEFROID
Aurore LATRAGNA
Simon VERMEIREN
Alba SABATÉ
Laurence RIS LAURENCE
Roberta GUALDANI
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Université Libre de Bruxelles
Université de Mons
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Publication of WO2022136344A1 publication Critical patent/WO2022136344A1/en

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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain

Definitions

  • the invention relates to modulation of the PRDM12 gene and PRDM12 gene products for use in the treatment or prevention of pain in a subject.
  • the finding is of particular interest to the fields of medical biotechnology and medicine.
  • the finding is applicable to numerous pain conditions and pain sensations.
  • Pain perception is an evolutionarily conserved warning mechanism that alerts us from environmental dangers and potential damaging stimuli, and is therefore essential for our survival. Pain is also causing untold misery and suffering when the pain-sensing system becomes over- or erroneously activated. Chronic inflammatory and neuropathic pain is a major public health burden, estimated to affect one fifth of the population worldwide.
  • the cell bodies of nociceptors as well as those of other somatic sensory neurons (proprioceptors, mechanoreceptors) are located in specialised ganglia of the peripheral nervous system (PNS): the trigeminal (TG), superior-jugular (SJG) and accessory ganglia in the head region, and the dorsal root ganglia (DRG) found in a metameric pattern on each side of the spinal cord in the body region.
  • PNS peripheral nervous system
  • TG trigeminal
  • SJG superior-jugular
  • DDG dorsal root ganglia
  • Nociceptors are slow conductive thinly myelinated A6- or unmyelinated C-fibers.
  • Nociceptors can be subdivided into several subtypes according to several features such as the stimuli they detect, their underlying molecular characteristics and innervation pattern. Two main groups can be distinguished: peptidergics and nonpeptidergics.
  • Peptidergic nociceptors secrete neuropeptides such as Substance P and CGRP, and express the Nerve Growth Factor receptor TrkA.
  • Non-peptidergic nociceptors express receptors such as Ret or P2X3.
  • TRP Transient Receptor Potential
  • PRDM12 is an evolutionarily conserved epigenetic transcriptional regulator whose mutation causes a rare disease, congenital insensitivity to pain (CIP) disorder, which is characterised by the inability to perceive pain from birth. Mutations in only a few other genes (NGF, TrkA,. . . ) have been shown to lead to CIP. New analgesics can be generated from the discovery of these CIP causing genes. However, until now, clinical trials that have been initiated targeting these other CIP gene products have had limited success due to severe side effects (Drissi et al., British Medical Bulletin, 2020).
  • the present invention identifies Prdml2 as an interesting pain target given its restricted expression in the nervous system suggesting that any compound targeting its action would have no side effect, its close link with NGF known to be involved in nociceptor sensitization, and the importance of epigenetic mechanisms in the induction and maintenance of chronic pain. Additionally, form the below, it can be concluded that in some pain disorders, it is the enhancement of Prdml2, rather than its blockade, that may have an analgesic effect.
  • Prdml2 functions as a transcriptional regulator in mature nociceptors of adult mice, controlling positively or negatively the expression of several receptors, ion channels and neuropeptides. Furthermore, loss of Prdml2 induced hypersensitivity in animals injected with formalin in the hindpaw during the second phase of the pain response, which is characterised by the involvement of inflammatory processes (Erami et al., (2017) Basic and Clinical neuroscience 8, 37-42).
  • RNA-seq analysis of the transcriptome of DRG of both Advil and Rosa26 icKO models shows that the loss of Prdml2 slightly deregulates the expression of many genes in nociceptors. Comparing the list of DEGs in the two models, a core set of 71 DEGs was identified, mostly neuronal genes, about half of them being downregulated and the other half upregulated by the loss of Prdml2, suggesting that Prdml2 may act as an activator or a repressor in mature nociceptors.
  • Prdml2 DEGs channel proteins and transmembrane receptors are overrepresented, providing first evidence that Prdml2 plays an essential role in the functional properties of adult nociceptors.
  • mice lacking Prdml2 exhibit normal response to thermal nociceptive stimuli under basal conditions. Unaltered responses to noxious cold is somehow surprising given the fact that we found that Trpm8 is reduced in Prdml2 icKO mice, as observed by Landy et al. (2021). This unaltered response to acute noxious cold may be due to the fact that Tprm8 expression is reduced, but not abolished in DRG neurons of Prdml2 icKO mice.
  • TRPV1 TRPV1 receptors that is rapidly internalized after stimulation.
  • capsaicin can be used as analgesic and has been showed to reduce lesion or inflammation-induced pain by triggering receptor endocytosis or nerve retraction. It could also be due to post-transcriptional modifications of TRPV1, its activity being modulated by kinases (CaMKII, PKA, PKC) and phosphatases (calcineurin) or lipids such as PIP2, themselves regulated by G-coupled membrane receptors. More conversingly, it was also shown here that Prdml2 icKO mice develop in contrast increased sensitivity to formalin-induced inflammatory pain.
  • the gene expression analysis of icKO mice revealed that the loss of Prdml2 dysregulates a set of genes known to contribute to formalin induced nociceptor sensitization. Among them Trpm8, Gfra3, Ntrkl, ASIC1 and Grikl that are downregulated, and Chrna6 and Cysltr2 that are upregulated in the absence of Prdml2 in basal conditions.
  • the upregulation of Chrna6 is unexpected as its overexpression is protective against tactile allodynia associated with inflammatory injuries.
  • the downregulation of Grikl and TrkA was also unpredicted since Grikl deletion reduces inflammatory pain and NGF acts as a mediator of inflammatory pain.
  • the electrophysiological data provided herein further supports the idea that that Prdml2 plays a role in the functional properties of mature nociceptors.
  • the increased excitability of cultured DRG neurons from icKO mice may provide some explanation to their hypersensitivity in the formalin test.
  • the deregulation of some potassium channels such as the calcium-activated potassium channel Kcnn3 and the calcium-activated potassium channel subunit beta-1 Kcnmbl could be involved in this increased excitability and increased response to formalin, as it was shown for Kcnn4. It is to note that their downregulation alone would also have increased the response to capsaicin. The opposite observed effect could therefore result from other concomitant gene dysregulations.
  • Prdml2 plays an important role in the transcriptional regulation of gene expression in mature nociceptors resulting in a complex modulation of pain-related behavior.
  • Prdml2 may have an analgesic effect, hereby providing the medical field with a new active pharmaceutical ingredient that is effective for managing pain responses, and thus pain sensation in subject. Indeed, in certain pain conditions, the enhancement of Prdml2, rather than Prdml2 blockade is beneficial for amelioration of the pain condition. Prdml2, or any modulator of Prdml2, may hence be used as a prophylactic or therapeutic component in pharmaceutical compositions.
  • pain responses related to or caused by inflammatory processes such as but not limited to skin or joint inflammation and pain related to itch are envisaged to be particularly suited for treatment through Prdml2 modulation.
  • Prdml2 function in pain responses is more complex than the role previously hypothesised in the art for this gene and related gene products.
  • Prdml2 function is dependent on previously disregarded parameters including different pain response phases and the specific pain condition that is induced.
  • the invention therefore relates to the following numbered aspects:
  • a modulator ofPrdm!2 activity foruse intreatment or prevention of a pain condition in a subject.
  • Aspect 2 The modulator for use according to aspect 1, wherein said modulator alters the expression level of Prdml2 in nociceptors and/or dorsal root ganglia of said subject.
  • Aspect 3 The modulator for use according to aspect 2, wherein said modulator increases Prdml2 expression in nociceptors and/or dorsal root ganglia of said subject, preferably wherein Prdml2 expression is increased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator.
  • Aspect 4 The modulator for use according to aspect 2, wherein said modulator decreases PRDM12 expression in nociceptors and/or dorsal root ganglia of said subject, preferably wherein PRDM12 expression is decreased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to PRDM12 expression in nociceptors and/or dorsal root ganglia of said subject before administration of said PRDM12 modulator.
  • Aspect 5 The modulator for use according to aspect 1, wherein the modulator alters the Prdml2-mediated G9a recruitment to histone H3, preferably wherein the modulator alters the methylation status of histone H3, preferably the methylation status of H3K9.
  • Aspect 6 The modulator for use according to aspect 5, wherein the modulator increases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to histone H3 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
  • modulator for use according to aspects 5 or 6, wherein said modulator decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes comprising: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, by at least 25%, preferably at least 50%, more preferably at least 75%, most preferably 100% when compared to their transcription rate in nociceptors and/or dorsal root ganglia before administration of said Prdml2 modulator.
  • modulators decreasing the activity of the gene product of any one of said genes, i.e. the protein is also envisaged in
  • Aspect 8 The modulator for use according to aspect 5, wherein the modulator decreases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, most preferably inhibits methylation of histone H3, when compared to histone H3 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
  • Aspect 9 The modulator for use according to aspects 5 or 8, wherein said modulator increases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes comprising: PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, and NTS by at least 25%, preferably at least 50%, more preferably at least 75%, most preferably 100% when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator.
  • modulators increasing the activity of the gene product of any one of said genes, i.e. the protein is also envisaged in this aspect.
  • Aspect 10 The modulator for use according to any one of aspects 1 to 9, preferably wherein the subject is suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or is a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, neuropathic itch, inflammatory itch such as atopic dermatitis, non-inflammatory itch such as pruritus-related pain, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
  • the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, neuropathic itch, inflammatory itch such as atopic dermatitis, non-inflammatory itch such as pruritus-related pain, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
  • Aspect 11 The modulator for use according to any one of aspects 1 to 10, wherein the subject is a mammalian subject, preferably a human subject.
  • Aspect 12 The modulator for use according to any one of aspects 1 to 11, wherein said modulator is a Prdml2 inhibitor, a Prdml2 activator, mutant Prdml2, or Prdml2, preferably wherein the modulator is anull loss-of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutantcaps.
  • said modulator is selected from the group comprising: Prdml2 binding molecules, Prdml2 protein or a polynucleotide encoding Prdml2, a functional fragment of Prdml2, or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene-expression or transcription targeting system, a transcription or expression inducer of the PRDM12 gene, a PRDM12 gene targeting system, inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof.
  • said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA
  • Aspect 13 The modulator for use according to any one of aspects 1 to 12, wherein said modulator alleviates pain in a subject by at least 1, preferably at least 2, at least 3, at least 4, more preferably at least 5, at least 6, at least 7, at least 8 scale points as reported by the treated subject when using a self-reporting pain scale, more preferably wherein the self-reporting pain scale is the numeric rating scale (NRS-11), Stanford pain scale, or visual numeric scale.
  • NRS-11 numeric rating scale
  • Stanford pain scale or visual numeric scale.
  • a pharmaceutical composition comprising Prdml2 and/or a Prdml2 modulator as described herein.
  • a pharmaceutical composition according to aspect 14, for use in treating or preventing a pain condition in a subject preferably wherein said subject is suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or is a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain (both inflammatory or noninflammatory), neuropathic itch, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
  • analgesic is selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal -inflammatory drugs, opioids, muscle-relaxants, anti-anxiety agents, antidepressants, anticonvulsants and corticosteroids.
  • a method for diagnosing a hypersensitivity to pain in a subject comprising a step of determining the expression of Prdml2 in a biopsy of said subject, wherein reduced expression of Prdml2 in nociceptors and/or dorsal root ganglia is indicative of a hypersensitivity to pain.
  • Aspect 18 The method according to aspect 17, wherein the hypersensitivity to pain is allodynia and/or hyperalgesia.
  • An analgesic comprising a modulator of Prdml2 activity.
  • modulator is PRDM12 or a functional fragment thereof, a Prdml2 inhibitor, a Prdml2 activator, mutant Prdml2, or Prdml2, preferably wherein the modulator is a null loss-of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutant.
  • said modulator is selected from the group comprising: Prdml2 binding molecules, Prdml2 protein or a polynucleotide encoding Prdml2, a functional fragment of Prdml2, or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene-expression or transcription targeting system, a transcription or expression inducer of the PRDM12 gene, a PRDM12 gene targeting system, inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof.
  • said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA
  • a method of diagnosing a hypersensitivity to pain in a subject comprising determining the level G9a recruitment to histone H3, preferably Prdml2-mediated recruitment to histone H3 in nociceptors and/or dorsal root ganglia in a biopsy of said subject.
  • Aspect 22 A method for diagnosing a hypersensitivity to pain in a subject, the method comprising determining the methylation status or representative methylation status of H3K9 in nociceptors and/or dorsal root ganglia in a biopsy of said subject.
  • Aspect 23 The method according to aspects 21 to 22, wherein the hypersensitivity to pain is allodynia and/or hyperalgesia.
  • a method of treating or preventing a pain condition in a subject comprising administering a modulator of Prdml2 activity as described herein or a pharmaceutical composition comprising a Prdml2 modulator as described herein, or an analgesic according to aspect 19 or 20 to a subject.
  • the subject is a subject suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, neuropathic itch, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
  • Aspect 25 Aspect 25.
  • the modulator of Prdml2 activity is an activator of Prdml2 activity. More preferably, when the modulator of Prdml2 activity is an activator, said pain condition is a pain condition induced by inflammation or itch, preferably inflammatory pain. More generally, said pain condition could be seen as a TRPV 1 -related pain condition, more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders (which include e.g. bladder pain syndrome (previously interstitial cystitis), irritable bowel syndrome (IBS) and fibromyalgia) and musculoskeletal pain. Many of these conditions are chronic pain conditions and many involve a burning sensation in the subject.
  • a pain condition induced by inflammation or itch preferably inflammatory pain.
  • said pain condition could be seen as a TRPV 1 -related pain condition, more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain,
  • Aspect 26 The method according to aspect 25, wherein said modulator is a polynucleotide encoding Prdml2, or a functional fragment or activated mutant of Prdml2, or wherein said activator is a transcription or expression inducer of the PRDM12 gene.
  • saidPrdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
  • said PRDM12 activator is a functional fragment of PRDM12 that retains a certain level of activity of the PRDM12 protein, such as 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the activity of the full-length PRDM12.
  • the PRDM12 activator is a fragment that comprises or consists of exons I-IV of the PRDM12 gene, resulting in a truncated protein with the conserved PR domain but lacking the zinc fingers. As it is thought that Prdml2 does not bind directly to DNA, this truncated protein may thus potentially retain some activity.
  • Aspect 27 The method according to any one of aspects 24 to 26, wherein the modulator is administered through gene therapy, or is administered systemically and/or topically.
  • Aspect 28 The method according to any one of aspects 24 to 27, wherein the modulator is comprised in a viral particle, preferably an adeno-associated viral particle.
  • AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAVPHPS, AAVrh20 and AAV9. Gene expression in DRG neurons has been shown using AAV vectors based on serotypes 1, 2, 3, 4, 5, 6, 8, 8.2, 9 and rh20.
  • Preferred vectors are AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self- complementary AAV9 vector (scAAV9).
  • said vector system comprises the PRDM12 mRNA (SEQ ID NO:2), or encodes the Prdml2 protein or a functional fragment thereof.
  • Aspect 29 The method according to aspect 24, wherein the modulator of Prdml2 activity is an inhibitor of Prdml2 activity. More preferably, when the modulator of Prdml2 activity is an inhibitor, said pain condition is heat induced pain or chemically induced pain, allodynia and/or hyperalgesia, or a pain that can be treated by capsaicin such as back pain, joint pain or headaches.
  • said inhibitor is selected from: inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof.
  • said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
  • the subject preferably is a subject suffering from heat-induced pain, operative pain, treatment-related pain, injury -related pain, or trauma-related pain.
  • Aspect 31 Use of a modulator of Prdml2 activity or PRDM12 expression as described herein for the manufacture of a medicament for the prevention or treatment of a pain condition as defined herein.
  • Aspect 32 Use of Prdml2, PRDM12, or a functional fragment thereof as described herein, or an activator of PRDM12 activity for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • a pain condition more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • Aspect 33 Use of an inhibitor of Prdml2 or PRDM12 activity, or a modulator thereof according to aspects 30 or 31, wherein said pain condition is heat induced pain.
  • Aspect 34 An in vitro method for identifying a molecule suitable as an analgesic, wherein said method comprises determining whether a candidate molecule modulates Prdml2 activity in nociceptors and/or dorsal root ganglia.
  • Aspect 35 The in vitro method according to aspect 34, wherein said method comprises contacting a nociceptor and/or a dorsal root ganglion with a candidate Prdml2 modulator, preferably wherein said nociceptor or dorsal root ganglion expresses Prdml2.
  • Aspect 36 The in vitro method according to aspects 34 or 35, wherein Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
  • Aspect 37 The in vitro method according to aspects 34 or 35, wherein Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
  • Prdml2 activity is assessed by measuring expression of one or more genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
  • genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, M
  • Aspect 38 The in vitro method according to aspect 37, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 activator when the expression of one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, Ntrk3, Cas
  • Aspect 39 The in vitro method according to aspect 37, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 inhibitor when the expression of one or more genes selected from the group consisting of: PRDM12, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425,
  • Aspect 40 The in vitro method according to any one of aspects 35 to 39, wherein the nociceptors and/or dorsal root ganglia are human nociceptors and/or dorsal root ganglia.
  • said Prdml2 modulator can have been identified by the method according to any one of aspects 34 to 40.
  • said Prdml2 activator can have been identified by the method according to any one of aspects 34 to 40; or alternatively, in any one of aspects 4, 5, 6, 7, 11, 12, 13, said Prdml2 inhibitor can have been identified by the method according to any one of aspects 34 to 40.
  • nucleotide sequence comprising a nucleotide sequence with a sequence identity of at least about 65%, preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 97%, at least about 99% to SEQ ID NO: 2 or a portion thereof.
  • a functional fragment of PRDM12 as defined above can be used.
  • the viral vector according to aspect 44 for use in gene therapy, preferably human gene therapy.
  • Aspect 46 The viral vector according to aspect 44 or 45, wherein said viral vector is an AAV vector, such as an AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self-complementary AAV9 vector (scAAV9) viral vector.
  • AAV vector such as an AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self-complementary AAV9 vector (scAAV9) viral vector.
  • a non-viral vector system encoding Prdml2 or encoding a modulator of Prdml2 activity as described herein, preferably wherein said modulator is a Prdml2 transgene, preferably wherein Prdml2 is human Prdml2 transgene, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or wherein the human PRDM12 transgene is defined by SEQ ID NO: 2.
  • a functional fragment of PRDM12 as defined above can be used.
  • said non-viral vector is selected from the group comprising: cationic lipids, liposomes, nanoparticles, PEG, PEI; plasmid vectors (e.g.
  • pUC vectors bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequencesO, a minicircle, an episomal vector, or a transposon-based vector, such as a PiggyBac-based vector or a Sleeping Beauty-based vector.
  • the Prdml2 modulator is a Prdml2 activator such as retinoic acid (RA) or zinc finger protein of the cerebellum 1 (Zicl).
  • RA retinoic acid
  • Zicl zinc finger protein of the cerebellum 1
  • said Prdml2 modulator is a Prdml2 inhibitor such as an inhibitor of alcohol and/or aldehyde dehydrogenases, for example citral.
  • Aspect 50 The method according to any one of aspects 24 to 29 comprising gene therapy using the viral or non viral vector according to any one of aspects 44 to 47.
  • Ppefl protein phosphatase with EF hand calcium-binding domain 1;
  • Mettl7a3 methyltransferase like 7 A3;
  • Kcnmbl potassium large conductance calcium-activated channel, subfam. M, beta member 1; Otoa: otoancorin;
  • Smr2 submaxillary gland androgen regulated protein 2
  • Ms4a3 membrane-spanning 4-domains, subfamily A, member 3.
  • Lrm4 leucine rich repeat neuronal 4
  • A3galt2 alpha 1,3-galactosyltransferase 2
  • klk5 kallikrein related-petidase 5.
  • a method of identifying pain modulating agents comprising the step of screening candidate agents for their ability to modulate any one or more of the following genes:; Ppefl; Ppmlj; Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3, Lrm4, A2galt2 or Klk5.
  • a method of identify agents for use in treating a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch comprising the step of screening candidate agents for their ability to restore or upregulate the expression, activity or function of any one or more of the following genes: Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3; Lrm4; A2galt2; or Klk5.
  • Aspect 54 The modulating agents identified in aspect 48 for use in treating a pain condition as defined herein.
  • a method of treating a pain condition as defined herein comprising administering to a patient in need thereof a modulating agent identified in aspect 48.
  • Aspect 56 Use of a modulating agent identified in aspect 48 for the manufacture of a medicament for the prevention or treatment of a pain condition as defined herein.
  • Aspect 57 Use of an agent able to restore or upregulate the expression, activity or function of any one or more of the following genes: Ppefl; Ppmlj; Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3, Lrm4, A2galt2 or Klk5.2 for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • a pain condition more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • a method of treating a pain condition comprising administering to a subject in need thereof a therapeutically effective amount of an agent able to restore or upregulate the expression, activity or function of any one or more of the following genes: Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3; Lrm4; A2galt2; or Klk5, more preferably wherein said pain condition is inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • a pain condition more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
  • Aspect 60 Use of an agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably in heat induced pain or chemically induced pain, allodynia and/or hyperalgesia, or pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches.
  • a method of preventing or treating of a pain condition comprising administering to a subject in need thereof a therapeutically effective amount of an agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj or Ppefl.
  • Aspect 62 An agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj or Ppefl for use in the prevention or treatment of a pain condition, more preferably in heat induced pain or chemically induced pain, allodynia and/or hyperalgesia or pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches.
  • FIG. 1 Validation of the AdvillinCreERT2; Prdml2 fl fl mice. Immunofluorescence with anti-Prdml2 antibodies on dorsal root ganglia (DRG) section of adult mice of the indicated genotype injected with Tamoxifen and sacrificed 4 days after injection for DRG dissection.
  • DRG dorsal root ganglia
  • Figure 4 Validation of the differential expression of selected up- and down-regulated genes identified in the RNA-seq of TAM injected Advillin-CreERT2; Prdml 2 1111 mice (icKO) by RT-qPCR. As expected, Prdml2 is knocked-out.
  • FIG. 7 Loss of Prdml2 increases sensitivity to inflammatory pain induced by subcutaneous injection of formalin in the hindpaw.
  • FIG. 8 Prdmll is expressed in nociceptors of adult mice and is downregulated upon CFA induced inflammation.
  • A X-Gal staining of the brain (ventral view) with attached trigeminal ganglia (TG) of an adult Prdml 2 LacZ/+ mouse. Superior jugular ganglia (SJG) and spinal cord (SC) with attached dorsal root ganglia (DRG) are shown on the right.
  • SJG Superior jugular ganglia
  • SC spinal cord
  • DDG dorsal root ganglia
  • RECTIFIED SHEET (RULE 91) ISA/EP immunostainings on transverse sections of TG, SJG and DRG of adult mice with the indicated markers.
  • FIG. 9 Loss of Prdml2 in nociceptor of adult mice does not alter diversity of DRG neuronal subpopulations.
  • A Representative images of DRG sections of Avil icKO and Rosa26 icKO injected with tamoxifen or with com oil as control immunolabelled for Peripherin, CGRP, Navi.8 and NF-200 or stained with IB4, with quantification showing that the number of cells positive for the different indicated markers is similar in DRG sections of Prdml2 icKO and control mice.
  • FIG. 1 Proportion of CGRP + neurons and of IB4 + neurons among Peripherin + neurons in DRG are similar in both icKO and control mice.
  • E Left panel, blll-Tubulin immunostainings on skin tissue of T AM-injected Rosa26 icKO and control mice. Arrows point skin epidermis innervation (E 5epidermis; D 5dermis). Scale bar: 50 mm.
  • FIG. 10 Transcriptomic analysis of the consequences of the loss of Prdml2 in DRG nociceptors of adult mice.
  • the genes were identified by bulk RNA-seq and selected based on adjusted p-value ⁇ 0.05. Genes with a log2FoldChange > 0,449 are indicated in red, genes below the threshold are in light grey.
  • C Proportion of neuronal genes among core Prdml2 DRGs. Neuronal genes up- and downregulated by the loss of Prdml2 are indicated in blue and red, respectively.
  • D Classification of the common DEG’s based on their expression in the major different subtypes of DRG neurons, with downregulated genes in red and upregulated genes in blue.
  • FIG. 11 Validation of some of the identified DEGs as Prdml2 targets in Advil icKO mice.
  • A RT-qPCR analysis of the indicated genes.
  • FIG. 12 Knockout of Prdml2 in mature nociceptors does not alter thermal and mechanical nocociception but affects responses to formalin and capsaicin.
  • A Avil icKO and control (TAM injected Prdml ⁇ M 1 mice) spent similar time on the test side in a two- temperature choice assay.
  • D Rosa26 icKO and control mice take show the same behavior in the Mechanical conflict avoidance test.
  • FIG. 1 Representative traces from a control DRG neuron, showing subthreshold responses and subsequent action potential induced by injection of 1310 pA (top), and action potential frequency induced by a one-second 1500 pA step (bottom). The same protocol was applied to a Prdml2 KO DRG neuron (B). The current threshold was 750 pA for this neuron. Arrows indicate the current amplitude used to elicit the labeled response.
  • FIG. 14 Validation of the Avil icKO and Rosa26 icKO mice.
  • Figure 15 Gene ontology analysis and KEGG pathway analysis of the Prdml2 core DEGs.
  • A Gene ontology analysis based in all categories of the 71 common DEGs indicating the 6 most enriched subclassifications.
  • B KEGG pathway analysis indicating the 3 most enriched pathways.
  • C Genes deregulated in the neuroactive ligand-receptor interaction pathway, in red the upregulated and in green the downregulated in the Prdml2 icKO’s.
  • FIG. Knockout of Prdml2 does not alter locomotor activity.
  • Values are means ⁇ SEM.
  • FIG. 18 Transcriptomic analysis of the consequences of the loss of Prdml2 in TG of adult mice.
  • A Volcano plots showing the -loglO adjusted P-value as a function of the log2 fold-changes of deregulated genes in TG of Rosa26 icKO mice (icKO mice, n 56 vs control mice, n 56) 1 month after tamoxifen injection. The genes were identified by bulk RNA-seq and selected based on adjusted P-value ,0.05. Genes with a log2 fold-change .0.31 are indicated in red, genes below the threshold are in light grey.
  • B Gene ontology classification of the DEGs. The graph shows the enriched biological molecular function associated with downregulated and/or upregulated genes.
  • FIG. 1 Venn diagram showing the overlap between the DEGs identified in TG and DRG of Rosa26 icKO mice.
  • the list of common DEGs in TG and DRG of Rosa26 icKO is shown in Table 5 of Example Y.
  • DRG dorsal root ganglia
  • TG trigeminal ganglia
  • icKO inducible conditional knockout.
  • FIG. 19 Conditional knockout of Prdml2 in mature nociceptors does not alter thermal and mechanical nociception but affects responses to formalin and capsaicin.
  • Avil icKO and control mice spent similar time on the test side in a 2-temperature choice assay.
  • B Rosa26 icKO (n 511) and control mice (n 59) show similar withdrawal latency in the tail flick test using a focus 30.
  • C Rosa26 icKO (n 510) and control mice (n 512) show no difference in withdrawal latency in the cold plantar assay.
  • Rosa26 icKO and control mice show the same behavior in the mechanical conflict avoidance test. They spent the same time to cross half of the second chamber with nails on the floor.
  • Results obtained with 2 different nail heights (2 and 5 mm) are shown.
  • E Intradermal cheek injection of N-met LTC4 elicits scratching bouts that are not significantly different between Rosa26 icKO and control mice.
  • F Rosa26 icKO mice (n 510) spent less time licking their paw than control mice (n 512) after capsaicin injection (P 50.011).
  • G Time course of the nocifensive response (licking time in seconds) of Avil icKO (n 511) and control mice (n 514) until 30 minutes after formalin injection. Response of individual Avil icKO and control mice in the first (0-5 minutes after formalin injection) and second phase (15-30 minutes after formalin injection) is shown on the right.
  • Avil icKO mice spent more time licking their paw when compared with control mice during the second phase (P 50.001). Values are represented as mean 6SEM. *P ,0.05, **P ,0.01 (Student t test, a 50.05). icKO, inducible conditional knockout.
  • the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter aha a reference to any one of said members, or to any two or more of said members, such as, e.g. any 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more etc. of said members, and up to all said members.
  • “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
  • numeric values indicated by “at least” as used herein are indicative for an interval between the lower value and an upper limit.
  • “at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% respectively indicates intervals from about 10% to about 100%, from about 25% to about 100%, from about 35% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%, from about 99% to about 100%.
  • the upper limit is not limited to 100%, and may be considerably higher than 100%, such as 150%, 200%, 250%, 500%, 1000%, or higher.
  • Amino acids are referred to herein with their full name, their three-letter abbreviation or their one letter abbreviation.
  • amino acid indicates naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D- and L-stereoisomers, provided their structure allows such stereoisomeric forms.
  • Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • a “naturally encoded amino acid” indicates an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy-lysine or selenocysteine.
  • the 20 common amino acids are: alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Vai), tryptophan (W or Trp), and tyrosine (Y or Tyr). Also envisaged by said term
  • nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g. the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g. RNA or RNA analogue) relationship.
  • a template-transcription product e.g. RNA or RNA analogue
  • Prdml2 is an interesting potential pain target given its biological function, epigenetic transcriptional regulation, and restricted expression in pain sensing neurons.
  • the present inventors were able to identify new insights in the functioning of Prdml2, both on a molecular level and on the level of the subject as a whole.
  • the findings expose numerous innovative strategies for interfering molecular pathways involved in pain sensation and/or pain signalling, and hereby provide a solution to the unmet need described above.
  • Subject may be used interchangeably herein and refer to animals, preferably warm-blooded animals, more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals, that have been the object of treatment, observation or experiment with a Prdml2 modulator.
  • Preferred subjects are human subjects including all genders and all age categories thereof. Both adult subjects, newborn subjects, and fetuses are intended to be covered by the term “subject”.
  • Preferred subject in the context of the invention are subjects that are experiencing pain, or suspected to experience pain either at a time of assessment or in an envisaged future point in time.
  • the subject is diagnosed with a condition or disorder that is known in the art to be associated with physical pain or discomfort.
  • the subject is an elderly subject.
  • “Elderly subject” refers to a subject of old age, i.e. the age nearing or surpassing the life expectancy of a subject.
  • An elderly subject is defined by an age of at least 65 years, preferably at least 70, at least 75, at least 80, at least 85, most preferably at least 85 years.
  • the subject is selected from the group consisting of: infants (i.e. juvenile subjects), adolescent subjects, and adult subjects.
  • the subject is diagnosed to be palliative or considered to be palliative.
  • the term is further intended to include transgenic species. It is further envisaged that the present invention is suitable to suppress pain responses and/or pain sensation in test animals.
  • animals that may benefit from the present invention include but are not limited to warm-blooded animals, more preferably vertebrates, and even more preferably non-human mammals such as those selected from the non-exhaustive list of examples of domestic animals, commercial animals, farm animals, zoo animals, sport animals, pet and experimental animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such
  • subjects particularly suited for administering the Prdml2 modulator to are subjects that have a medical history of intolerance to traditional analgesics and/or may have experienced an analgesic addiction or susceptibility to analgesic addition, such as but not limited to opioid addiction, alcohol addiction, cannabis, any substance classified by authorities as “hard drug”, or any combination thereof.
  • a preferred subject may be a subject that is allergic to known analgesics.
  • a skilled person is aware of the physical and physiological symptoms of opioid addiction and a healthcare practitioner is readily capable of establishing whether a subject is susceptible for, or is experiencing an opioid use disorder, all of which are described in detail in throughout the art (e.g. according to the DSM-5 criteria, Diagnostic and Statistical Manual of Mental Disorders Fifth Edition, 2013).
  • the subject is a recovering opioid addict.
  • Pain as defined by the International Association for the Study of Pain, is a submodality of somatic sensation and has been defined in the art as a complex constellation of unpleasant sensory, emotional and cognitive experiences provoked by real or perceived tissue damage and manifested by certain autonomic, psychological, and behavioral reactions (Terman and Bonica, Bonica’s management of pain, 2003).
  • the term “pain” as used within the context of the present disclosure refers to physical pain.
  • physical pain may additionally cause secondary unwanted emotional states that are associated with non-physical, i.e. mental pain.
  • said modulator may additionally exert an improvement in the emotional state of the subject.
  • Particularly relevant in this context is the alleviation of chronic pain, which is known to be harmful and often detrimental for the mental well-being of a subject (described in a.o. Sheng et al., Neural Plasticity, 2017).
  • the pain may be acute (i.e. temporary), chronic, intermittent, or increasing over time.
  • pain includes but is not limited to pain in subjects suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or wherein said is a palliative subject. More preferably said pain condition is nociceptive pain, inflammatory pain, non-inflammatory pain, neuropathic pain, itch-related pain, inflammatory itch-related pain caused by e.g. atopic dermatitis, non-inflammatory itch-related pain caused by e.g. pruritus, neuropathic itch, or any combination thereof, wherein in some embodiments said pain condition is induced by nerve injury or inflammation or is itch-induced (cf. Wong et al., (2017), Int J Mol Sci. 2017 Jul; 18(7): 1485).
  • Emerging methods to assess pain include but are not limited to techniques such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). Any of these methods may be suitable for determining whether a person will, or is suspected to benefit from Prdml2 modulation.
  • the pain experienced by a subject is assessed by one or more methods selected from the group of methods consisting of: self-reporting scales, fMRI, EEG, or any combination thereof.
  • analgesic as used herein is to be interpreted in its broadest interpretation and may therefore refer to any compound, substance, or composition that is able to reduce pain or suppress pain in a subject, and hence any product that is able to achieve analgesia in a subject.
  • any analgesic may interchangeably be referred to as “painkiller” or “pain reliever”.
  • the analgesic may be used in the treatment of pain and/or in pain prevention (i.e. pain prophylaxis).
  • An analgesic may act on the peripheral and/or central nervous system.
  • analgesics are commonly classified according to the mechanism of action. Different classes of analgesics include but are not limited to acetaminophen (i.e.
  • NSAIDs nonsteroidal -inflammatory drugs
  • opioids opioids
  • muscle-relaxants anti-anxiety agents
  • antidepressants anticonvulsants
  • corticosteroids corticosteroids
  • any NSAID referred to herein may be selected from an NSAID group consisting of salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, enolic acid derivatives (oxicam), anthranilic acid derivatives (fenamates), (selective) COX-2 inhibitors (coxibs), sulfonanilides, etc.
  • Non limiting examples of NSAIDs include: aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid and salts thereof, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam, isoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxi
  • Opioids may be classified according to the degree of chemical modification.
  • an opioid referred to herein may be selected from the group consisting of natural opiates, morphine esters, semi-synthetic opioids, fully synthetic opioids, endogenous opioid peptides.
  • opioid as referred to herein further encompasses substances or molecules classified in the art as “opioidergics”, said opioidergics are commonly referred to in the art to define groups of chemicals that modulate the opioid neuropeptide systems.
  • Examples of natural opioids include alkaloids such as morphine, codeine, thebaine, Mitragyna speciosa (or leaves thereof), and salvorin A from Salvia dinorum.
  • morphine esters include morphine prodrugs such as but not limited to diacetylmorphine, nicomorphine, dipropanoylmorphine, desomorphine, acetylpropionylmorphine, dibenzoylmorphine, and diacetyldihydromorphine.
  • semi-synthetic opioids include but are not limited to hydromorphone, hydrocodone, oxycodone, oxymorphone, ethylmorphine, buprenorphine.
  • fully synthetic opioids include fentanyl, pethidine, levorphanol, tramadol, tapentadol, and dextropropoxyphene.
  • Endogenous opioid peptides include but are not limited to endorphins, enkephalins, dynorphins, and endomorphins.
  • Alternative opioids or opioid-like compounds include tramadol, tapentadol, nefopam, orphenadrine, phenyltoloxamine, and antihistamines.
  • opioid as referred to herein may optionally refers to one or more of the opioids of the group consisting of: codeine, morphine, thebaine, oripavine, diacetylmorphine (morphine diacetate; heroin), nicomorphine (morphine dinicotinate), dipropanoylmorphine (morphine dipropionate), diacetyldihydromorphine, acetylpropionylmorphine, desomorphine, methyldesorphine, dibenzoylmorphine, dihydrocodeine, ethylmorphine, heterocodeine, buprenorphine, etorphine, hydrocodone, hydromorphone, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl, alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl, pethidine (meperidine), ketobemidone, MPPP,
  • Nono-limiting examples of allosteric modulators include BMS-986121 and BMS-986122 (both modulators of the p-opioid receptor, as described in Burford at al. PNAS USA, 2013), ignavine (modulator of the p-opioid receptor, Ohbuchi et al.
  • Oxytocin p-opioid receptor, Meguro et al., Journal of pharmacological sciences, 2018), 6-PAM (6-opioid receptor, Burford et al., Journal of Medicinal Chemistry, 2015), cannabidiol and tetrahydrocannabinol (6-opioid receptor, Kathmann et al., 2006), sodium (capable of modulating opioid receptors by sodium ions, Shang et al., 2014).
  • corticosteroids as referred to herein is representative for a class of steroid hormones, derivatives and related molecules that may have an effect on a plethora of processes in a subject such as stress responses, immune responses, inflammation regulation, metabolism, protein catabolism, electrolyte concentration in e.g. the blood, and behavior.
  • Corticosteroids are commonly classified in the art in two classes, glucocorticoids (i.e. corticosteroids that bind to the glucocorticoid receptor) and mineralocorticoids, both of which are envisaged herein by the term corticosteroids.
  • corticosteroids may be classified as natural or synthetic corticosteroids, said synthetic corticosteroids consisting of the group of corticosteroids selected from: progesterone-type, hydrocortisone-type, methasone-type, acetonides, cortivazol, and RU-28362, all of which are envisaged herein by the term “corticosteroids”.
  • corticosteroids may be selected from the group of corticosteroids consisting of: 11 -dehydrocorticosterone, 11- deoxy corticosterone, 11 -deoxy cortisol, 11 -ketoprogesterone, l ip-hydroxypregnenolone, l ip- hydroxyprogesterone, 1 ip,17a,21-trihydroxypregnenolone, 17a, 21 -dihydroxypregnenolone, 17a-hydroxypregnenolone, 17a-hydroxyprogesterone, 18-hydroxy-l 1 -deoxycorticosterone, 18-hydroxy corticosterone, 18-hydroxyprogesterone, 21 -deoxy cortisol, 21 -deoxy cortisone, 21- hydroxypregnenolone (prebediolone), aldosterone, corticosterone (17-deoxy cort
  • Muscle-relaxants as referred to herein indicates a class of pharmaceutically active ingredients that are able to affect skeletal muscle function and decrease the muscle tone. Muscle-relaxants are commonly stratified into to categories: neuromuscular blockers and spasmolytics (the latter often being referred to as antispasmodics).
  • Non-limiting examples of known muscle-relaxants include chlorzoxazone, carisoprodol, methocarbamol, cyclobenzaprine, orphenadrine, metocurine, botulinum toxin type A, botulinum toxin type B, baclofen, succinylcholine, cisatracurium, tizanidine, rocuronium, hexafluronium, doxacurium, chlormezanone, tubocurarine, dantrolene, mivacurium, pancuronium, pipecuronium, vecuronium, eperisone, thiocolchicoside, tolperisone, bromazepam, diazepam, clonazepam, flunitrazepam, lorazepam, nitrazepam, temazepam, eszopiclone, quinine, fludiazepam, etc.
  • Anti-anxiety agents are interchangeably annotated in the art as “antipanics” or “anxiolytic agents” and in addition to medication for preventing or reducing anxiety, several of them also display analgesic properties, and several anti-anxiety agents are therefore prescribed for both anxiety and pain conditions.
  • Non-limiting exemplary classes of anti-anxiety agents include barbiturates, benzodiazepines, carbamates, antihistamines, opioids, antidepressants, sympatholytics (i.e.
  • beta blockers alpha blockers, and alpha-adrenergic agonists
  • phenibut mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, pregabalin, menthyl isovalerate, propofol, and racetams.
  • antidepressant is well known to a person in the art, and several antidepressants have been additionally used to treat pain, in particular chronic pain.
  • Antidepressants have been categorised in several classes, including but not limited to selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors, and NMDA receptor antagonists.
  • anticonvulsant indicates a class of antiepileptic and/or antiseizure drugs, which are increasingly used as pain treatment medication, particularly for treatment of neuropathic pain (Rogawski et al., Nature Reviews Neuroscience, 2004). Numerous anticonvulsant drugs have been developed and may be classified according to chemical structure.
  • Anticonvulsants as envisaged herein include aldehydes, aromatic allylic alcohols, barbiturates, benzodiazepines, bromides, carbamates, fatty acids, fructose derivatives, hydantoins, oxazolidinediones, propionates, pyrimidinediones, pyrrolidines, succinimides, sulfonamides, triazines, valproylamides, and additionally anticonvulsants that are not generally classified under one of the former categories, including but not limited to perampanel, stiripentol, and pyridoxine.
  • the subject is insensitive or considered to be insensitive to traditional analgesics, preferably wherein the subject is insensitive or considered insensitive to one or more analgesics selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal-inflammatory drugs (NSAIDs), opioids, muscle-relaxants, anti-anxiety agents, antidepressants, and corticosteroids.
  • analgesics selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal-inflammatory drugs (NSAIDs), opioids, muscle-relaxants, anti-anxiety agents, antidepressants, and corticosteroids.
  • NSAIDs nonsteroidal-inflammatory drugs
  • opioids i.e. paracetamol
  • muscle-relaxants i.e. paracetamol
  • anti-anxiety agents i.e. where the pain conditions persists, optionally with a mild improvement despite treatment with traditional anal
  • protein is to be interpreted according to the commonly accepted meaning in the art, and therefore generally encompasses macromolecules comprising one or more polypeptide chains, i.e, polymeric chains of amino acid residues linked by peptide bonds.
  • the term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins.
  • protein is a collective name to indicate polypeptide chains as such but also encompasses proteins that carry one or more co- or post-expression-type modifications of at least one polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc.
  • the term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis corresponding canonical version of said protein, such as, e.g. amino acid deletions, additions and/or substitutions.
  • full-length proteins and functional protein fragments, e.g. naturally- occurring protein parts or domain incorporated in the full-length protein which exerts or maintains a certain functionality when isolated from the full-length protein or used separately as part of a larger polypeptide chain distinct form the full-length protein.
  • protein protein
  • polypeptide and even “peptide” are commonly used interchangeably, particularly in the context of relative short peptides. None of the terms is limited to any minimum length of the polypeptide chain. However, a consensus in the art is maintained that a peptide at least comprises two amino acids, which may be identical or different amino acids.
  • a non-limiting production method of proteins, polypeptides and/or peptides is recombinant production, wherein a suitable host or host cell expresses by translation the envisaged protein, polypeptide, or peptide (i.e. wherein the host cell(s) function(s) as an expression system for an expression vector), and wherein said translated product is in a subsequent step isolated therefrom.
  • suitable expression systems have been described in the art and non-limiting examples thereof are bacterial, yeast, fungal, plant or animal cells or cell expression system).
  • the proteins, polypeptides and/or peptides may be produced recombinantly by cell-free transcription and/or translation, or non- biological protein, polypeptide or peptide synthesis.
  • a suitable peptide synthesis method known in the art that does not involve a live expression system is solid phase peptide synthesis (reviewed in detail in e.g. Jaradat, Amino Acids, 2018).
  • nucleic acid typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units.
  • a nucleoside unit commonly includes a heterocyclic base and a sugar group.
  • Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5- propynylcytosine.
  • 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • Sugar groups may include inter aha pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxy arabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-O-alkylated, e.g., 2'-O-methylated or 2'-0-ethylated sugars such as ribose; 2'-O-alkyloxy alkylated, e.g., 2’ -O-methoxy ethylated sugars such as ribose; or 2'-O,4'-C-alkylene-linked, e.g., 2'-O,4'-C-methylene-linked or 2'-O,4'-C-ethylene- linked sugars such as ribose; 2 ’-fluoro-arabinose,
  • Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA.
  • Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g., by a methyl, ethyl, alkyl, or alkyloxyalkyl).
  • ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units.
  • Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA.
  • deoxyribonucleoside unit(s) comprise 2'-H.
  • deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units.
  • Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter aha phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate- based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3
  • nucleic acid also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)).
  • PNA peptide nucleic acids
  • PONA peptide nucleic acids with phosphate groups
  • LNA locked nucleic acids
  • PMO morpholino phosphorodiamidate-backbone nucleic acids
  • CeNA cyclohexene nucleic acids
  • tcDNA tricyclo-DNA
  • Alkyl as used herein particularly encompasses lower hydrocarbon moi eties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2- propenyl, and isopropyl.
  • Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof.
  • a modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.
  • nucleic acid sequence and the likes further encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids.
  • RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA).
  • a nucleic acid can be naturally occurring, e.g. present in or isolated from nature, can be recombinant, i.e. produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised.
  • a naturally occurring variant of a given sequence refers to all variants of the sequence which encode the same functional protein and that are present in or can be isolated from nature. Typically, this includes all variants of the sequence encountered in mammals, more particularly humans. It will be understood that variants from closely related species will have a higher sequence identity than variants from evolutionary more distant species.
  • nucleic acids can be produced recombinantly by a suitable host or host cell expression system and isolated therefrom (e.g. a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription, or non-biological nucleic acid synthesis.
  • a suitable host or host cell expression system e.g. a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system
  • a nucleic acid can be double-stranded, partly double stranded, or single-stranded.
  • nucleic acid can be the sense strand or the antisense strand.
  • nucleic acid can be circular or linear.
  • nucleic acid further indicates any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (reviewed in e.g. Kurreck, European Journal of Biochemistry, 2003).
  • Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof.
  • Prdml2 short for “PR domain zinc finger 12” is indicative for a protein that in humans is encoded by the PRDM12 gene which is located on chromosome 9 (9q34.12). Prdml2 is known to be a transcriptional regulator of sensory neuronal specification that plays a critical role in pain perception. Through this function, Prdml2 is involved in the development of nerve cells that assist in perception and sensation of pain. At least in vertebrates, Prdml2 has been shown to repress different genes, such as the non-limiting examples of the DBX1 and NKX6 genes, hereby acting as a general determinant of VI cell fate.
  • Prdml2 VI interneurons in turn play an essential role in vertebrate locomotion (Thelie et al., development, 2015). Mutations in Prdml2 are associated in the art with a congenital insensitivity to pain (abbreviated herein and in the art as “CIP”). Further, Prdml2 mutations are associated with hereditary sensory and autonomic neuropathies (HSAN’s) affecting peripheral sensory and autonomic neurons (e.g. HSAN VIII), and anhidrosis. Additional consequences of Prdml2 mutations are an early loss of teeth, soft tissue injuries, dental carries, submucosal abscesses, and hypomineralisation of primary teeth, and mandibular osteomyelitis.
  • HSAN hereditary sensory and autonomic neuropathies
  • the PRDM12 gene and/or gene product may alternatively be annotated by one or more alternative names including but not limited to PFM9, HSAN8, PR domain 12, PR/SET domain 12, PR-containing protein 12, PR domain containing 12.
  • Prdml2 denotes the Prdml2 peptide, polypeptide, protein, or nucleic acid as will be evident to a skilled person from the context of the specific disclosure.
  • the full length human Prdml2 protein comprises an N-terminal PRDI-BF1 and RIZ homology (PR) domain, a SET domain, and three C-terminal C2H2 zinc finger DNA-binding domains (Chen et al. Nature Genetics, 2015). It has been described in the art that the human PRDM12 gene comprises 5 exons (Chen et al., Nature Genetics, 2015).
  • the human PRDM12 gene is annotated and publicly available under NCBI Genbank (www.ncbi.nlm.nih.gov) accession number (NCBI reference sequence NG_053081.1).
  • NCBI Genbank www.ncbi.nlm.nih.gov
  • NCBI reference sequence NG_053081.1 The messenger RNA (mRNA) sequence of Homo sapiens PRDM12 is publicly accessible under NCBI reference sequence: NM_021619.3.
  • the reference Human Prdml2 protein sequence is annotated and publicly available as NCBI reference sequence NP_067632.2 or alternatively retrievable on UniProt (www.uniprot.org) under accession number Q9H4Q1 (PRD12 HUMAN) and is by means of example reproduced below (SEQ ID NO: 1): MMGSVLPAEALVLKTGLKAPGLALAEVITSDILHS FLYGRWRNVLGEQLFEDKSHHAS PKTA FTAEVLAQS FSGEVQKLSSLVLPAEVI IAQSS I PGEGLGI FSKTWIKAGTEMGPFTGRVIAP EHVDICKNNNLMWEVFNEDGTVRYFIDASQEDHRSWMTYIKCARNEQEQNLEWQIGTS I FY KAIEMI PPDQELLVWYGNSHNTFLGI PGVPGLEEDQKKNKHEDFHPADSAAGPAGRMRCVIC HRGFNSRSNLRSHMRIHTLDKPFVCRFCNRRFSQSSTLRNHVRLHTGERPYKCQVC
  • any sequences represented in sequence databases or in the present specification may be of precursors of peptides, polypeptides, proteins, or nucleic acids and may include parts which are processed away from mature molecules commonly known under the respective designations in the art.
  • the terms PRDM12 and Prdml2 encompass such peptides, polypeptides, proteins, or nucleic acids, of any subject where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
  • Non-limiting examples of such methods and tools are Protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/), SIM alignment tool (https://web.expasy.org/sim/), TranslatorX (http://translatorx.co.uk/) and T-COFFEE (https://www.ebi.ac.uk/Tools/msa/tcoffee/).
  • the percentage of identity between two sequences may show minor differences depending on the algorithm choice and parameters.
  • sequence identity refers to the relationship between sequences at the nucleotide or amino acid level.
  • % identical is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences.
  • the reference sequence does not comprise insertions or deletions.
  • a reference window is chosen and the “% identity” is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence.
  • modulator as used herein may be used interchangeably with synonymous terms such as “regulator” and indicate any moiety that may affect Prdml2 activity, either by affecting the function of a Prdml2 molecule, by affecting the expression level of Prdml2, or by both affecting the function and expression of Prdml2 or its gene PRDM12 in cells of a subject.
  • certain modulator disclosed herein might exclusively alter expression levels of Prdml2 or PRDM12, while others might exclusively alter the intrinsic activity effected by Prdml2.
  • certain modulators will alter both the function and the expression level of Prdml2, wherein both parameters may be upregulated or downregulated by said modulator, or wherein each parameter may be affected in a different direction.
  • modulation of the activity of a gene and/or its gene products may function by direct hybridisation or binding to the said target or to any of the molecules said target engages in an interaction with.
  • the modulator may influence Prdml2 activity by binding to an element governing the expression of Prdml2, non-limiting examples hereof being certain genetic sequences including PRDM12 promoter or enhancer sequences. Nevertheless, a skilled person will appreciate that each of the above groups of modulators will affect the nett Prdml2 activity.
  • a moiety is only to be considered a modulator when a nett change in Prdml2 activity upon or after administration of said moiety is observed in a cell, tissue, and/or subject.
  • a Prdml2 modulator is a moiety that by any mechanism causes a deviation or Prdml2 activity in a target cell, tissue, and/or subject from a baseline value, said baseline value being a predetermined Prdml2 activity level when said cell tissue, and/or subject is not subjected to, or contacted with, a Prdml2 modulator.
  • the assay to quantify said expression, or change in expression is selected from the group consisting of: PCR (e.g. RT-PCR or qPCR), mass spectrometry analyses, spectrophotometric assays (e.g. UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays), ELISA, or a combination thereof.
  • PCR e.g. RT-PCR or qPCR
  • mass spectrometry analyses e.g. UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays
  • spectrophotometric assays e.g. UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays
  • RT-PCR real-time polymerase chain reaction
  • qPCR real-time polymerase chain reaction
  • dye-based and probe-based detection Two main methodologies to perform quantitative PCR have been described: dye-based and probe-based detection. Each of these methods relies on calculating the initial (zero cycle) DNA concentration by extrapolating back from a reliable fluorescent signal. Protocols and related methods that rely on RT-PCR have been described in detail in the art (Ary a et al., Expert Review of Molecular Diagnostics, 2015).
  • spectrophotometric assays include but are by no means limited to mass spectrometry analyses, spectrophotometric assays and enzyme -linked immunosorbent assays (ELISA).
  • targeted proteomics experiments include selective reaction monitoring and multiple reaction monitoring.
  • Mass spectrometry approaches to measure protein expression levels have been extensively described in the art (Shi et al., Proteomics, 2016).
  • spectrophotometric assays have been described and include UV light absorption spectroscopy, dye-based protein assays, Coomassie blue (Bradford) assays, and Lowry alkaline copper reduction assays (Noble and Bailey, Methods in Enzymology, 2009).
  • ELISA assays suitable for protein quantitation have also been described (Pamas and Linial, Brain research protocols, 1998).
  • nociceptor refers to any sensory neuron developed from neural-crest or placodal stem cells that respond to, or is considered to be able to respond to harmful (i.e. potentially damaging to the subject or tissue) stimuli.
  • the term may interchangeably be used with the term “pain receptor”.
  • Nociceptors constitute a key component of nociception (i.e. perceiving of pain). Nociception in turn may optionally induce one or more physiological and/or behavioural responses in a subject. In any of the embodiments and aspects described herein, the term may refer to the total amount of nociceptors in the subject, or alternatively to a certain subgroup of nociceptors.
  • the subgroup may be defined by their localisation, molecular characteristics, innervation pattern or a combination thereof.
  • the Prdml2 modulators as described herein may exert their function in peptidergic nociceptors and/or nonpeptidergic nociceptors. Alternatively, the Prdml2 modulators as described herein may exert their function in external nociceptors and/or internal nociceptors.
  • Dorsal root ganglia and the singular form “dorsal root ganglion” may be used interchangeably with terms such as “spinal ganglion” and “posterior root ganglion”.
  • the terms indicate a cluster of neurons (i.e. a ganglion) in a dorsal root of a spinal nerve that carry sensory signals to the central nervous system from the peripheral nervous system.
  • Dorsal root ganglion axons are commonly considered “afferents” in the art.
  • the right and left spinal nerve pairs in humans form out of afferent sensory dorsal axons (the dorsal root) and motor ventral efferent axons (the ventral root).
  • dorsal root ganglion which is a group of cell bodies responsible for the transmission of sensory messages from receptors such as thermoreceptors, nociceptors, proprioceptors, and chemoreceptors, to the central nervous system for a response.
  • the cell bodies of dorsal root ganglia are separated by layers of satellite glial cells that inhibit the interaction between somas.
  • Dorsal root ganglion neurons are accepted in the art to be, or behave as pseudo-unipolar neurons, with one axon that bifurcates into two separate branches resulting in a distal process and proximal process.
  • dorsal root ganglia are active participants in the signalling process; they sense specific molecules and produce molecules needed to regulate the process (Ahimsadasan et al., StatPearls, 2020).
  • Dorsal root ganglia are relatively easily accessible from the exterior of the epidural space through the neuroforamina and from the epidural space to the outside.
  • dorsal root ganglion stimulation is enjoying increased popularity as neuromodulation therapy, having an effectiveness comparable to spinal cord stimulation in reducing certain pain conditions, including but not limited to chronic postsurgical pain, regional pain syndromes, and pain from failed back surgery syndrome (Liem, Progress in neurological surgery, 2015).
  • external nociceptors include nociceptors with endings localised in tissues such as skin, cornea, and mucosa.
  • Internal nociceptors in contrast have projections that are localised in or in near proximity of organs including but not limited to: muscles, joints, bladder, visceral organs, and the digestive tract.
  • the detection and transduction of noxious stimuli occurs in the peripheral terminal of the nociceptor.
  • Noxious stimuli are commonly categorised in the art as: thermal, mechanical, and chemical (Loeser et al., Pain, 2008).
  • Nociceptors may respond to a single category of noxious stimuli, more than one category of noxious stimuli (i.e.
  • the Prdml2 modulators as described herein may selectively act on nociceptors consisting of the group of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, polymodal nociceptors, or any combination thereof.
  • the Prdml2 modulator may act exclusively on sleeping nociceptors.
  • the Prdml2 modulators as described herein may affect pain sensation in a subject by modulation of the peripheral sensitisation.
  • Prdml2 modulator administration may induce a change in one or more groups of nociceptors as described herein from acting as a noxious stimulus detector to an inactive nociceptor, or alternatively change an inactive, silent, or defective nociceptor to a noxious stimulus detector.
  • the Prdml2 modulator may affect the function of A6 fiber axons, C fiber axons, or both.
  • A6 fiber axons conduct action potentials faster than C fiber axons (about 20 meter/second compared to about 2 meter/second), which is mainly due to light myelination of A6 fiber axons, while C fiber axons are not myelinated (Williams et al. Neuroscience, 2001).
  • A6 fiber axons attribute mainly to a first pain phase (i.e. an initial sharp pain sensation), while C fiber axons contribute mainly to a second pain phase (i.e. a more prolonged and less intense pain sensation).
  • the Prdml2 modulators as described herein may affect the first pain phase, the second pain phase, or both pain phases.
  • control or “baseline” conditions, levels, and/or activity intend to specify one or more comparative values (i.e. reference values) which may be used as a benchmark (i.e. standard) to assess any influence of aPrdm!2 modulator, or candidate Prdml2 modulator on Prdml2 activity.
  • Control conditions and baseline values may indicate a Prdml2 expression level or activity on the molecular level in absence of any Prdml2 modulator.
  • a control condition or baseline value may refer to a certain metric on the subject as a whole, a non-limiting example hereof being the quantification of hind paw licking by a rodent when not subjected to treatment comprising the administration of a Prdml2 modulator or candidate Prdml2 modulator.
  • a person skilled in the art readily appreciates that the above examples of baseline values and/or control conditions are non-limiting and is able to devise alternative yet equally appropriate control conditions or baseline values at any measurement level given a certain context.
  • behavioral methods to measure nociception in animals may be divided into stimulus-evoked (e.g. mechanical, heat, cold, etc.) and nonstimulus evoked methods.
  • stimulus-evoked e.g. mechanical, heat, cold, etc.
  • nonstimulus evoked methods e.g. mechanical, heat, cold, etc.
  • methods including the manual Von Frey test, the electronic Van Frey test, the Randall-Selitto test and variations of each of these tests may be used.
  • suitable nonlimiting methods to assess heat stimuli are the tail flick test, the Hargreaves test, and the thermal probe test. Routinely used tests for assessing cold stimuli are the cold plate test, the acetone evaporation test, and the cold plantar test.
  • nonstimulus evoked nociception can be quantified by parameters such as grimace scales, burrowing, wait bearing, gait analysis, or any combination thereof.
  • Relevant parameters relating to gait analysis in freely walking test animals include paw intensity (a measure of paw pressure or weight bearing), paw print parameters (e.g. toe spread, print length, print width, print area), dynamic parameters (e.g. stance phase, swing phase, duty cycle, stride length, swing speed) and regularity index (a measure of interlimb coordination).
  • Inflammatory responses are, in accordance with the examples of the present disclosure, routinely assessed by licking of the inflamed area, for example hind paw licking when said hind paw is inflamed.
  • Suitable control conditions differ depending on the experimental design of an assay.
  • a suitable baseline value may be derived from a point in time before Prdml2 modulator administration, and/or in a point in time after Prdml2 administration if the pain condition is or considered to be temporary and reversible.
  • the baseline value may be a measure of centre (non-limiting examples being a mean value or median value) in a control population of subjects, wherein the treatment population (i.e. the population subjected to Prdml2 modulator administration) may be the control population in a different point in time or a different subject population.
  • a Prdml2 modulator is a substance that induces a significant change vis-a-vis a suitable baseline value.
  • the Prdml2 modulator as described herein may alleviate pain in a subject by at least 1, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 as reported by the treated subject when using a self-reporting pain scale.
  • the self-reporting pain scale is the numeric rating scale (NRS 11), Stanford pain scale, or visual numeric scale.
  • the self-reported pain sensation experienced by a subject and expressed in a numeric value is halved by administration of the Prdml2 modulator.
  • the Prdml2 modulator may significantly alleviate pain in said subject as measured by objective quantitation methods including but not limited to functional magnetic resonance imaging (fMRI) and electroencephalography (EEG).
  • fMRI functional magnetic resonance imaging
  • EEG electroencephalography
  • the pain alleviation in a subject is assessed by at least two methods selected from the group consisting of: self-reporting scales, fMRI, and EEG, which are all techniques suitable to study or measure pain as indicated above.
  • the modulators as described herein may have an immediate or near immediate effect and/or a delayed effect on the pain sensation of the subject to which said modulator is administered.
  • the (near) immediate effect may markedly differ from the delayed effect of said modulator.
  • Certain modulators as described herein may be characterised by a dosage-dependent effect, while others may display a consistent effect over time as long as a certain threshold concentration in the target cell, tissue, or subject is equalled and/or surpassed.
  • treatment encompass both the therapeutic treatment of an already developed pain condition, such as the therapy of an already developed inflammatory pain, nociceptive pain, or neuropathic pain, as well as prophylactic or preventive measures to prevent an envisaged pain condition in a future point in time, wherein the aim is to prevent or lessen the chances of incidence of pain, such as to prevent occurrence, development and progression of an inflammatory pain, nociceptive pain, or neuropathic pain.
  • Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of pain, stabilised (i.e. not worsening) pain, delay or slowing of the pain sensation, amelioration or palliation of the pain condition, and the like.
  • Treatment can also indicate an increased quality of life when compared to the quality of life without Prdml2 modulator.
  • therapeutic treatment refers to treatments wherein the object, or goal, is to change a subjects body or a part of a subjects body from an undesired physiological state, disease or disorder, such as any pain condition described herein, to a desired state, such as a less severe state (e.g. amelioration or palliation), or even back to its normal, healthy state (e.g. restoring a pain-free or near pain-free status of a subject), to keep it (i.e. not worsening) at said undesired physiological status (e.g.
  • Measurable lessening includes any statistically significant decline in a measurable marker or symptom.
  • the terms encompass both curative pain treatments and pain treatments directed to reduce pain and/or slow progression and/or stabilise the pain condition over time.
  • prevention or “prevent” as used in the context of the invention refers to an aversion of manifestation of a pain condition in a subject, i.e. the establishment of preventive measures or prophylactic measures.
  • Preventive treatment refers to treatments wherein the object is to avoid a subject’s body showing a physiological pain response and/or avoid a subject’s perception of a pain sensation.
  • predicting generally refer to a statement, declaration, indication, anticipation or foretelling of a pain condition in a subject not (yet) showing any, or a limited, clinical indication of said pain condition, or more relevant in the context of the present invention a pain-inducing condition.
  • a prediction of a pain condition in a subject may indicate a probability, chance or risk that the subject will develop said condition, for example within a certain time period (expressed in minutes, hours, days, weeks, months, and/or years), or by a certain age.
  • This probability, chance or risk may be formulated or phrased as any suitable qualitative or quantitative expression, optionally including both an expected or predicted time interval and additionally a measure of the predicted severity of the pain condition or pain sensation.
  • this probability, chance, or risk may be indicated relative to a suitable control subject or subject population (such as, e.g. relative to a general, normal or healthy subject or subject population).
  • a suitable control subject or subject population such as, e.g. relative to a general, normal or healthy subject or subject population.
  • the probability, chance or risk that a subject will develop a pain condition may be advantageously indicated as increased or decreased, or as fold- increased or fold-decreased relative to a suitable control subject or subject population.
  • the term “prediction” of the conditions or pain-inducing diseases as taught herein in a subject may also be expressed as the subject having a 'positive' prediction of such, i.e. that the subject is at risk of having such (e.g. the risk is significantly increased vis-a-vis a control subject, control subject population, or an earlier examination of said subject).
  • the invention relates to a modulator of Prdml2 activity, for use in treatment or prevention of a pain condition in a subject.
  • the Prdml2 modulator is a Prdml2 activator. In further embodiments, the Prdml2 activator is a conditional Prdml2 activator.
  • the Prdml2 modulator is a Prdml2 inhibitor.
  • the Prdml2 inhibitor is a conditional Prdml2 inhibitor.
  • the Prdml2 modulator may have an opposite effect on Prdml2 activity in distinct temporal pain responses.
  • the Prdml2 modulator may have an activating effect on Prdml2 activity in the primary or first pain response and an inhibiting effect on Prdml2 activity in a subsequent pain response.
  • the Prdml2 modulator may have an inhibiting effect on Prdml2 activity in the primary or first pain response and an activating effect on Prdml2 activity in a subsequent pain response.
  • the Prdml2 modulator as disclosed herein may be used for any type of pain.
  • the Prdml2 modulator described herein is used for treatment of one or more occupational injuries.
  • the Prdml2 modulator described herein is used for treatment of disease-related pain or age-associated pain, preferably chronic disease-related pain or chronic age-related pain.
  • the Prdml2 modulator is used as a prophylactic, optionally in a subject, wherein said subject is envisaged to be or scheduled to be undergoing a medical procedure, such as but not limited to invasive medical procedures such as an operation.
  • the Prdml2 modulator as described herein may be used as a topical analgesic or systemic analgesic.
  • the Prdml2 modulator for use in treatment or prevention of a pain condition in a subject, in support of a distinct analgesic known in the art is intended.
  • the effect of the Prdml2 modulator is juxtaposed to the effect of the distinct analgesic.
  • administration of the Prdml2 modulator exerts a synergistic effect with said analgesic.
  • Prdml2 activity as used throughout the present disclosure is to be interpreted according to its broadest interpretation. Hence, Prdml2 activity may intent to denote any parameters of Prdml2 that may influence or alter the nett resulting activity of Prdml2 in a subject, preferably in a nociceptor of said subject and therefore includes but is not limited to parameters such as Prdml2 expression (subject to both transcription and translation rates), half-life, functionality, localisation, capacity to bind to other proteins (including but not limited to G9a and histones), or any combination thereof.
  • a Prdml2 modulator is considered a Prdml2 inhibitor when said modulator negatively effects the activity of Prdml2.
  • a Prdml2 modulator as disclosed herein is considered a Prdml2 inhibitor when said modulator gives rise to a measured Prdml2 activity value that is lower than the baseline value.
  • inhibitor is to be interpreted by its commonly accepted meaning in the art, and the associated verb “to inhibit” may be annotated by non-limiting synonyms such as to reduce, to diminish, to render void, to nullify, to negate, to mitigate, to downregulate, to turn off, to decrease, to reduce, to minimise, to lessen, to weaken, to attenuate, to constrain, to hinder, to impede, to deter, to stop, etc. the activity of a certain target molecule, here Prdml2.
  • Prdml2 inhibitors as envisaged herein may inhibit Prdml2 activity either partially (i.e. to a certain degree) or completely.
  • a Prdml2 modulator is considered a Prdml2 activator when said modulator positively affects the activity of Prdml2.
  • a Prdml2 modulator as described herein is considered a Prdml2 activator when said modulator leads to a Prdml2 activity value that is higher than the baseline value.
  • activator is to be interpreted by its commonly accepted meaning in the art, and the associated verb “to activate” may be annotated by non-limiting synonyms such as to induce, to actuate, to exaggerate, to amplify, to elevate, to upregulate, to turn on, to increase, to ameliorate, to enhance, to improve, to stimulate, to effectuate, etc. the activity of a certain target molecule, here Prdml2.
  • a Prdml2 inhibitor may have a positive or negative effect on the resulting pain sensation, optionally even differing in time within one pain response.
  • a Prdml2 activator may also have a positive or negative effect on the resulting pain sensation, optionally even differing in time within one pain response.
  • the Prdml2 modulator alters the expression of Prdml2 in nociceptors of a subject.
  • the alteration in expression level of Prdml2 may be limited in time e.g. while the Prdml2 modulator is present in said nociceptors or permanent, i.e. until a point in time wherein no detectable levels of Prdml2 modulator are present in said nociceptors).
  • the Prdml2 modulator may alter Prdml2 expression by binding or modification of the Prdml2 promoter sequence.
  • a “promoter” as defined herein is a region of DNA that initiates transcription of a particular gene and hence enables a gene to be transcribed.
  • a promoter is recognised by RNA polymerase, which then initiates transcription.
  • a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase.
  • a promoter sequence may also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster.
  • the enhancer(s) while typically at the 5’ end of a coding region, can also be separate from a promoter sequence, e.g. can be within an intronic region of a gene or 3’ to the coding region of the gene.
  • Promoters may be located in close proximity of the start codon of genes, in preferred embodiments on the same strand and typically upstream (5’) of the gene. Promoters may vary in size and are preferably from about 100 to 1000 nucleotides long, although this length is not to be interpreted as a strict limitation.
  • the Prdml2 modulator may conditionally alter the expression of Prdml2 in nociceptors of a subject.
  • a person skilled in the art is aware of inducible expression and/or inducible activation systems, the Tet-on and Tet-off systems being typical, yet non-limiting examples hereof (e.g. Das et al. Current Gene therapy, 2016).
  • the Prdml2 modulators as described may affect the Prdml2 expression in nociceptors by impacting the subcellular localisation of Prdml2 in nociceptors, optionally in a conditional manner.
  • the Prdml2 modulator may sequester Prdml2 away from the nucleus of a nociceptor (inducing an extranuclear or mainly extranuclear localisation), resulting in decreased expression of Prdml2 in the nucleus of said nociceptor.
  • the Prdml2 modulator increases the expression of Prdml2 in nociceptors of a subject, preferably by at least about 10%, at least about 25%, at least about 35%, more preferably at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator.
  • the Prdml2 modulator increases the expression of Prdml2 in nociceptors in a subject by at least 1.5-fold, at least 2-fold, at least 5-fold, or even at least 10-fold when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator.
  • the increased expression of Prdml2 as described above may be vis-a-vis a reference Prdml2 expression value of a group of subjects that are not administered said modulator, e.g. a mean or median value.
  • the reference value is the Prdml2 expression level during said pain sensation or pain response before Prdml2 administration.
  • the increased Prdml2 expression occurs selectively in sleeping nociceptors.
  • the Prdml2 modulator may increase the half-life of Prdml2 by binding to Prdml2 or inhibiting proteasomal degradation of Prdml2 in said nociceptors.
  • the Prdml2 modulator may increase the Prdml2 expression by upregulation of transcription and/or translation of Prdml2.
  • Prdml2 modulators that increase Prdml2 expression are described further below. It is to be understood that Prdml2 modulators that increase Prdml2 expression may for examples also be exogenous oligonucleotide sequences that express additional Prdml2 protein in the nociceptors, or even functional mutants of Prdml2 with increased intrinsic potency, as discussed further below.
  • the Prdml2 modulator reduces the expression of Prdml2 in nociceptors of a subject, preferably by at least about 10%, at least about 25%, at least about 35%, more preferably at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably reduces the expression of Prdml2 in nociceptors of a subject with about 100%, i.e. to a level below the quantification and/or detection limit by suitable techniques for expression measurement available in the art.
  • the reduction in expression level is expressed in view of the Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator.
  • the reduction of Prdml2 expression occurs selectively in sleeping nociceptors.
  • the mode of action of the Prdml2 modulator may rely on binding with the Prdml2 protein or Prdml2 RNA, or alternatively by binding and/or modification of the Prdml2 encoding genomic sequence.
  • the Prdml2 modulator may act as a so-called road block for Prdml2 expression or induce a change in the genomic Prdml2 sequence or Prdml2 promoter sequence which reduces or eliminates expression of a (fully) functional Prdml2 protein.
  • the half-life of Prdml2 may be shortened by the Prdml2 modulator.
  • suitable Prdml2 modulators that decrease Prdml2 expression are described further below.
  • Suitable techniques for expression measurement include but are not limited to (reverse transcription polymerase chain reactions (rt-PCR), qPCR, and/or enzyme-linked immunosorbent assays (ELISA).
  • rt-PCR reverse transcription polymerase chain reactions
  • qPCR qPCR
  • ELISA enzyme-linked immunosorbent assays
  • the Prdml2 modulator for use in treatment or prevention of a pain condition in a subject, wherein the modulator alters the Prdml2-mediated G9a recruitment to histone H3.
  • the human G9a protein has been described in detail in the art (e.g.
  • human G9a is annotated under NCBI Genbank Gene ID: 10919 and the canonical human G9a amino acid sequence is by means of example reproduced below (SEQ ID NO: 29):
  • the Prdml2-mediated G9a recruitment to histone H3 is reduced by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 99% or even about 100%.
  • the Prdml2-mediated G9a recruitment to histone H3 is eliminated or reduced to levels below quantification or even detection by methods known to a skilled person.
  • the above reduction in Prdml2-mediated G9a recruitment occurs in one or more classes (i.e.
  • the reduction in G9a recruitment is selectively, or even exclusively observable in sleeping nociceptors.
  • the Prdml2- mediated G9a recruitment to histone H3 is increased by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, most preferably at least about 100%.
  • the Prdml2-mediated G9a recruitment to histone H3 is increased by 1.5-fold, 2-fold, 2.5-fold, 5- fold, 10-fold, or higher.
  • the above described increase in Prdml2- mediated G9a recruitment occurs in one or more classes (i.e. groups) of nociceptors consisting of the group of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, and polymodal nociceptors.
  • the increase in G9a recruitment is selectively, or even exclusively observable in sleeping nociceptors.
  • G9a histone H3 methylation by G9a is mediated by recruitment of G9a to histone H3 by Prdml2, and it is established that this recruitment occurs by binding, i.e. physical interaction of Prdml2 with G9a (e.g. Yang and Shinkai, Cell Structure and Function, 2013).
  • a skilled person can assess the level of G9a recruitment by known means to detect protein-protein interactions, which include non-limiting exemplary assays such as yeast- two-hybrid assays, complementation assays, immunofluorescence assays such as FiSH, etc (Rao, International Journal of Proteomics, 2014).
  • the Prdml2 modulator affects the expression level of the canonical Prdml2 protein in a subject, preferably the human Prdml2 protein as identified by SEQ ID NO: 1, then a skilled person may reasonable assume that G9a recruitment to H3 will be correlated to Prdml2 expression, optionally linearly correlated.
  • G9a recruitment to H3 will be correlated to Prdml2 expression, optionally linearly correlated.
  • the recruitment of G9a to histone H3 is modified by modifying the physical interaction between Prdml2 and G9a
  • the (changed) affinity of Prdml2 for G9a will determine the extent of, and impact on, Prdml2- mediated G9a recruitment.
  • the Prdml2 modulator alters the methylation status of histone H3, preferably at position Lysine 9 (K9). In preferred embodiments the Prdml2 modulator alters the methylation status of H3K9.
  • Methods to evaluate methylation at specific locations are readily available in the art (reviewed in Kurdyukov and Bullock, Biology, 2016). “Methylation of H3K9” as referred to herein may indicate a change in monomethylation status (H3K9mel), dimethylation (H3K9me2) or a combination thereof.
  • a general method suitable to assess methylation routinely used in the art is bisulfite sequencing.
  • At least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even about 100% of H3K9 is methylated in nociceptors of a subject in the effective temporal time window after administration of the Prdml2 modulator.
  • the at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, most preferably at least about 95%, or even about 100% of H3K9 is methylated in nociceptors and/or dorsal root ganglia of a subject in the effective temporal time window after administration of the Prdml2 modulator.
  • the Prdml2 modulator increases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, more preferably at least about 100%, when compared to histone H3 methylation, preferably H3K9 methylation in respectively nociceptors or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
  • the Prdml2 modulator decreases the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 by at least about 10%, at least about 25%, preferably at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even reduces said transcription rate by 100% (i.e.
  • the transcription rate of all the genes CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, and optionally PRDM12 is reduced by at least about 10%, preferably at least about 25%, preferably at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even most preferably reduces said transcription rate by 100%,
  • the Prdml2 modulator decreases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, more preferably at least about 100%, when compared to histone H3 methylation, preferably H3K9 methylation in respectively nociceptors or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
  • the Prdml2 modulator increases the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, NTS by at least about 10%, preferably at least 25%, at least 35%, preferably at least about 50%, at least about 60%, more preferably at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%, when compared to their expression levels in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator.
  • the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene is at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, NTS is at least about 1.5-fold, at least about 2- fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold increased when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator.
  • the Prdml2 modulator decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or most
  • nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, more preferably at least about 95%, or most preferably even completely inhibits said transcription rate (i.e.
  • the change in methylation status and related hereto the transcription rate may be specific for one or more classes of nociceptors selected from the group consisting of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, and polymodal nociceptors.
  • the change in H3K9 methylation induced by the Prdml2 modulator occurs at least in, or even occurs exclusively in sleeping nociceptors.
  • transcription rate as used herein is to be interpreted in line with the generally accepted meaning thereof in the art.
  • any of the above embodiments concerning the increase or decrease of one or more genes is associated with an increase or decrease of their expression level in the modulated nociceptors or dorsal root ganglia.
  • the transcription rate and expression level may be positively correlated, preferably linearly positively correlated or are alternatively characterised by an exponential correlation.
  • the term “transcription rate” may relate to the nascent transcription rate and/or the rate of synthesis of mature mRNA (Perez-Ortin, BioEssays, 2013).
  • Prdml2 activators as disclosed herein or as identified by the methods disclosed herein will generally be of use for treating pain caused or aggravated by reduced or lost Prdml2 expression.
  • Preferred examples of such pain conditions are pain condition induced by inflammation or itch, preferably inflammatory pain.
  • said pain condition could be seen as a TRPV1 -related pain condition, more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders (which include e.g. bladder pain syndrome (previously interstitial cystitis), irritable bowel syndrome (IBS) and fibromyalgia) and musculoskeletal pain.
  • a TRPV1 -related pain condition more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders (which include e.g. bladder pain syndrome (previously interstitial cystitis), irritable bowel syndrome (IBS) and fibromyalgia) and musculoskeletal pain.
  • IBS irritable bowel syndrome
  • fibromyalgia fibromyalgia
  • Prdml2 inhibitors as disclosed herein or as identified by the methods disclosed herein will generally be of use for treating pain caused or aggravated by increased Prdml2 expression.
  • Preferred examples of such pain conditions are pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches.
  • pain may be classified in two categories, i.e. acute and chronic pain.
  • the Prdml2 modulators may be used for both acute and chronic pain, as the Prdml2 modulators described herein may be used to treat any pain condition.
  • different classifications of pain and pain perceptions have been defined in the art, and these pain responses and pain conditions are well known to a person skilled in the art and when referred to herein.
  • nociceptive pain may be an acute pain and a somatic pain.
  • the pain condition is selected from the group consisting of: acute pain, chronic pain, somatic pain, visceral pain, neuropathic pain, nociceptive pain, inflammatory pain, neuropathic pain, radicular pain, or any combination thereof.
  • the pain condition is a nociceptive pain condition, an inflammatory pain condition, a neuropathic pain condition, or any combination thereof.
  • said pain condition is nerve injury, skin inflammation, joint inflammation, or allergy-related inflammation.
  • the pain condition is an inflammation-induced pain condition wherein said pain is related to, or caused by an inflammatory disease having a main inflammatory localisation selected from the group consisting of: the nervous system, the cardiovascular system, the respiratory system, the digestion system, the integumentary system, the musculoskeletal system, the urinary system, the reproductive system, the reproductive system, the endocrine system, the lymphatic system, or any combination thereof.
  • the Prdml2 modulator is used for treating or preventing allodynia and hyperalgesia in a subject.
  • Preferred inflammatory diseases related to or causative of pain conditions that benefit from any of the aspects of the invention described herein are selected from the group consisting of: encephalitis, myelitis, meningitis, arachnoiditis, neuritis, dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, otitis externa, otitis media, labyrinthitis, mastoiditis, carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, capillaritis, sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis, mediastinitis,
  • parotitis parotitis
  • cheilitis pulpitis, gnathitis, esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis, proctitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, dermatitis, folliculitis, cellulitis, hi dradenitis, arthritis, dermatomyositis, myositis, synovitis (i.e.
  • Chronic pain is defined in the art by a sudden onset which is caused by a specific noxious stimulus, of which non limiting examples are given below when defining “nociceptive pain”.
  • the arbitrary cut-off to define “acute” in the field of pain management is 6 months.
  • chronic pain is commonly described as an ongoing pain that continues after 6 months, wherein the pain sensation perceived by the subject may be constant, increasing, or intermittent in nature.
  • Non-limiting examples hereof are headache, arthritis, cancer, nerve pain, back pain, and fibromyalgia pain.
  • certain chronic pain conditions are representative for a disease state or may be considered a disease state (Grichnik and Ferrante, The Mount Sinai Journal of Medicine, 1991).
  • nociceptive pain is used herein and in the art as a collective name to indicate pain that arises, or is activated in response to underlying tissue damage, injury or a stimulus that is capable of causing an injury (Dubin and Patapoutian, The Journal of Clinical Investigation, 2010).
  • Subject experiencing nociceptive pain typically describe the pain as sharp, achy, throbbing, or a combination thereof.
  • the final pain sensation or pain sensation may depend on the local injury site environment that may alter the specific characteristics of nociceptors, central connections and the autonomous nervous system.
  • the peripheral nociceptors are directly activated by a stimulus and transmit a signal (i.e.
  • nociceptive pain has a defined cause, wherein removal of said cause or recovery from a causative injury resolves the pain.
  • Acute nociceptive pain lasts for less 6 months.
  • Non-limiting examples of nociceptive pain are sprains, strains, contusions, post-operative pain, tissue trauma, bum pain, vascular pain, and ischemic pain. While nociceptive pain is by definition a distinct pain from inflammatory pain, the two pain conditions may often co-occur by inflammation of the injury site.
  • Neuroneuropathic pain is a pathological state of pain that is triggered by changes of the somatosensory nervous system. Where nociceptive pain and inflammatory pain may be considered protective responses, neuropathic pain is perceived throughout the art as a disease state wherein pain occurs spontaneously or apparent spontaneously in response to innocuous stimuli. Neuropathic pain is indicative of a dysfunction of the nervous system which may include defect in circuitry, signal transmission and/or altered action potentials. Neuropathic pain is commonly described by patients as a burning or shooting sensation that optionally may fluctuate in intensity over time (Costigan et al., Annual Review of Neuroscience, 2009). Neuropathic pain may arise as a consequence of, or have an increased chance to develop following a series of diverse (disease) conditions.
  • Non-limiting conditions associated with (the onset of) neuropathic pain include: addictions (e.g. alcoholism), limb removal (e.g. amputation), treatment-induced pain (e.g. chemotherapy), metabolic disorders (e.g. diabetes), infection (e.g. herpes infection, or HIV infection), neurodegenerative diseases (e.g. multiple sclerosis), damage to the nervous system (e.g. nerve compression such as carpal tunnel syndrome or herniated disc, nerve trauma), sexual transmitted infections (e.g. syphilis), hormonal disturbance (e.g. hypothyroidism, hyperthyroidism), post-herpetic neuralgia (e.g.
  • addictions e.g. alcoholism
  • limb removal e.g. amputation
  • treatment-induced pain e.g. chemotherapy
  • metabolic disorders e.g. diabetes
  • infection e.g. herpes infection, or HIV infection
  • neurodegenerative diseases e.g. multiple sclerosis
  • damage to the nervous system e.
  • Varicella zoster virus (often referred to in the art as shingles)), chronic physical pain (e.g. back pain), cancer neuropathy (e.g. multiple myeloma), phantom limb pain, and channel opathies.
  • Chronic physical pain e.g. back pain
  • cancer neuropathy e.g. multiple myeloma
  • phantom limb pain e.g. multiple myeloma
  • Channelopathies have been described in detail in the art and a skilled person is therefore capable of identifying this heterogenous group of disorders that are the result of ion channel dysfunction in the membranes and cellular organelles (Kim, Korean Journal of Pediatrics, 2014).
  • Neuropathic pain is associated with a decreased quality of life, including amongst others sleep impairment, lost workdays, and psychological distress.
  • Allodynia and “hyperalgesia” are two specific pain conditions that are associated with, or considered related to neuropathic pain and have a generally accepted meaning within the technical field (Jensen and Finnerup, The Lancet Neurology, 2014). A skilled person readily appreciates that allodynia refers to pain due to a stimulus that does not usually provoke pain, and hyperalgesia refers to increased pain (sensation) from a stimulus that usually provokes pain.
  • inflammatory pain indicates both inflammatory pain as such and pain arising from inflammation in a subject. Inflammation is associated with the release of several immune mediators including but not limited to bradykinin, histamine, 5- hydroxytryptamine, adenosine triphosphate, and nitric oxide. Further downstream activation by these mediators leads to the recruitment of immune cells that release additional molecules such as cytokines, chemokines that are considered algogenic. During inflammation, the ongoing exposure to these ligands will cause hypersensitivity of sensory neurons at or near the site of initial insult (known in the art as peripheral sensitisation). Continued peripheral sensitisation may in turn lead to sensitisation of the (near) complete nociceptive pathway (i.e. central sensitisation).
  • “Radicular pain” as used herein is caused by irritation of the sensory root or dorsal root ganglion of a spinal nerve and generally is used in the art to indicate back pain that is caused by a combination of compression sensitising the nerve root to mechanical stimulation, stretching, and a chemically mediated noncellular inflammatory reaction (often as a consequence of a herniated disc in the subject). Radicular pain is typically perceived as sharp, shooting, piercing, or stabbing pain down the length of the leg. Depending on the severity, loss of sensation and/or loss of motor function may be observed (Dydyk and Das, StatPearls, 2020; Govind, Australian Family Physician, 2004). The Prdml2 modulators as described herein may be used to treat or prevent cervical radicular pain, lumbar radicular pain, spinal radicular pain, or a combination thereof.
  • Somatic pain indicates pain as the result of activation of pain receptors in tissues and may be further stratified into superficial somatic pain and deep somatic pain, which both can be constant or intermittent in nature.
  • Superficial pain occurs by activation of nociceptors in for examples skin, mucus, and mucous membranes. Common injuries such as skin cuts may be considered a non-limiting example of superficial somatic pain.
  • deep somatic pain is typically described as a localised pain which is considered as aching, gnawing, throbbing, or cramping by the subject.
  • Deep somatic pain occurs when stimuli active pain receptors deeper in the body of a subject such as those part of tendons, joints, bones, and muscles. Typical deep somatic pain is often experienced by but not limited to subjects having cancer and bone metastasis (Carver and Foley, Holland-Frei Cancer Medicine, 6 th edition).
  • visceral pain is caused by activation of nociceptors in for example the cardiovascular, respiratory, gastrointestinal, and genitourinary systems and is typically described by subjects as a dull, deep, squeezing pressure or colicky pain. Visceral pain is often difficult to localise and is often referred to a distinct and typically superficial structure (i.e. referred pain) due to the dual innervation of somatic and visceral structures by common afferent fibers that converge in the dorsal horn in the spinal cord. There is no pathology or cause for experiencing the pain at the referred somatic localisation. The referred pain is sharper in nature and less likely to be co-occurring with autonomic or emotional distress.
  • Prdml2 modulator as described herein may broadly refer to any chemical (e.g. inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g. biological macromolecule), a combination or mixture thereof, a sample of undetermined composition, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues.
  • modulators include nucleic acids, oligonucleotides, ribozymes, peptides, polypeptides, proteins, peptidomimetics, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof.
  • modulator may denote a “therapeutic agent”, “drug”, or “active pharmaceutical ingredient”, useful for or used in the treatment, cure, prevention, or diagnosis of a pain condition as taught herein.
  • the Prdml2 modulator is selected from the group consisting of: Prdml2 binding molecules, Prdml2 or a polynucleotide encoding Prdml2, a functional fragment of Prdml2 or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene targeting system, or any combination thereof.
  • the PRDM12 gene targeting system is a PRDM12 antisense agent or a PRDM12 gene editing system.
  • Prdml2 binding molecule refers to any protein or non-protein chemical molecule that binds to Prdml2 (either the PRDM12 gene, PRDM12 RNA, or Prdml2 protein), preferably human Prdml2.
  • the Prdml2 modulator is a Prdml2 or PRDM12 binding molecule, selected from the group consisting of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a aptamer, a aptamer, a aptamer, a photoaptamer, a apta a apta a aptamer, a aptamer, a photoaptamer, a aptamer, a aptamer, a aptamer, a aptamer, a aptamer, a photoaptamer, a aptamer, a aptamer, a aptamer, a aptamer, a photoaptamer, a aptamer, a aptamer, a aptamer, a aptamer, a apt
  • the Prdml2 modulator is selected from the group consisting of a chemical substance, a Prdml2- binding antibody, a PRDM12 RNA antisense agent, and a PRDM12 gene editing system.
  • the Prdml2 modulator specifically recognises and/or binds to one or more specific Prdml2 C-terminal polyalanine stretch variants, Prdml2 gene isoforms, or nucleotides encoding said variants and/or isoforms.
  • Prdml2 modulator specifically recognises and/or binds to one or more specific Prdml2 C-terminal polyalanine stretch variants, Prdml2 gene isoforms, or nucleotides encoding said variants and/or isoforms.
  • binding preferably indicates the binding of a first moiety to a second moiety, for example the binding of a transcription factor to a DNA sequence, the binding of a protein to another protein, the binding of a chemical substance to Prdml2 protein, etc.
  • hybridizing is indicative for a specific interaction between two nucleotide sequences based on sequence complementarity.
  • a suitable synonym for “hybridizing” is “annealing”, as appreciated by a person skilled in the art.
  • a specific yet non-limiting example of hybridizing in the context of the invention is the hybridisation of an antisense agent to PRDM12 RNA.
  • nucleic acid sequences that are suitable for hybridisation are not limiting, and therefore encompass DNA-DNA hybridisation, DNA-RNA hybridisation, and RNA-RNA hybridisation.
  • the term “specifically” in the context of “binding”, “hybridizing to” or “targeting” implies that the modulator binds to, influences, or targets the Prdml2 protein sequence or PRDM12 DNA/RNA sequence, without substantially binding or hybridizing to the sequence of another protein or DNA/RNA.
  • binding or “specifically interacting” is meant that a modulator of Prdml2 activity binds to, influences, or targets Prdml2 or a nucleotide sequence encoding Prdml2 or associated sequences (e.g.
  • an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g. at least about 1000-fold or more greater, at least about lxl0 4 -fold or more greater, or at least about lxl0 5 -fold or more greater, than its affinity for a non-target molecule.
  • the binding or interaction between the modulator of Prdml2 activity and Prdml2 may be covalent (i.e. mediated by one or more chemical bonds that involve the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e. mediated by non-covalent forces, such as for example, hydrogen bridges, dipolar interactions, van der Waals interactions, etc.).
  • KA affinity constant
  • KA Determination of KA can be carried out by methods known in the art, such as for example, using equilibrium dialysis and Scatchard plot analysis (Attie and Raines, Journal of Chemical Education, 1995).
  • the binding of a modulator of Prdml2 activity as described herein to Prdml2 and the affinity and specificity of said binding may be determined by any methods known in the art. Non-limiting examples thereof include co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, label transfer, phage display, proximity ligation assay (PLA), Tandem affinity purification (TAP), in silico docking and calculation of the predicted Gibbs binding energy and competition binding assays (e.g. review in Hunter and Cochran, Methods in Enzymology, 2016).
  • the chemical substance is an organic molecule, preferably a small organic molecule.
  • small molecule refers to organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, e.g. up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g. up to about 900, 800, 700, 600 or up to about 500 Da.
  • antibody is used herein in its broadest sense and hence generally refers to any immunologic binding agent, such as a whole antibody or antibody fragments, including without limitation a chimeric, humanised, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest.
  • the term antibody thereby includes a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanised antibody, a human antibody, or an immunologically effective fragment of any of these. The term thus specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g.
  • antibody is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g. a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo.
  • CDR complementarity-determining region
  • the term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g. birds and mammals.
  • the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant.
  • the antibodies may be human, murine (e.g. mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g. cynomolus monkeys), camel (e.g. Camelus bactrianus and Camelus dromaderius) also including camel heavy-chain antibodies, llama (e.g. Lama paccos, Lama glama or Lama vicugna) also including llama heavy-chain antibodies, or horse.
  • llama e.g. Lama paccos, Lama glama or Lama vicugna
  • antibody as used herein also encompasses “chimeric antibodies” which originate from at least two animal species. More specifically, the term “chimeric antibody” or “chimeric antibodies” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, non-human primate, canine, equine, or feline constant regions.
  • Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (Morrison et al., PNAS USA, 1984). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibody-producing DNA from another species, e.g. human.
  • the modulator of Prdml2 activity is a Prdml2 affinity ligand such as an antibody
  • affinity ligand recognises and binds to at least one epitope of Prdml2, preferably an epitope unique for Prdml2 within the relevant proteomic background, a preferred proteomic background being the human proteome.
  • epitope includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • An antibody is said to specifically bind an antigen when it preferentially recognises its target antigen in a complex mixture of proteins and/or macromolecules.
  • binding region indicates a certain site, part, domain or stretch of amino acid residues that is responsible for binding to an antigen of interest.
  • binding region essentially consists of specific amino acid residues of the affinity ligands such as antibodies described herein, which residues are in contact with Prdml2.
  • the term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an antibody) molecule can bind.
  • the specificity of an antigen-binding protein can be determined based on affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD) is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD).
  • affinity can be determined in a manner known per se, depending on the specific antigen of interest.
  • Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an antibody) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.
  • antigenbinding proteins such as antibodies will bind with a dissociation constant (KD) of 10' 5 to 10’ 12 moles/liter (M) or less, and preferably 10' 7 to IO 42 moles/liter (M) or less and more preferably 10' 8 to IO 42 moles/liter, and/or with an association constant(KA) of at least 10 7 M’ ⁇ preferably at least 10 8 M 4 , more preferably at least 10 9 M 4 , such as at least 10 12 M 4 .
  • KD dissociation constant
  • KD dissociation constant
  • M moles/liter
  • M moles/liter
  • KA association constant
  • Any KD value greater than 10 4 M is generally considered to indicate non-specific binding.
  • an antibody will bind to the desired antigen with an KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.
  • a full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds.
  • the amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
  • CDRs complementarity determining regions
  • the carboxy -terminal portion (i.e. C-terminal portion) of each chain defines a constant region primarily responsible for effector function.
  • the CDRs are interspersed with regions that are more conserved, termed framework regions (FR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.”
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well- known Kabat numbering convention, which refers to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, 1991 ).
  • Kabat numbering convention refers to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody.
  • Light chains are classified as kappa or lambda, and are characterised by a particular constant region as known in the art.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively.
  • IgG antibodies can be further divided into subclasses, e.g. IgGl, IgG2, IgG3, IgG4.
  • Each heavy chain type is characterised by a particular constant region with a sequence well known in the art.
  • the Prdml2 binding molecule is an antibody selected from the group consisting of: IgA, IgD, IgE, IgG and IgM antibodies, preferably an IgG class antibody.
  • the antibody may be a polyclonal antibody, e.g. an antiserum or immunoglobulins purified there from.
  • the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies preferably exist in a homogeneous or substantially homogeneous population.
  • Monoclonal antibodies and antigenbinding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g. CDR- grafting, or combinations of such technologies, or other technologies known in the art.
  • monoclonal antibodies may be made by the hybridoma method (initially described by Kohler et al. Nature, 1975), or may be made by recombinant DNA methods. Monoclonal antibodies may also be made using phage antibody libraries (Clackson et al., Nature, 1991).
  • antibody fragment or “antigen -binding moiety” comprises a portion or region of a full length antibody, generally the antigen binding or variable domain thereof.
  • antibody fragments include Fab, Fab', F(ab)2, Fv , scFv fragments, single domain (sd)Fv, such as VH domains , VL domains and VHH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g. dibodies, tribodies, and multibodies.
  • the above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning.
  • antigen-binding portion or “antigen-binding region” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 1989); which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 1988).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are known to a person skilled in the art and are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger et al., PNAS, 1993).
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas, 1995) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., Molecular Immunology, 1994).
  • Antibody portions such as Fab and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques.
  • the Prdml2 antibody fragment may be a Nanobody®.
  • the terms “Nanobody®” and “Nanobodies®” are trademarks of Ablynx NV (Belgium).
  • the term “Nanobody” is well-known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a naturally occurring heavy-chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanisation” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanised VHH domain; (4) by "camelisation” of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelised VH domain; (5) by "camelisation”
  • the amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as “Framework region 1" or “FR1”; as “Framework region 2" or “FR2”; as “Framework region 3" or “FR3”; and as “Framework region 4" or “FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's”, which are referred to in the art as "Complementarity Determining Region For "CDR1”; as “Complementarity Determining Region 2" or “CDR2”; and as “Complementarity Determining Region 3" or “CDR3", respectively.
  • CDR's complementary determining regions
  • the total number of amino acid residues in a Nanobody can be in the region of 110-120, and preferably 112-115. It should however be noted that parts, fragments, analogs or derivatives of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.
  • variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”).
  • VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins.
  • VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein.
  • the anti-Prdm!2 antibody fragment is an domain antibody (dAb) (described in e.g. Ward et al., Nature, 1989).
  • Single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", as described in Streltsov et al., Protein Science, 2005).
  • the antibody or antibody fragment may be multispecific (such as a bispecific, trispecific, etc. antibody) comprising at least two (such as two, three, etc.) binding sites, each directed against a different antigen or antigenic determinant.
  • the therapeutic agent may be a dual variable domain immunoglobulin (DVD-IgTM) (described in e.g. DiGammario et al., Methods in Molecular Biology, 2012).
  • the anti-Prdm!2 antibody may be a “fully human antibody”.
  • the term “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Fully human antibodies may be derived from phage human antibody libraries as described above, or they may be obtained through immunisation of transgenic mice which have been engineered to replace the murine immunoglobulin encoding region as described in Lonberg and Husznar 1995 (Int. Rev. Immunol. 13 (1): 65-93). Fully human antibodies that are made using phage display are preferably produced by recombinant expression in a human cell line resulting in antibodies with a human glycosylation pattern. Non-limiting examples of fully human antibodies are HuCAL® antibodies (Morphosys).
  • the genetic information for constructing a HuCAL® antibody is extracted from the HuCAL® antibody library (Morphosys) and introduced into human PER.C6® cells in the form of a vector (i.e. transfection).
  • the transfected cells translate the genetic information into protein.
  • the protein is further modified by glycosylation and the resulting antibody molecule is finally secreted by the cells into the culture medium.
  • the term antibody as used further encompasses “humanised antibodies”, which are antibodies derived from non-human species whose protein sequence have been modified so as to increase their similarity to antibodies produced naturally in humans. More particularly, the term “humanised antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g.
  • VH and/or VL sequence has been altered to be more “human-like”, i.e. more similar to human germline variable sequences.
  • One type of humanised antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.
  • the humanised antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody.
  • a humanised antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e. donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanised antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a humanised antibody may contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanised antibody may only contain a humanised light chain, or a humanised heavy chain.
  • An exemplary humanised antibody contains a humanised variable domain of a light chain and a humanised variable domain of a heavy chain.
  • humanised antibodies may be derived from conventional antibodies (i.e.
  • an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds) from the family Camelidae, in particular from the llama (e.g. Lama paccos, Lama glama or Lama vicugna), whose variable domains exhibit a high degree of amino acid sequence identity with the variable domains of human antibodies.
  • llama e.g. Lama paccos, Lama glama or Lama vicugna
  • a suitable platform for the production of such humanised antibodies is the SIMPLE AntibodyTM platform (ArGEN-X, WO 2011080350).
  • the animals to be immunised may include any animal species, preferably warmblooded species, more preferably vertebrate species, including birds and mammals.
  • the antibody capable of modulating Prdml2 activity is a bird antibody, preferably wherein said antibody is selected from the group consisting of chicken, turkey, goose, duck, guinea fowl, quail, or pheasant antibody.
  • the antibody capable of modulating Prdml2 activity is a mammalian antibody, preferably wherein said antibody is selected from the group consisting of: human, murine (e.g. mouse, rat), porcine, donkey, rabbit, goat, sheep, guinea pig, camel, llama or horse antibody.
  • host cell and “host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals.
  • Contemplated as host organisms or cells for the production of antibodies include inter alia unicellular organisms, such as bacteria (e.g. E. coli), and (cultured) animal cells (e.g. mammalian cells or human cells).
  • bacteria e.g. E. coli
  • animal cells e.g. mammalian cells or human cells.
  • Bacteria are particularly suitable for the production of antibody fragments with a simple structure.
  • the bacterial cells may be transformed with at least two nucleic acids each encoding a different portion of the antibody fragment or the immunoglobulin, e.g. the heavy chain or the light chain, as described in WO 2009021548 for full-length immunoglobulins.
  • the genetic information encoding the antibody is read and translated into a protein.
  • the resulting antibodies accumulate in the periplasmic space and can be harvested upon lysis of the bacterial cells. A further separation step may be performed to purify the antibodies.
  • WO 2009021548 describes an E.
  • Coli-based secretion system wherein the bacteria release the antibodies in the surrounding culture medium due to the introduction of a signal sequence into the antibody encoding construct.
  • This enables the easy and convenient purification of the antibodies from the cell culture medium.
  • An exemplary mammalian cell line that can be used for the production of antibodies is the Chinese hamster ovary (CHO) cell line.
  • An exemplary human cell line suitable for the production of antibodies includes the PER.C6® cell line as deposited under EC AC no. 96022940. Particularly preferred for the production of fully human antibodies are human cell lines due to their capacity to produce antibodies that contain a human glycosylation pattern.
  • PRDM12 gene targeting system encompasses both PRDM12 antisense agents and PRDM12 gene editing systems.
  • the Prdml2 modulator is an antisense agent or a gene editing system
  • the modulator is directed to a portion of a nucleotide sequence encoding Prdml2 in a genomic sequence of a subject.
  • the modulator of Prdml2 is an antisense agent or a gene editing system
  • the inhibitor is directed to a portion of the PRDM12 promoter sequence.
  • the inhibitor comprises a nucleotide sequence that has a sequence identity of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% compared to a portion of a naturally occurring genomic sequence of PRDM12, SEQ ID NO: 2, or PRDM12 promoter sequence in a subject, preferably a human subject.
  • the modulator of Prdml2 inhibits Prdml2 activity by direct hybridisation to PRDM12 RNA.
  • the inhibitor of Prdml2 inhibits Prdml2 activity by direct hybridisation to PRDM12 RNA and additionally targeting it for degradation, wherein said degradation is optionally mediated by an RNA-induced silencing complex.
  • the inhibitor specifically targets and hybridises to a sequence comprised within SEQ ID NO: 2, or a sequence encoding SEQ ID NO: 1 or a naturally occurring variant thereof.
  • the antisense agent is a sequence of between 8 and 50 nucleotides in length which is a sequence which is complementary to a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% sequence identity to an mRNA encoding SEQ ID NO: 1 or a portion thereof, or wherein the antisense agent is complementary to a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% sequence identity to a portion of SEQ ID NO: 2 and is able to hybridise to said sequence.
  • antisense agents indicates an oligonucleotide configured to specifically anneal with (i.e. hybridise to) a given sequence in a target nucleic acid, typically an mRNA and in the context of the present invention in particular PRDM12 mRNA.
  • An PRDM12 antisense agent in the context of the present invention comprises, consists essentially of or consists of a nucleic acid sequence that is complementary or essentially complementary to said target PRDM12 nucleic acid sequence.
  • Antisense agents suitable for use as a PRDM12 modulator are able to hybridise to the PRDM12 target nucleic acid sequences at high stringency conditions, and are able of hybridising specifically to PRDM12 mRNA under physiological conditions. Synthesis, manipulation and introduction into cells of antisense agents have been described in the art and are therefore known to a skilled person (for example Dias and Stein, Molecular Cancer Therapeutics, 2002).
  • an antisense agent does not need to be a perfect “match” (i.e. 100% complementary to the PRDM12 nucleic acid sequence) to that of its target sequence to anneal or hybridise specifically with the latter.
  • An antisense agent may be said to be specifically hybridisable when interaction of the agent to a target nucleic acid molecule interferes with the normal function of the target nucleic acid such as to attain an intended outcome (e.g. loss of utility), and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense agent to non-target sequences under conditions in which specific hybridisation is desired, i.e.
  • the PRDM12 antisense agent is an inhibitor of Prdml2 activity, wherein said antisense agent reduces the Prdml2 activity by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, preferably at least about 75%,; more preferably at least about 80%, at least about 85%, at least about 90%, at least about 95% when compared to the Prdml2 activity in absence of said antisense agent.
  • the antisense agent reduces the Prdml2 activity by about 100%, i.e. below a detection threshold.
  • the reduction in Prdml2 activity may be limited to nociceptors and/or dorsal root ganglia, to one or more tissues or organs, or on the level of the subject as a whole.
  • the Prdml2 inhibitor is selected from the group of antisense agents consisting of: RNAi (RNA interference), shRNA (short hairpin RNA), siRNA (silencer RNA), and miRNA (micro RNA).
  • nucleic acids refer to the normal binding (i.e. hybridisation) of single-stranded nucleic acids under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson- Crick base pairing.
  • base pairing preferably Watson- Crick base pairing.
  • complementary Watson-Crick base pairing occurs between the bases A and T, between A and U, and between G and C.
  • the nucleotide sequence of the antisense agent may comprise at least one modification, preferably a terminal modification that may optionally inhibit or reduce degradation of the PRDM12 antisense nucleotide sequence by cellular machinery of a host cell.
  • a non-limiting example of a modification that inhibits or reduces degradation of the antisense nucleotide sequence is a phosphorothioate bond in the phosphate backbone of an oligo wherein a sulfur atom is substituted for a non-bridging oxygen.
  • the at least one phosphorothioate bond is introduced at the 5’ or 3’ terminus. In certain embodiments, the at least one phosphorothioate bond is present internally.
  • said nucleotide sequence may be physically coupled (i.e. conjugated) to other moi eties or conjugates that alter other characteristics of said nucleotide sequence, or even impart additional functions to said nucleotide sequence.
  • Such characteristics are altered activity, cellular distribution, immunogenicity and/or cellular uptake.
  • modifications include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.
  • dodecandiol or undecyl residues a phospholipid, e.g. di-hexadecyl-rac-glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecyl amine or hexylamino-carbonyl- oxy cholesterol moiety.
  • a phospholipid e.g. di-hexadecyl-rac-glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
  • a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an oc
  • Gene editing system indicates any molecular tool or system that is able to induce one or more targeted nucleic acid modifications in the sequence of PRDM12, PRDM12 promoter sequence and/or optionally PRDM12 enhancer sequence(s) within an intact and living cell.
  • the PRDM12 sequence may be the genomic PRDM12 sequence or PRDM12 (m)RNA sequence.
  • modulator of Prdml2 activity is a (endo)nuclease or a variant thereof having altered or modified activity.
  • (endo)Nucl eases known in the art comprise programmable, sequence-specific DNA- or RNA-binding modules linked to a nonspecific DNA or RNA cleavage domain (review in e.g.
  • these nucleases When targeting DNA, these nucleases create site-specific double-strand breaks at desired locations in the genome. The induced double-stranded breaks are repaired through non-homologous end-joining or homologous recombination, resulting in targeted mutations in the PRDM12 sequence. Alternatively modified, i.e. mutated forms of the endonucleases may be used to generate DNA “nicks” instead of double stranded breaks.
  • DNA “nick” is intended herein a double stranded DNA sequence wherein only one of the two strands contains a breaks, while the second strand remains intact, or may be separately nicked by a complementary nicking endonuclease optionally at a location proximal to the location of the nick location on the first strand.
  • endonucleases are restriction enzymes, meganucleases, zinc-finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and CRISPR-associated (Cas)-based nucleases.
  • the Prdml2 modulator is selected from the group of endonucleases consisting of restriction enzymes, meganucleases, ZFNs, TALENs, CRISPR-Cas. In preferred embodiments, the Prdml2 modulator is selected from the group of endonucleases consisting of ZFNs, TALENs, CRISPR-Cas. In further preferred embodiments, the Prdml2 modulator is a CRISPR-Cas endonuclease, preferably a CRISPR-Cas9 endonuclease.
  • the Prdml2 modulator is an endonuclease
  • said endonuclease specifically binds, or has a preference of binding to the human promoter sequence of Prdml2 or at least one exon selected from one of the 5 human PRDM12 exons.
  • the endonuclease specifically binds, or has a preference of binding to exon 1, 2, or 3 of human PRDM12.
  • restriction enzyme as used herein can be used interchangeably with “restriction endonuclease” or “restrictase” and refers to a class of endonucleases that cleave DNA at or in close proximity to specific recognition sites, which are commonly referred to as “restriction sites” in the art. Numerous restriction enzymes have been identified in the art. Methods, tools, and databases have been described in the art and are freely available to find information on both restriction enzyme activity and restriction sites (Roberts et al., Nucleic Acids Research, 2007). “Meganucleases” are a class of nucleases that are characterised by a larger recognition site than traditional (i.e.
  • Meganucleases have been identified in a considerable number of organisms, including but not limited to Archaea, bacteria, phages, fungi, yeast, algae, and plants. Additionaly, methodologies and tools are available in the art that allow the design and production of artificial meganucleases (Bartsevich, et al., Molecular Therapy, 2016).
  • the Prdml 2 modulator is a meganuclease targeting a PRDM12 sequence
  • the meganuclease is selected from the group consisting of one of the following families (based on sequence and structure motil): LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)XK.
  • Zinc-finger nucleases are artificial restriction enzymes that comprise a zinc finger DNA binding domain fused to a DNA cleavage domain.
  • Zinc finger domains can be engineered to target specific desired DNA sequences which allows for a skilled person to design zinc finger nucleases that are able to target unique sequences within a given genomic sequence, such as but not limited to the PRDM12 genomic sequence or PRDM12 promoter sequence.
  • Traditional zinc finger DNA binding domains contain between about three and six zinc finger repeats which will each recognise 9 to 18 nucleotides.
  • a suitable non-specific cleavage domain is the obligate dimeric endonuclease FokI domain, and engineered FokI domains with enhanced cleavage activity such as Sharkey (Guo et al., Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases, Journal of molecular biology, 2010).
  • Transcription-activator like effector nucleases or short “TALENs” are another class of artificial restriction enzymes that comprise a Transcription Activator Like (TAL) effector DNA binding domain which is fused to a DNA cleavage domain. Similar to ZFNs, it is possible to engineer TAL effector domains to specifically bind any given DNA sequence present in a genome, such as but not limited to (a portion ol) the PRDM12 genomic sequence or PRDM12 promoter sequence. TAL effector domains comprise a repeated conserved of about 33 or 34 amino acid sequence with variable amino acids at the 12th and 13th position, commonly annotated as repeat variable diresidues (RVDs).
  • RVDs repeat variable diresidues
  • TALENs determine the specific nucleotide recognition pattern of the TALEN.
  • Tools and protocols to generate TAL effector domains specific for a desired sequence are publicly available (Heigwer et al., Nucleic acids research, 2013; Neff et al., BioMedCentral Bioinformatics, 2013).
  • TALENs may be based on the use of a (modified) FokI domain as DNA cleavage domain, but can in theory include any DNA cleavage domain.
  • CRISPR-associated (Cas)-based nucleases which may be used interchangeably with “CRISPR/Cas nuclease”, “CRISPR-Cas” is indicative for a genome engineering system comprising an endonuclease that relies on the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences to recognise and cleave specific strands of nucleotide sequences complementary to the CRISPR sequence.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas proteins include Cas3, Cas 8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5, CaslO, Csxll, CsxlO, Csfl, Cas9, Csn2, Cas4, C2cl, C2c3, Casl2 (i.e. Cpfl), Casl3a, Casl3b, Casl3c, and Casl3d. in accordance to what is described in the art, a skilled person appreciated that different Cas proteins require different CRISPR sequences.
  • CRISPR-Cas9 comprises two nuclease domains, a RuvC domain and a HNH nuclease domain, which are responsible for cleavage of the nontarget DNA strand and the target strand respectively.
  • CRISPR-Cas9 can be specifically or preferentially to virtually any DNA sequence that comprises a protospacer adjacent motif (PAM), with the PAM being the nucleotide sequence NGG, wherein N may be any nucleotide.
  • PAM protospacer adjacent motif
  • the Cas9 protein is a modified Cas9 protein comprising a mutagenised HNH and/or RuvC catalytic domain.
  • said Cas9 protein is a nickase Cas9 (nCas9).
  • said Cas9 protein is a catalytically inactive Cas9 (i.e. dead Cas9, or dCas9) (cf. also https://www.origene.com/products/gene-expression/crispr-cas9/crispra-crispri).
  • double stranded breaks in a genomic PRDM12 sequence or PRDM12 promoter sequence may be achieved by fusing the catalytically inactive Cas9 protein to a DNA cleavage domain such as the non-limiting example Fokl.
  • the crRNA sequence or target specific portion of the gRNA sequence has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
  • the single guide RNA or crRNA comprises a portion of the nucleotide-encoded Prdml2 sequence.
  • the single guide RNA or crRNA comprises a portion of a sequence contained in exon 1, 2, or 3 of the PRDM12 sequence, preferably the human PRDM12 sequence.
  • the single guide RNA or crRNA comprises a portion of the endogenous PRDM12 promoter sequence, preferably the human PRDM12 sequence.
  • the gene-editing system comprises a guide RNA that is complementary to, and hence able to hybridise to, a sequence within the human PRDM12 genomic sequence or PRDM12 promoter sequence.
  • the Cas9-based PRDM12 modulator comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% complementary to a nucleotide sequence encoding SEQ ID NO: 1 or a nucleotide sequence comprised in the sequence of SEQ ID NO: 2.
  • gene editing systems are suitable for inhibiting or deleting the PRDM12 target sequence
  • these gene editing systems may be further mutagenised or modified to act as a transcriptional repressor or transcriptional activator of PRDM12 embodiment.
  • a catalytically inactive endonuclease specifically targeting the promoter sequence of Prdml2 may be coupled or genetically fused to a transcriptional activator or repressor domain.
  • transcriptional activators suitable for coupling include VP16, VP64, MyoD, FoxA, VPR, TET, p300, SMYD3, DOT1L, and PRDM9.
  • Non limiting examples of transcriptional repressors suitable for coupling include the KRAB domain, DNMTs, LSD1, HDAC3, and EZH2 (Hirai et al., The International Journal of Developmental Biology, 2012; and Xu et al., International Journal of Molecular Sciences, 2020).
  • Prdml2 as such or a mutant Prdml2 protein is used as a modulator of Prdml2 activity.
  • the Prdml2 has a sequence having at least about 65%, preferably at least about 75%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, more preferably 100% identity to the Prdml2 amino acid sequence of SEQ ID NO: 1. It is appreciated to a skilled person that when using a Prdml2 protein that mimics the activity of naturally occurring Prdml2, the Prdml2 modulator will exaggerate or increase the function of the naturally occurring Prdml2.
  • Prdml2 a nucleotide sequence wherefrom Prdml2 is translated and/or transcribed from
  • the additional Prdml2 may act therefore act as an inhibitor or enhancer of the pain response and/or pain sensation.
  • increasing the Prdml2 expression or activity will hence have an analgesic effect, which is unexpected based on what has been described in the art.
  • the modulator of Prdml2 activity is a mutant Prdml2 protein or nucleotide sequence encoding a mutant Prdml2 protein that exerts a higher intrinsic activity than the endogenous Prdml2 protein.
  • the modulator of Prdml2 activity is a mutant Prdml2 protein or nucleotide sequence encoding a mutant Prdml2 protein that exerts a lower intrinsic activity than the endogenous Prdml2 protein, or is an inactive Prdml2 protein.
  • the mutant Prdml2 protein is selected from the group of Prdml2 mutants consisting of: null loss-of-function Prdml2 mutants, leaky loss-of function Prdml2 mutants, and gain-of-function Prdml2 mutants.
  • the Prdml2 mutant is a gain-of-function mutant.
  • a skilled person is capable to identify different classes of mutant proteins in accordance to information readily available in the art (Griffiths et al., An Introduction to Genetic Analysis, 7th edition, 2000).
  • said mutant Prdml2 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations when compared to the canonical sequence of Prdml2, preferably the human Prdml2 sequence of SEQ ID NO: 1, which may optionally be encoded by a nucleotide sequence as described by SEQ ID NO: 2 or a portion thereof.
  • the modulator of Prdml2 activity may be a fusion protein of at least two distinct proteins, at least two functional fragments of proteins, at least two concatenated proteins, or any combination thereof.
  • fusion protein or “fusion polypeptide” and “protein conjugate” or “polypeptide conjugate” as used interchangeably in the art denote hybrid or chimeric molecules comprising at least two proteins or polypeptides linked, connected or joined together in a manner not normally found in nature.
  • the molecules may be suitably denoted as amino acid-based compounds, i.e. as substances or molecules as including primarily but not necessarily exclusively amino acid residues. Any recombinantly, semi-synthetically or synthetically produced fusion proteins or proteins conjugates are encompassed.
  • fusion proteins as envisaged herein may be modified by glycosylation, phosphorylation, sulfonation, methylation, acetylation, lipidation, pegylation or the like.
  • the terms “fusion protein” or “fusion polypeptide” indicate genetic fusions, whereby two or more proteins, polypeptides or variants or fragments thereof are joined by a co-linear and/or covalent linkage via their individual polypeptide backbones, through genetic expression of a single contiguous oligonucleotide molecule encoding the fusion product.
  • two or more open reading frames (ORFs) each encoding a given polypeptide segment are joined to form a continuous longer ORF in a manner that maintains the correct reading frame for each original ORF.
  • ORFs open reading frames
  • the two or more polypeptide segments encoded by the original ORFs are joined in the same polypeptide molecule, whereas they are not normally so joined in nature. While the reading frame is thus made continuous throughout the fused genetic segments, the so fused polypeptide segments may be physically or spatially separated by, for example, an in-frame polypeptide or peptide linker.
  • protein conjugate or “polypeptide conjugate” denote substances or molecules in which two or more proteins, polypeptides or variants or fragments thereof are joined by non-genetic means, whereas they are not normally so joined in nature.
  • the polypeptide segments may be joined via their individual polypeptide backbones or via one or more of their respective amino acid side chains, or one protein segment may be joined via its polypeptide backbone to an amino acid side chain of another polypeptide segment.
  • the Prdml2-binding molecule is a mutant G9a protein, preferably a mutant G9a protein that has either reduced or increased activity when compared to the native G9a protein.
  • a hyperactive G9a protein can be introduced or expressed in nociceptors or dorsal root ganglia to induce increased methylation of H3K9
  • an inactive or attenuated G9a protein can be introduced or expressed in nociceptors or dorsal root ganglia to induce a decrease in H3K9 methylation.
  • the mutant G9a protein is a mutant human G9a protein, more preferably a mutant human G9a protein characterised by a sequence having at least about 65%, preferably at least about 75%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 99%, identity to the G9a amino acid sequence of SEQ ID NO: 3.
  • a further aspect of the invention is directed to a nucleotide sequence encoding Prdml2 or encoding a modulator of Prdml2 activity as described herein, wherein said nucleotide sequence encodes Prdml2 or a molecule capable of binding to human Prdml2, or wherein said modulator is a nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2, preferably wherein Prdml2 is human Prdml2 as defined by SEQ ID NO: 1 and/or wherein said nucleotide sequence encoding the Prdml2 protein is the PRDM12 gene (SEQ ID NO: 2).
  • the nucleotide sequence is a DNA sequence.
  • the DNA sequence is a single stranded or double stranded DNA sequence.
  • the nucleotide sequence is an RNA sequence.
  • the nucleotide sequence may contain one or more modifications as described herein.
  • the nucleotide sequence consists of a modulator of Prdml2 activity.
  • the nucleotide sequence comprises, consists or, or essentially consists of a nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2 (i.e.
  • a PRDM12 antisense agent preferably the human nucleotide sequence encoding Prdml2 as identified by SEQ ID NO: 2, or a nucleotide sequence having at least about 65%, preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 97%, at least about 99% to SEQ ID NO: 2 or a portion thereof.
  • viral or non-viral vectors encoding Prdml2 or a modulator of Prdml2 activity as described herein, preferably wherein Prdml2 is human Prdml2, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or encoded by SEQ ID NO: 2.
  • the viral or non-viral vector is designed for and/or used for gene therapy, preferably human gene therapy.
  • the viral or non-viral vector comprises at least one additional encoded protein or at least one nucleotide sequence encoding capable of influencing a pain condition.
  • the viral or non-viral vector is designed to insert one or more nucleotide sequences comprised in said vector into the genome of the host cell, host tissue, or host organism.
  • the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 further comprises a human promoter sequence.
  • the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 further comprises an inducible promoter sequence.
  • the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 comprises a tissue-specific promoter sequence, preferably a human tissue-specific promoter sequence.
  • tissue-specific promoter sequences have been described in the art (e.g. in Zheng and Baum, Methods in Molecular Biology, 2009).
  • the tissue specific promoter sequence is a native promoter sequence.
  • the tissue specific promoter sequence is a composite promoter sequence.
  • native promoter sequences comprise a single fragment of a 5’ region of a gene, preferably wherein said gene is naturally expressed in a specific group of tissues or even one single tissue or cell type, while composite promoter sequences originate from the combination of promoter elements of different origins.
  • a native promoter particularly suited in the context of neurons and thus in the context of the present invention is the neuron-specific enolase (NSE) promoter sequence.
  • the viral vector comprises at least two distinct modulators or Prdml2 activity.
  • the term 'vector' as used in the application refers to nucleic acid molecules which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule.
  • the vector is used to transport the insert nucleic acid molecule into a suitable host cell.
  • a vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism.
  • the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated.
  • the vectors can be episomal vectors (i.e. , that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome.
  • the term 'vector' may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors.
  • Non- viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI, plasmid vectors (e.g.
  • Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector.
  • viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis.
  • Virosomes are a nonlimiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003).
  • Another example encompasses viral vectors mixed with cationic lipids.
  • the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector, more preferably an AAV vector.
  • AAV vectors are preferably used as self-complementary, double-stranded AAV vectors (scAAV) in order to overcome one of the limiting steps in AAV transduction (i.e. single-stranded to doublestranded AAV conversion) (McCarty, 2001, 2003; Nathwani et al, 2002, 2006, 2011; Wu et al., 2008), although the use of single-stranded AAV vectors (ssAAV) are also encompassed herein.
  • AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAVrh20 and AAV9.
  • Gene expression in DRG neurons has been achieved using AAV vectors based on serotypes 1, 2, 3, 4, 5, 6, 8, 8.2, 9 and rh20.
  • Preferred vectors are AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self- complementary AAV9 vector (scAAV9).
  • the vector comprises viral and non-viral elements.
  • AAV5 and AAV1 are preferred as well as AAV6, AAV8 and AAV9.lt is understood by the skilled person that expression systems as described herein encoding for Prdml2 or a functional fragment thereof are typically considered as activators of Prdml2, i.e. of modulators that restore or increase the expression of Prdml2 in the subject.
  • Such activators can typically be used for treating pain conditions caused or aggravated by reduced or lost expression of Prdml2 (as compared to the expression level in subjects not suffering from said pain condition) in DRG or TG neuronal cells as defined herein elsewhere.
  • antisense agents and Prdml2 binding molecules as disclosed herein are understood to typically be negative modulators of Prdml2, or inhibitors of Prdml2.
  • Such inhibitors can typically be used for treating pain conditions caused or aggravated by increased expression of Prdml2 (as compared to the expression level in subjects not suffering from said pain condition) in DRG or TG neuronal cells as defined herein elsewhere.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising Prdml2, a modulator of Prdml2 activity as described herein, or a combination thereof.
  • said pharmaceutical composition is used for treatment or prevention of a pain condition in a subject.
  • compositions in the context of the invention are indicative for those compositions that comprise a therapeutically or prophylactically effective amount of Prdml2 and/or Prdml2 modulator.
  • the Prdml2 of Prdml2 modulator present in the pharmaceutical composition is considered as at least one of the pharmaceutical active ingredients of the pharmaceutical composition.
  • the modulator of Prdm activity may be administered as such, as a nucleotide sequence encoding said modulator, as an nucleotide expression vector encoding said modulator, or as a viral vector encoding said modulator.
  • “Pharmaceutical active ingredient” or “API” as referred to herein is to be interpreted according to the definition of the term by the World Health organisation: “a substance used in a finished pharmaceutical product (FPP), intended to display pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings”.
  • FPP finished pharmaceutical product
  • therapeutically effective dose” or “therapeutically effective amount” as used herein refers to an amount of Prdml2, or an amount of Prdml2 modulator (i.e.
  • Prdml2 inhibitor or Prdml2 activator as described herein, that when administered brings about a clinical positive response with respect to treatment of a subject afflicted by a pain condition, e.g. a patient having been selected (e.g. diagnosed) to have or is expected to have or be exposed to a certain pain condition.
  • a pain condition e.g. a patient having been selected (e.g. diagnosed) to have or is expected to have or be exposed to a certain pain condition.
  • the patient is diagnosed with a nociceptive, neuropathic or inflammatory pain condition, wherein said pain condition may be chronic in nature.
  • prophylactically effective dose or “prophylactically effective amount” refers to an amount of Prdml2 modulator that inhibits or delays in a subject the onset of a pain conditions as being sought by a researcher, veterinarian, medical doctor or other clinician. While a skilled person appreciates that occupational injury cannot be predicted, the prediction of pain in the context of the present invention relates to the prediction of a pain-inducing disease or condition. By means of example and not limitation, a subject diagnosed with cancer, such as multiple myeloma is expected to perceive pain in a future point in time (i.e. treatment-related pain, pain due to disease progression, or a combination thereol).
  • a person scheduled for surgery can be expected to perceive pain in a future point in time, i.e. when recovering from the surgery.
  • a skilled person understands that the required dosage or amount of Prdml2 modulator that is needed to arrive at a therapeutically effective or prophylactically effective dose needs to be assessed on a case-by-case and subject-to-subject basis. It is standard practice to adapt a dosage to a certain individual to obtain an optimal, i.e. ideal effect or response.
  • carrier may be indicative for any solvent, diluent, buffer (including but not limited to neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubiliser (including but not limited to Tween 80 or Polysorbate 80), colloid, dispersion medium, vehicle, filler, chelating agent (including but not limited to EDTA or glutathione), amino acid, protein, disintegrant, binder, lubricant, wetting agent, stabiliser, emulsifier, sweetener, colorant, flavoring, aromatiser, thickener, any agent suitable to achieve a depot effect, coating, antifungal agent, any preservative (including but not limited to ThimerosalTM, benzalkonium chloride, or benzyl alcohol), antioxidant (including but not limited to ascorbic acid, sodium metabisulfite), tonicity controlling agent, ab
  • one or more excipients may be used in the pharmaceutical formulation on condition that the one or more excipient is compatible with the one or more pharmaceutical ingredient (i.e. in the context of the present invention at least the modulator of Prdml2 activity) and that a pharmaceutically acceptable formulation is obtained.
  • “Pharmaceutically acceptable” as used herein is indicative for the ingredients to be compatible with each other and that the combination of ingredients in the pharmaceutical formulation does not lead to deleterious effects (including but not limited to toxicity) to the subject receiving the formulation.
  • the excipient may be an active pharmaceutical ingredient excipient, binder excipient, carrier excipient, co-processed excipient, coating system excipient, controlled release excipient, diluent excipient, disintegrant excipient, dry powder inhalation excipient, effervescent system excipient, emulsifier excipient, lipid excipient, lubricant excipient, modified release excipient, penetration enhancer excipient, permeation enhancer excipient, pH modifier excipient, plasticiser excipient, preservative excipient, preservative excipient, solubiliser excipient, solvent excipient, sustained release excipient, sweetener excipient, taste making excipient, thickener excipient, viscosity modifier excipient, filler excipient, compaction excipient, dry granulation excipient, hot melt extrusion excipient, wet granulation excipient, rapid release agent excipient, increased bioavailability
  • the excipients should be non-toxic in the concentration contained in the final pharmaceutical composition and should not negatively interfere with the activity of the one or more pharmaceutically active ingredients, in this context at least the Prdml2 activity modulator.
  • more than one excipient which a skilled person would classify as belonging to the same group of excipients is added to the pharmaceutical composition.
  • more than one excipient wherein the different excipients belong to different groups is added to the pharmaceutical composition.
  • the excipients may fulfill more than one function and/or be classified by a skilled person as belonging to different groups or classes of excipients.
  • the formulation may comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, preservatives, complexing agents, tonicity adjusting agents, wetting agents and the like, nonlimiting examples include sodium acetate, sodium lactate, sodium phosphate, sodium hydroxide, hydrogen chloride, benzyl alcohol, parabens, EDTA, sodium oleate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • at least one additional component is combined with the pharmaceutical formulation prior to administration.
  • the additional component is combined with the pharmaceutical formulation immediately prior to administration.
  • the additional component may be part of the solvent used to reconstitute the pharmaceutical composition.
  • Aqueous solutions suitable for reconstitution of pharmaceutical compositions are known to a person skilled in the art.
  • a non-limiting example of a suitable aqueous solution is water for injection.
  • the amount of the additional component added to the pharmaceutical formulation is calculated based on certain patient parameters including but not limited to age, weight, gender, severity of the disease condition, and other known disease conditions of the patient or disease conditions the patient is suspected to be afflicted with.
  • any of the pharmaceutical compositions described to herein may be formulated into a unit dosage form, including but not limited to hard capsules, soft capsules, tablets, coated tablets such as lacquered tablets or sugar-coated tablets, granules, aqueous or oily solutions, syrups, emulsions, suspensions, ointments, pastes, lotions, gels, inhalants or suppositories, which may be provided in any suitable packaging means known in the art, non-limiting examples being troches, sachets, pouches, bottles, films, sprays, microcapsules, implants, rods or blister packs.
  • the pharmaceutical composition is suitable for direct administration to a subject.
  • Preferred administration routes include oral, rectal, bronchial, nasal, topical, buccal, sublingual, transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intra-arterial, intracerebral, intracerebroventricular intraocular injection or intravenous infusion) administration, or in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration.
  • the composition is comprised in an aqueous solution which is preferably pyrogen- free and additionally has a suitable pH, isotonicity and stability.
  • the aqueous solution optionally contains at least one of the following: sugars, alcohols, antioxidants, buffers, bacteriostatics (bacteriostats), solutes, suspending agents or thickening agents.
  • the pH of the pharmaceutical composition to be administered parentally is adjusted to a physiologic pH in the region of 7 to 9. In certain embodiments, the pH of the pharmaceutical composition to be administered parentally is from about 7.35 to about 7.45.
  • the pharmaceutical composition may be formulated as an immediate release formulation dosage form.
  • the pharmaceutical formulation may be comprised in a delayed release dosage form.
  • the pharmaceutical composition may be comprised in a controlled release formulation dosage form.
  • immediate release the pharmaceutical composition is about immediately, or near immediately released from a dosage form to a body of a subject or patient.
  • delayed release dosage forms the pharmaceutical composition is delivered in the body, or its contents contacted or exposed to the body or part thereof with a delay after administration.
  • sustained release or controlled release dosage forms the dosage form is designed to release a pharmaceutical composition at a predetermined rate in order to maintain a constant drug concentration for a specific period of time.
  • the release profile of a dosage form can be assessed as described in the major pharmacopeias published by government authorities or medical or pharmaceutical societies.
  • immediate release is defined by the European Medicines Agency as dissolution of at least 75% of the active substance within 45 minutes (European Pharmacopeia (Ph. Eur.) 9th edition).
  • European Medicines Agency as dissolution of at least 75% of the active substance within 45 minutes
  • suitable tests and time windows may vary depending on therapeutic ranges, solubility and permeability factors of the drug substance.
  • Techniques regarding the formulation and administration of pharmaceutical compositions are known to a skilled person and have been described in the art (e.g. the reference book: Remington: The Science and Practice of Pharmacy, periodically revised).
  • solid dosage forms may be manufactured by mixing the one or more active ingredients with a portion of the excipients or all excipients followed by wet or dry granulation, or direct compression.
  • the pharmaceutical composition comprises in addition to Prdml2 and/or one or more Prdml2 modulator a further pharmaceutically active ingredient, preferably wherein said further pharmaceutically active ingredient is an analgesic.
  • the further analgesic pharmaceutical active ingredient is an analgesic selected from the group consisting of: acetaminophen (i.e.
  • the further analgesic pharmaceutical active ingredient is an NS AID, preferably an NS AID disclosed in the present disclosure, more preferably an NSAID selected from the group consisting of: salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, enolic acid derivatives (oxicam), anthranilic acid derivatives (fenamates), (selective) COX-2 inhibitors (coxibs), sulfonanilides, or any combination thereof.
  • the further analgesic pharmaceutical active ingredient is an opioid, preferably an opioid disclosed in the present disclosure, more preferably an opioid selected from the group consisting of natural opiates, morphine esters, semi-synthetic opioids, fully synthetic opioids, endogenous opioid peptides, or any combination thereof.
  • the further analgesic pharmaceutical active ingredient is corticosteroid as disclosed in the present disclosure, more preferably a glucocorticoid and/or a mineralocorticoid.
  • the further analgesic pharmaceutical active ingredient is an anticonvulsant, preferably an anticonvulsant selected from the group consisting of: aldehydes, aromatic allylic alcohols, barbiturates, benzodiazepines, bromides, carbamates, fatty acids, fructose derivatives, hydantoins, oxazolidinediones, propionates, pyrimidinediones, pyrrolidines, succinimides, sulfonamides, triazines, valproylamides, perampanel, stiripentol, pyridoxine, or any combination thereof.
  • an anticonvulsant selected from the group consisting of: aldehydes, aromatic allylic alcohols, barbiturates, benzodiazepines, bromides, carbamates, fatty acids, fructose derivatives, hydantoins, oxazolidinediones, propionates, pyrimidinedione
  • the further analgesic pharmaceutical active ingredient is an muscle-relaxant as disclosed herein, more preferably a neuromuscular blocker or a spasmolytic.
  • the further analgesic pharmaceutical active ingredient is an anti-anxiety agent, preferably an anti-anxiety agent selected from the group consisting of: barbiturates, benzodiazepines, carbamates, antihistamines, opioids, antidepressants, sympatholytics (i.e.
  • beta blockers alpha blockers, and alpha-adrenergic agonists
  • phenibut mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, pregabalin, menthyl isovalerate, propofol, racetams, alcohol, inhalants, and combinations thereof.
  • the further analgesic pharmaceutical active ingredient is an antidepressant, preferably an antidepressant selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrinedopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors, NMDA receptor antagonists, and combinations thereof.
  • an antidepressant selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrinedop
  • a therapeutic effect may be achieved by incorporating doses or concentrations of said Prdml2 modulator and/or the further analgesic which are not effective in isolation.
  • a modulator of Prdml2 activity as described herein may be combined in a pharmaceutical composition with an opioid, wherein both the Prdml2 modulator and the opioid are present in amounts or concentrations that are below the therapeutically effective dose when used in isolation. Yet, is envisaged by the inventors that such a pharmaceutical composition would nevertheless result in a favorable clinical outcome, i.e. a diminishment or cessation of the pain condition and/or pain sensation.
  • an analgesic comprising of, or essentially consisting of a modulator of Prdml2 activity.
  • the analgesic comprises or essentially consists of Prdml2 and/or a mutant Prdml2, preferably human Prdml2 and/or human mutant Prdml2.
  • the analgesic comprises or essentially consists of a Prdml2 activator.
  • the analgesic comprises or essentially consists of a Prdml2 inhibitor.
  • the analgesic may comprise or essentially consist of a nucleotide sequence encoding Prdml2 and/or mutant Prdml2.
  • the analgesic may comprise a DNA sequence encoding Prdml2 and/or mutant Prdml2.
  • the analgesic is a null loss- of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutant.
  • the analgesic may be administered to a subject by use of a normally exogenous living delivery vehicle.
  • the living delivery vehicle is a prokaryotic or eukaryotic cell.
  • the living delivery vehicle is a human cell, such as a stem cell.
  • the living delivery vehicle is a bacterial cell, for example a bacterial cell capable of surviving and/or propagating within the gut of the subject.
  • An alternative aspect is the use of a modulator of Prdml2 activity as additive to pharmaceutical compositions comprising a traditional analgesic as described herein.
  • the modulator of Prdml2 activity is added to, and optionally suspended and/or dissolved in a liquid pharmaceutical analgesic composition suitable for administration by injection.
  • a further aspect of the invention relates to a method of diagnosing a hypersensitivity to pain in a subject, wherein the method comprises determining the expression of Prdml2 in a biopsy of said subject, wherein reduced expression of Prdml2 in dorsal root ganglia cells and/or nociceptors is indicative of a hypersensitivity to pain.
  • the method is directed to diagnosing allodynia and/or hyperalgesia in a subject.
  • the Prdml2 expression in the biopsy of the subject is determined by a method selected from the group consisting of: PCR (preferably RT-PCR or qPCR), mass spectrometry analyses, spectrophotometric assays (preferably UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays), ELISA, or a combination thereof.
  • PCR preferably RT-PCR or qPCR
  • mass spectrometry analyses preferably UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays
  • spectrophotometric assays preferably UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays
  • ELISA ELISA
  • a Prdml2 expression level of at least about 75%, at least about 60%, preferably at least about 50%, preferably at least about 25%, preferably at least about 10% when compared to a suitable control or baseline value as defined herein is indicative of allodynia and/or hyperalgesia.
  • Biopsy refers to a medical method comprising a step of extracting sample cells or tissues from a subject by a procedure that allows further examination of said cells or tissues.
  • the nature of the further examination is not limited, and may be for example chemical analysis or histological analysis.
  • the biopsy is performed on inflamed tissue or tissue suspected or expected to be inflamed.
  • biopsies include excisional biopsies, incisional biopsies (also referred to in the art as core biopsy), and needle aspiration biopsy.
  • the biopsied site is selected from the group consisting of: bone tissue, bone marrow tissue, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, nervous system tissue, urogenital tissue, lymph node tissue, muscle tissue, skin tissue.
  • a method of diagnosing a hypersensitivity to pain (such as allodynia and/or hyperalgesia) in a subject comprises determining the level of G9a recruitment to histone H3 in dorsal root ganglia cells and/or nociceptors, preferably Prdml2-mediated recruitment to histone H3 in dorsal root ganglia cells and/or nociceptors, wherein a reduction in G9a recruitment indicates decreased Prdml2 activity and indicates hypersensitivity to pain of the subject.
  • a method of diagnosing a hypersensitivity to pain (such as allodynia and/or hyperalgesia) in a subject comprises determining the methylation status, or representative methylation status of H3K9 in dorsal root ganglia cells and/or nociceptors in a biopsy of a subject, wherein reduced methylation indicates reduced Prdml2 activity and a hypersensitivity to pain of the subject.
  • the method of diagnosing provides information for prognosticating the severity of pain that may be experienced by a subject, and/or pain that is expected to be experienced by a subject.
  • diagnosis is indicative for a process of recognizing, deciding on or concluding on a pain-inducing disease or pain condition in a subject on the basis of symptoms and signs and/or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and/or quantity of one or more biomarkers of or clinical symptoms characteristic for the diagnosed disease or condition).
  • diagnostic procedures such as, for example, from knowing the presence, absence and/or quantity of one or more biomarkers of or clinical symptoms characteristic for the diagnosed disease or condition.
  • Particularly relevant tools for diagnosing a pain condition in the context of pain are self-reporting pain scales. “Diagnosis of’ the diseases or conditions as taught herein in a subject may particularly mean that the subject has such disease or (pain) condition.
  • a subject may be diagnosed as not having such despite displaying one or more conventional symptoms or signs pronounced of such.
  • Prognosticating in the context of the invention is indicative for anticipation on the progression of a paininducing disease or pain condition condition and the prospect (e.g. the probability, duration, and/or extent) of recovery, and/or the severity of experiencing or amelioration of pain.
  • the term "a good prognosis of generally encompass anticipation of a satisfactory partial or complete recovery from a diagnosed pain-inducing disease or pain condition, optionally within an acceptable time period. Alternatively, the term may encompass anticipation of not further worsening or aggravating of such, preferably within a given time period.
  • a poor prognosis of the disease or condition typically encompass an anticipation of a substandard recovery and/or unsatisfactorily slow recovery, or no recovery at all, or further worsening of said pain-inducing disease or pain condition, or any clinical manifestation associated with said disease or condition.
  • the pain is selected from nociceptive pain, neuropathic pain, inflammatory pain, or any combination thereof.
  • a further aspect of the invention relates to a method of treating or preventing a pain condition as described herein, comprising administering a modulator of Prdml2 activity as described herein or an analgesic comprising a modulator of Prdml2 activity as described herein, or a pharmaceutical composition comprising a modulator of Prdml2 activity as described herein to a subject.
  • An alternative method of treating or preventing a pain condition as described herein comprises administering Prdml2, a functional fragment thereof, or mutant Prdml2 to a subject.
  • the administration is intravenous administration.
  • the Prdml2 modulator may be administered in combination with a further analgesic to the subject.
  • the further analgesic that is administered to the subject is administered before, after, simultaneously, or intermittently to the subject.
  • the administration may be topical and/or systemic.
  • the modulator of Prdml2 activity is administered daily during a treatment period.
  • the modulator of Prdml2 activity is administered at least once a day during the treatment.
  • the modulator of Prdml2 activity is administered at least twice a day during the treatment.
  • the modulator of Prdml2 activity is administered at least three times a day during the treatment.
  • the modulator of Prdml2 activity may be administered continuously during the treatment for instance in an aqueous drinking solution or in an intravenous solution.
  • different administrations of the modulator of Prdml2 activity each contain an about equal amount and/or concentration of said modulator.
  • the methods of treatment comprise a single administration moment.
  • the Prdml2, functional fragment thereof, or mutant Prdml2 is expressed in the subject after administration of a nucleotide sequence as described herein encoding said protein.
  • the nucleotide sequence is a DNA sequence, which is optionally part of an expression vector.
  • Said expression vector may comprise the endogenous Prdml2 promoter sequence, a constitutive heterogenous promoter sequence, an inducible heterogenous promoter sequence, or a combination thereof.
  • the nucleotide sequence is an RNA sequence.
  • the Prdml2, functional fragment of Prdml2, mutant Prdml2, Prdml2 inhibitor, or Prdml2 modulator may be packaged in a delivery vector, preferably a viral delivery particle prior to administration to the subject.
  • a suitable viral delivery particle is an adeno-associated viral particle.
  • the use of a modulator of Prdml2 activity as described herein is intended for the manufacture of a medicament for the prevention or treatment of a pain condition.
  • the use of Prdml2, functional fragment of Prdml2, mutant Prdml2, or a nucleotide sequence as described herein encoding one or more of the preceding moieties is intended for the manufacture of a medicament for the prevention or treatment of a pain condition.
  • said use is intended for the manufacture of a medicament for the prevention or treatment of nociceptive pain, neuropathic pain, inflammatory pain, or any combination thereof.
  • said use is intended for the manufacture of a medicament for the prevention or treatment of inflammatory pain or nerve injury pain, preferably wherein said inflammatory pain condition is selected from the group consisting of: skin inflammation, joint inflammation, and allergy-related inflammation.
  • kit of parts comprising means to assess Prdml2 expression and/or activity levels in a subject, or in a biopsy of a subject.
  • kit of parts and “kit” as used herein refer to a product containing components necessary for carrying out the methods (e.g. the diagnosis methods), packed so as to allow their transport and storage.
  • Materials suitable for packing the components comprised in a kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, flasks, vials, ampules, paper, envelopes, or other types of containers, carriers or supports.
  • kits comprises a plurality of components
  • at least a subset of the components e.g., two or more of the plurality of components
  • all of the components may be physically separated, e.g., comprised in or on separate containers, carriers or supports.
  • kits may be sufficient or may not be sufficient for carrying out the specified methods, such that external reagents or substances may not be necessary or may be necessary for performing the methods, respectively.
  • kits are employed in conjunction with standard laboratory equipment, such as liquid handling equipment, environment (e.g., temperature) controlling equipment, analytical instruments, etc.
  • kits may also include some or all of solvents, buffers (such as for example but without limitation histidine-buffers, citrate-buffers, succinate-buffers, acetate- buffers, phosphate-buffers, formate buffers, benzoate buffers, TRIS (Tris(hydroxymethyl)- aminomethan) buffers or maleate buffers, or mixtures thereof), enzymes (such as for example but without limitation thermostable DNA polymerase), detectable labels, detection reagents, and control formulations (positive and/or negative), useful in the specified methods.
  • buffers such as for example but without limitation histidine-buffers, citrate-buffers, succinate-buffers, acetate- buffers, phosphate-buffers, formate buffers, benzoate buffers, TRIS (Tris(hydroxymethyl)- aminomethan) buffers or maleate buffers, or mixtures thereof
  • enzymes such as for example but without limitation thermostable DNA polymerase
  • detectable labels such as for example but
  • kits include instructions for use thereof, non-limiting examples hereof being a printed insert and/or a computer readable medium.
  • the terms may be used interchangeably with the term “article of manufacture”, which encompasses any man-made tangible structural product, when used in the present context.
  • nucleotide sequence encoding a modulator of Prdml2 activity as described herein.
  • said nucleotide sequence may be introduced into a host cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle.
  • said nucleotide sequence encoding the modulator of Prdml2 activity is incorporated or part of an expression vector.
  • expression vectors may act as autonomous expression vectors (i.e. the expression vector is maintained extrachromosomally in the target cell) or as integrative expression vectors (i.e. the expression vector is integrated into the target.
  • Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g. URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and/or selection of those cells transformed with the desired nucleic acids.
  • Expression vectors can also include an autonomous replication sequence (ARS).
  • the ARS may comprise a centromere (CEN) and an origin of replication (ORI).
  • CEN centromere
  • ORI origin of replication
  • the ARS may be ARS 18 or ARS68.
  • Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment.
  • the first and second insertable DNA fragments are each about 200 (e.g. about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed.
  • a nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene.
  • Integrative vectors can be linearised prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome.
  • the vectors Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g. amplified) in bacterial cells such as Escherichia coli (E. coli).
  • E. coli Escherichia coli
  • the vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu.
  • the purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells.
  • the host cell may be a any cell deemed suitable by a skilled person.
  • host cell may be used interchangeably with the term “host organism” and is indicative for cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals.
  • Contemplated as host cells are inter aha unicellular organisms, such as bacteria (e.g. E. coli, Salmonella tymphimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g. Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g. from Arabidopsis thaliana or Nicotiana tobaccum) and (cultured) animal cells (e.g.
  • vertebrate animal cells mammalian cells, primate cells, human cells or insect cells.
  • Contemplated as host organisms are inter aha multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are nonhuman.
  • the Prdml2 modulator may be isolated from a host cell or natural environment.
  • isolated with reference to a particular component (such as for instance a nucleic acid, protein, polypeptide or peptide) generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment.
  • an isolated human or animal protein or complex may exist in separation from a human or animal body where it naturally occurs.
  • isolated as used herein may preferably also encompass the qualifier “purified”.
  • purified with reference to peptides, polypeptides, proteins, or nucleic acids does not require absolute purity.
  • a discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc.
  • Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.
  • Purified peptides, polypeptides or proteins may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment. Protein content may be determined, e.g. by the Lowry method (Lowry et al. Journal of Biological Chemistry, 1951). Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.
  • nucleic acid can be directly injected into the target cell or target tissue.
  • Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun” method), infection with viral vectors (i.e.
  • nucleic acid molecules derived from lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like.
  • AAV adeno-associated virus
  • Other techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilised) NA molecule(s) into micropumps delivering the product.
  • Another possibility is the use of implantable drugreleasing biodegradable microspheres.
  • NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; and co-inj ection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used.
  • the invention is directed to an in vitro method for identifying a molecule suitable as an analgesic, wherein said method comprises determining whether a candidate molecule modulates Prdml2 activity in nociceptors and/or dorsal root ganglia.
  • the in vitro method comprises a step of contacting a nociceptor or dorsal root ganglion with one or more candidate Prdml2 modulators and a step of measuring Prdml2 activity in the nociceptor or dorsal root ganglion.
  • the Prdml2 activity is measured by assessing the Prdml2 expression level before and after contacting the nociceptor and/or dorsal root ganglion with a candidate molecule. In alternative embodiments the Prdml2 activity is measured by assessing H3K9 methylation.
  • the in vitro method is a computer-implemented method. In one or more embodiments of the in vitro method, the Prdml2 is human Prdml2, preferably human Prdml2 as defined by SEQ ID NO: 1. In certain embodiments, the method is an in vitro method for identifying a molecule suitable as an analgesic in pain-inducing inflammatory conditions and/or pain conditions induced by nerve injury.
  • the Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
  • Exemplary assays can be tailor made sequencing kits, PCR-based assays, or ChIP assays.
  • the suitability of a molecule as an analgesic will depend on the influence on Prdml2 activity combined with the specific pain condition.
  • the molecule is considered an analgesic when G9a recruitment to histone H3 and/or H3K9 methylation is increased.
  • the molecule is considered an analgesic when G9a recruitment to histone H3 and/or H3K9 methylation is decreased.
  • the molecule is considered prima facie a molecule suitable as an analgesic when the G9a recruitment to histone H3 and/or the H3K9 methylation is altered by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, most preferably at least about 100% when compared respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
  • a suitable means to assess Prdml2 activity is by measuring expression of one or more genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, MRGPRB5, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6
  • the expression is assessed at the RNA level. In alternative embodiments, the expression is assessed at the protein level. In yet alternative embodiments, the expression is assessed at the RNA and protein level.
  • said genes are the human PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, MRGPRB5, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3,
  • the candidate modulator of Prdml2 activity is considered a Prdml2 activator when the expression of one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb
  • the candidate modulator of Prdml2 activity is considered a Prdml2 inhibitor when the expression of one or more genes selected from the group consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng
  • the gene expression is assess by PCR, preferably qPCR or RT-PCR. In one or more of these embodiments, the gene expression is expressed as a value normalised to the expression level of one or more housekeeping genes, preferably one or more housekeeping genes included in the HRT Atlas Database (https://www. housekeeping.unicamp.br, Hounkpe et al., Nucleic Acids Research, 2020).
  • Prdml2 is selectively expressed in sensory ganglia in developing pain sensing neurons, termed nociceptors, and that it is required for their development (Desiderio et al., 2019; Bartesaghi et al., 2019). Interestingly, earlier published data of the inventors also indicated that Prdml2 remains expressed in mature nociceptors in adult animals (Desiderio et al., 2019; Fig. 1, bottom panels), suggesting it could modulate the function of mature nociceptors.
  • mice All mice were maintained on a C57BL/6J background and mice of either sex were used. Mice were provided ad libitum with standard mouse lab pellet food and water and housed at room temperature with a 12h light/dark cycle. The experimental protocols were approved by the CEBEA (Comite d’e thique et du bien etre animal) of the IBMM-ULB and conformed to the European guidelines on the ethical care and use of animals.
  • CEBEA Compite d’e thique et du bien etre animal
  • Prdmn ⁇ (Desiderio et al, 2019), Advillin-CreERT2 (Lau et al., 2011) and Rosa CreERT2 (JAX# 008463)(Ventura et al., Nature 445, 661-665, 2007).
  • Females carrying Prdml2 conditional alleles were crossed to Advillin-CreERT2 males to generate tamoxifen (TAM)-inducible Prdml2 conditional knockout offspring in which Prdml2 is specifically deleted in somatosensory neurons following treatment with tamoxifen.
  • TAM tamoxifen
  • Tamoxifen has been administrated (1,9 mg/25g body weight) intraperitoneally 2 times in a week in more than 8 week old Advillin-Cre-ERT2; Prdml2fl ; fl mice and, as control, Advillin-Cre-ERT2; Prdml2fl ; A mice injected with com oil, or Prdml2fl ; fl mice injected with tamoxifen.
  • Tamoxifen dissolved in com oil has been administrated (1,9 mg/25g body weight) intraperitoneally in more than 56-days old ⁇ V il CreERT2 -Prdml2f l: f l mice two times in a week, and four times in a week in the case of Rosa CreERT2 ;Prdml2fl : fl mice.
  • Injected animals were sacrificed 28-35 days after the second tamoxifen injection for analysis. For tissue recovery, mice were anesthetized with Domitor (Img/Kg) and Ketamine (75mg/Kg) or with Ketamine (75mg/kg) and Rompun (Img/kg).
  • Prdml2 floxed allele was detected using primers forward 5’- GCTGATCGAGTCCAGGAGAC-3 (SEQ ID NO: 25) and reverse 5’ CCAAACATCCACAACCTTCA-3.
  • mice injected with tamoxifen were used as controls.
  • Formalin behavior assays were performed on 11 conditional mutants and 15 WT mice, both male and female, between 5-7 months of age. Experimenter was blind to the genotype of the test subjects. Animals were habituated the day before the test for 5 minutes in a plexiglass cage with mirrors on the walls and the floor covered with litter. On the day of the test, the right hind paw of the mouse was injected with 25ul of a 3% formalin solution. The mouse was observed for 30 minutes after the injection. The duration of the licking time of the injected paw was recorded at 5 min intervals until 30 minutes.
  • mice were euthanized with Domitor (Img/Kg) and Ketalar (75mg/kg. Intracardiac perfusion with PBS was then carried out, and their DRG collected for validation of their genotype by IF and/or RT-qPCR. Plantar incision in the hindpaw is currently be used as model to test whether Prdml2 also affect neuropathic pain (Brennan et al., 2004).
  • BIOSEB BIO-TC2T device that is composed of two joined thermal plates and is equipped with a camera to follow the transitions of the mouse from one plate to another and monitor the time spent on each plate.
  • mice were habituated for 180 seconds on the device and both plates were set at 30°C, the reference temperature for all tests. Mice were recorded for 180 seconds for each temperature tested.
  • the temperatures of 22°C, 10°C, 43°C and 52°C have been tested sequentially, with a day off in between hot and cold temperatures. Each day, the reference and the test plates were reversed.
  • mice were habituated for 5 minutes the day before the test in a plexiglass cage such as for the formalin test.
  • the day of the test the mouse was injected with 10 pl of a 0,35pg/pl capsaicin solution (0,9% saline, 10% ethanol, 10% Tween-20). Mice were recorded for 15 minutes after injection and the time licking the injected paw was measured.
  • Tail flick was also tested in mice using the Bioseb device BX-TF. The mouse was restrained in a cylinder with the tail outside of the cylinder in a notch and the tail was subjected to a radiant heat source.
  • the focus 30 was selected for this test meaning that the temperature applied on the tail was at 50°C after 5 seconds and at 60°C after 10 seconds. The withdrawal latency from the heat stimulus applied to the tail was therefore measured. In total, 3 measurements were carried out with a rest time of 15 seconds between measurement.
  • mice Cold plantar assay was used to evaluate the cold sensitivity in mice. Mice were habituated in small cages on a 3mm thick glass plate for 20 minutes. A 5ml syringe cut at the end and filled with powdered dry ice was applied under the hind paw against the glass plate and the withdrawal latency was measured. Three measurements were taken per paw and both paws were tested with a time interval of 5 minutes between measurements. The mechanical conflict avoidance test was used to evaluate the response to mechanical stimuli. For the habituation, the mouse was placed on the device for 5 minutes and was free to move around the different parts of the box. For the test, the mouse was placed in the first part which had a separation with the second part, after 10 seconds the lamp was turned on and after 15 seconds the separation was removed.
  • mice went through the second part of this box with nails to get to the last part which was dark. The time it took for the mouse to cross half of the part with nails was measured. Two heights were tested: 2mm and 5mm on two different days and 3 measurements were taken per height with a rest time of 30 minutes between each measurement. Open field was used to analyse the activity of mice and their exploratory behaviour. Mice were placed for 10 minutes in a plexiglass cage (44x44cm) with a camera placed above the cage and the behaviour was analysed with a Noldus software. Several parameters have been analysed such as the distance travelled, the velocity, the time moving and the time spent in the center and on the borders. Statistical analysis was conducted with the student’s t-test. All graphs were processed with GraphPad.
  • RNA-sequencing and data processing Dissected lumbar DRG (8 DRG from one animal) from 5 mutants (2 males and 3 females) and 4 WT mice where immediately frozen at -80°C and the frozen pellet placed after in 1 ml Trizol (Thermo Fisher, ref 15596026) for RNA extraction. RNA-seq analysis and data processing has been done at the NGS Integrative Genomics platform of the University of Gotingen as described previously described (Desiderio et al, 2019).
  • the sequence of the primers used are listed in Table 4 below:
  • Tissue dissection for ISH and immunostaining was performed as previously described (Thelie et al., 2015; Desiderio et al., 2019). Plasmids used for generating probes for Prdml2, Ntrkl and Trpm8 were as described (Desiderio et al., 2019). Grikl,Mrgprb5 and Cysltr2 cDNA used for probes generation were obtained by PCR from mouse embryonic dorsal root ganglia using the following primers:
  • ISH experiments were performed as previously described using antisense digoxy genin-labeled riboprobes (Thelie et al., 2015; Desiderio et al., 2019).
  • Secondary antibodies used were: goat Anti-rabbit Alexa 594 (1:800, Invitrogen A11012), donkey anti-goat Alexa 594 (1:2000, Invitrogen A 11058), goat anti-guinea pig Alexa 488 (1:2000, Invitrogen A11073), donkey anti-guinea pig Alexa 488 (1:800, Bio connect) and donkey a-chicken Alexa 488 (1:1000, Bio connect - Jackson).
  • Piezoelectric Burleigh micromanipulator was used to lower the electrode on cells. Liquid junction potential was not corrected. Cells were excluded from analysis if the resting membrane potentials was more positive than -40 mV. Neurons were recorded within 24 hours of culture to prevent neurite outgrowth that degrades space clamp.
  • electrodes had a resistance of 2.5-3.5 M.Q when filled with the pipette solution of the following composition (in mM): 140 K-Aspartate, 10 NaCl, 10 EGTA, 1 MgC12, 10 HEPES pH 7.20, with KOH (290 ⁇ 5 mOsm).
  • the extracellular solution was composed of (in mM): 150 NaCl, 5 KC1, 1 MgC12, 2 CaC12, 10 HEPES, 10 Glucose, pH 7.40 with NaOH (290 ⁇ 5 mOsm).
  • Whole-cell configuration was obtained in voltage-clamp mode before proceeding to the current-clamp recording mode.
  • Threshold current was determined by the first action potential elicited by a series of depolarizing current injections (300 ms) that increased in 20 pA increments.
  • Action potential frequency was determined by quantifying the number of action potentials elicited in response to depolarizing current injections (2 s, 1500pA). Any modification in basal excitability will be evidenced by change in the threshold current and/or action potential frequency.
  • mice were deeply anesthetized with Ketamin (75mg/kg) and Rompun (Img/kg) and intracardiacally perfused with ice-cold PBS.
  • Whole lumbar DRG from inducible knockout animals were harvested 4 weeks after the last tamoxifen injection.
  • Controls were either Cre negative TAM injected Prdml2 Fl/Fl or Prdml2 Fl/Fl mice or com oil injected Cre expressing Prdml2 Fl/Fl mice.
  • RNA-seq libraries were performed using 100 ng total RNA of a non stranded RNA Seq, massively -parallel mRNA sequencing approach from Illumina (TruSeq RNA Library Preparation Kit v2, Set A; 48 samples, 12 indexes, Cat. N°RS-122- 2001).
  • Sequence images were transformed to BCL files with the Illumina software BaseCaller software which were demultiplexed to fastq files using bcl2fastq v2.20.0.422. Sequencing quality was asserted using FastQC software (http://www. bioinformatics, babraham. ac.uk/proj ects/fastqc/)(version
  • Gene ontology and pathway analysis was performed in RStudio using Bioconductor version 3.12 selecting all DEG with an FDR- adjusted? value ⁇ 0.05 and log2FoldChange>0.449.
  • ClusterProfiler and enrichGO were used for gene ontology analysis based on molecular function, biological process and cellular component using a pvalueCutOff for the enriched categories of 0.03.
  • KEGG pathways analysis was performed using enrichKEGG. Analysis and sensory neuron classification of deregulated genes was performed using the gene expression visualization tools developed by Usoskin et al., 2015
  • Deregulated genes were classified as ‘’Neuron enriched” if they had significant expression in at least one neuronal cell type. Deregulated genes were manually classified as ‘’aspecific” if they had unknown expression pattern, no significant expression at least in one neuronal cell type or significant expression in at least in one non-neuronal cell type.
  • mice (3 to 7-month-old) were anesthetized with a mixture of Domitor (Img/kg) and Ketamine (75mg/kg) and an intracardiac perfusion with PBS was performed.
  • DRG from thoracic and lumbar parts were collected and then digested for 35 minutes at 37°C in an enzyme mixture containing Collagenase (12.5mg/mL) and Dispase (3mg/mL). DRG were quickly centrifuged at 150G after digestion and then the enzyme mix was removed and replaced by DMEM/F12. Dissociation using a P 1000 was carried out and the cell suspension was then filtered through a 0,2pm filter followed by a 7 minute centrifugation at 150G.
  • Cells were then resuspended in complete medium containing DMEM/F12, 10% of fetal bovine serum and 1% of Penicillin- Streptomycin. Cells were plated in 4-well plates on glass coverslips coated with 0,lmg/mL of Poly-L-lysine and lOpg/mL of laminin and kept in complete medium at 37°C.
  • Prdml2 is a conserved epigenetic transcriptional regulator whose mutation in human leads to congenital insensitivity to pain and that is required for the development of the nociceptors. Here we show that Prdml2 continues to display restricted expression in nociceptors in the peripheral nervous system of adult mice (Fig. 8).
  • Prdml2 is required at adulthood in mature nociceptors for pain perception
  • the inventors crossed Prdml ⁇ fl mice with Advillin-Cre-ERT2 BAC transgenic mice to generate Advillin-CreERT2; Prdml ⁇ M 1 (Prdml2 icKO) in which Prdml2 is specifically deleted in somatosensory neurons following treatment with tamoxifen or with Rosa26-CreERT2 mice that express Cre ubiquitously after treatment with tamoxifen, to generate Rosa26-CreERT2,' PrdmnAA (Rosa26 icKO).
  • Controls were Prdml2f l/ f l or Prdm ⁇ mice with no Cre transgene injected with tamoxifen; Cre expressing Prdml2f l/ f l mice injected with com oil,; or Cre-expressing Prdml2 +/+ mice injected with tamoxyfen.
  • mice lacking Prdml2 exhibit normal responses to thermal and mechanical nociceptive stimuli but a reduced response to capsaicin and hypersensitivity to formalin-induced inflammatory pain (Figure 7 and 12,17,19). Together, this data indicate that Prdml2 regulates pain-related behavior in a complex way by modulating gene expression in adult nociceptors and controlling their excitability. They suggest that, depending on the type of pain condition, increasing or reducing its expression and/or activity, may provide analgesic effect.
  • Prdml2 appears interesting given its restricted expression in the nervous system suggesting that any compound targeting its action would have no side effect, its close link with NGF known to be involved in nociceptor sensitization, and the importance of epigenetic mechanisms in the induction and maintenance of chronic pain. Additionally, it can be concluded from these data that in some pain disorders, it is the enhancement of Prdml2, rather than its blockade, that may have an analgesic effect. Overexpression of Prdml2 could be obtained using recombinant viruses encoding Prdml2 or encoding a modified version of Prdml2 that functions as a stronger transcriptional regulator.
  • Example 1 Prdml2 expression in mature nociceptors is dispensable for DRG nociceptor survival
  • Fig. 8A shows that /Wm/2 remains expressed in all trunk and cranial ganglia containing nociceptors.
  • Prdml2 is absent from mechanoreceptors (TrkB + ) and proprioceptors (TrkC + ), but is expressed in both peptidergic (TrkA + , CGRP + ) and non-peptidergic (Ret + ) nociceptors (Navl.8 + , TrpVl + ), as well as in some itch-mediating Somatostatin (Sst + ) neurons and c-fiber low-threshold mechanoreceptors (C-LTMRs) expressing tyrosine hydroxylase (TH + ) (Fig. 8B). Altered epigenetic mechanisms are known to contribute to inflammation- induced pain hypersensitivity.
  • CFA Complete Freund’s adjuvant
  • mice with two Rosa26 CreERT2 alleles were used (Sandlesh et al., PlosOne 2018).
  • Avil CreERT2 The resuitingconditional knockout offspring, Avil CreERT2 ; Prdml ⁇ A mice and Rosa26 CreERT2 ,-Prdml2fl / fl mice are here designated Avil icKO and Rosa26 icKO.
  • Avil CreERT2 The resuitingconditional knockout offspring, Avil CreERT2 ; Prdml ⁇ A mice and Rosa26 CreERT2 ,-Prdml2fl / fl mice are here designated Avil icKO and Rosa26 icKO.
  • Fig. 16 shows the validation of some of the identified DEGs as Prdml2 targets in Rosa26 icKO mice by RT-qPCR.
  • Prdml2 plays a role in the control of gene expression in mature nociceptors
  • the inventors performed a transcriptomic analysis by bulk RNA-seq on dissected lumbar DRG of both Advil and Rosa26 icKO models and control mice one month after TAM injection.
  • Using an FDR-adjusted p-value ⁇ 0.05 and an absolute log2 Fold-Change cutoff of >0.449 (based on the Fold-Change of Ntrkl, recently reported as reduced in adult Prdml2 knockout mice (Landy et al., 2021) we obtained a list of 140 genes in Avil icKO and 134 in Rosa26 icKO that are dysregulated in the absence of Prdml2 (Fig. 3 and 10A and Table 2 and 3).
  • KEGG pathway analysis revealed that the three most significantly deregulated pathways are neuroactive ligand-receptor interaction, calcium signaling and cholinergic synapse (Fig. 15B). Focusing on the neuroactive ligand-receptor interaction category, 6 genes appear upregulated while 3 appear downregulated (Fig. 15C).
  • Prdml2 among the core DEGs are genes encoding membrane proteins such as the NGF receptor Ntrkl/TrkA, the sensory specific G protein-coupled receptor Mrgprb5 (Liu et al., Cell 139, 13531365, 2019), the a 6 subunit of the nicotinic acetylcholine receptor Chrna6 that inhibits P2X2/3 receptors, the ionotropic glutamate receptor kainate 1 (Grikl/Glur5), the RET coreceptor GFRa3, the leukotriene receptor Cysltr2, the neuronal specific leucine-ricjh protein Lrm4 and the glycosylphosphatidylinositol-anchored protein OTOA (Zwaenepoel et al., PNAS 30, 6240- 6245, 2002), ions channel proteins such as the acid-sensing channel ASIC1, the cold-activated channel Trpm8, the potassium channels Kcn
  • extracellular proteins such as the secreted glycoprotein CREG2 (Kunita et al., Genmics 80, 456-460, 2002) and the secretory neuropeptide CALCB (Calcitonin Related Polypeptide Beta) (Wimalawansa et al., Endocr. Rev 1996, 17, 533-585, 1996) and enzymes such as A3galt2, Mettl7a3 and Stk32a.
  • the differential expression of some of these genes has been confirmed by RT-qPCR and in situ hybridization on DRG section in Advil icKO and by RT-qPCR alone in Rosa26 icKO (Fig. 4-6 and 11 and 16).
  • the consistency of the results obtained in the two mouse lines strongly support a role for Prdml2 in the transcriptional regulation of gene expression in mature nociceptors.
  • TG have a more complex embryonic origin than DRG, being derived from both neural crest and placodal cells
  • the inventors also performed bulk RNA-seq analysis on TG samples from TAM-injected Rosa26 icKO and control mice.
  • Fig. 18A and Table 6 As in DRG, most (87%) of these DEGs are nociceptive neuronal genes, with many of them encoding proteins with channel activity (Fig. 18B).
  • icKO mice and controls were injected with tamoxifen and used one month after TAM injection in different nociceptive assays. Given the difficulties encountered to generate Advil icKO mice due to non Mendelian tramsmission of the Cre and based on the similar transcriptional changes observed in DRG using the two mouse models, both Avil icKO and Rosa26 icKO mice were used. All behavior tests were performed using both male and female mice. To evaluate thermal nociception, the inventors performed thermal preference place in Avil icKO and tail flick test and cold plantar test in Rosa26 icKO. Mechanical nociception was evaluated in Rosa26 icKO in the mechanical conflict avoidance test.
  • Prdml2 expression is decreased in a model of joint inflammation
  • the inventors verified whether loss of Prdml2 might lead to changes in inflammation-related nociceptive behavior of adult mice, the inventors thus examined behavior responses of Advil icKO mice in the formalin test, a common model for tissue injury-induced pain, which produces paw swelling and an inflammatory response. This acute inflammation is caused by cell damage that provokes the production of endogenous mediators and subsequently the release of inflammatory mediators in the paw. In this test, two distinct periods of high licking activity of the injected paw can be identified.
  • Avil icKO mice showed similar responses to control animals during the first phase. However, they spent more time licking when compared to control mice during the second phase, suggesting a role for Prdml2 in mediating inflammatory pain (Fig. 7, 12F and 19). An increase of sensitivity in the second phase of the test was also observed in Rosa26 icKO mice (Fig. 12G).
  • Table 2 List of DEG comparing Avil CreERT2 icKO with control, FDR-adjusted p-value > 0.05 and log2FoldChange > 0.449. In bold genes in common with the list of DEG from Rosa26 CreERT2 icKO.
  • Table 3 List of DEG comparing Rosa26 CreERT2 icKO with control, FDR-adjusted p-value > 0.05 and log2FoldChange > 0.449. In bold genes in common with the list of DEG from Avil CreERT2 icKO.
  • Table 4 List of primer pairs used for RT-qPCR analysis. _
  • Table 5 List of transcripts common to both ganglia, designed here as Prdml2 core DEGs
  • Table 6 List of DEG identified in TG of Rosa26 icKO. Genes in common with the list of DEGs from DRG of Rosa26 icKO are indicated in bold. Genes in common with the list of DEGs from DRG of Avil icKO are in italic. References
  • PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis. Cell Reports, 26(13), 3484-3492.
  • Prdml2 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA. Cell Reports, 26(13), 3522-3536.
  • Prdml2 specifies VI interneurons through cross-repressive interactions with Dbxl and Nkx6 genes in Xenopus. Development, 142(19), 3416-3428.

Abstract

The present invention concerns the modulation of PRDM12 activity for prevention or treatment of pain in a subject. Both means to modify the expression level of PRDM12 and means to modify the PRDM12 activity are envisaged. The invention further relates to pharmaceutical compositions comprising a modulator of PRDM12 and to methods for diagnosing hypersensitivity to pain.

Description

MODULATION OF PRDM12 FOR USE IN TREATMENT OF PAIN CONDITIONS
FIELD OF THE INVENTION
The invention relates to modulation of the PRDM12 gene and PRDM12 gene products for use in the treatment or prevention of pain in a subject. The finding is of particular interest to the fields of medical biotechnology and medicine. The finding is applicable to numerous pain conditions and pain sensations.
BACKGROUND OF THE INVENTION
Pain perception is an evolutionarily conserved warning mechanism that alerts us from environmental dangers and potential damaging stimuli, and is therefore essential for our survival. Pain is also causing untold misery and suffering when the pain-sensing system becomes over- or erroneously activated. Chronic inflammatory and neuropathic pain is a major public health burden, estimated to affect one fifth of the population worldwide.
Pain is a complex unpleasant experience that results from the initial activation of a subset of sensory neurons called nociceptors. The cell bodies of nociceptors as well as those of other somatic sensory neurons (proprioceptors, mechanoreceptors) are located in specialised ganglia of the peripheral nervous system (PNS): the trigeminal (TG), superior-jugular (SJG) and accessory ganglia in the head region, and the dorsal root ganglia (DRG) found in a metameric pattern on each side of the spinal cord in the body region. Nociceptors are slow conductive thinly myelinated A6- or unmyelinated C-fibers. They are pseudo-unipolar neurons with a unique process attached to the cell body that bifurcates into two branches. One branch, referred to as the distal process, projects to cutaneous or deep peripheral tissues. The other one, referred to as the proximal process, stereotypically projects onto interneurons in specific laminae in the dorsal hom of the spinal cord or in sensory nuclei of the brainstem to process and transmit the nociceptive signal to higher brain centers. Nociceptors can be subdivided into several subtypes according to several features such as the stimuli they detect, their underlying molecular characteristics and innervation pattern. Two main groups can be distinguished: peptidergics and nonpeptidergics. Peptidergic nociceptors secrete neuropeptides such as Substance P and CGRP, and express the Nerve Growth Factor receptor TrkA. Non-peptidergic nociceptors express receptors such as Ret or P2X3. Anatomically, non-peptidergic nociceptors preferentially innervate the skin whereas peptidergics innervate most parts of the body (F. C. Yang et al., Cell reports, 2013). They can be further classified on the basis of their functional specialisation. Their ability to detect various specific, generally intense stimuli, of thermal, mechanical or chemical nature, is conferred by the combinatorial expression of modality specific receptors. Among them the Transient Receptor Potential (TRP) ion channels that respond to specific ranges of temperature and also to some chemicals (Table 1; Basbaum et al., Cell, 2009; Emery & Emfors, The Oxford Handbook of the Neurobiology of Pain, 2018; Usoskin et al., Nature Neuroscience, 2015; Zeisel et al., Cell, 2018). While nociceptor sensitisation after injury drives behavioral changes that enhance survival, the persistent and excessive excitation of nociceptors is also a key process in the development of neuropathic and chronic pain syndromes. However, currently available analgesics mostly target central nervous system (CNS) neurons, and are often inappropriate or inefficient (Colloca et al., (2017) Neuropathic pain. Nature Reviews Disease Primers, 3, 17002).
PRDM12 is an evolutionarily conserved epigenetic transcriptional regulator whose mutation causes a rare disease, congenital insensitivity to pain (CIP) disorder, which is characterised by the inability to perceive pain from birth. Mutations in only a few other genes (NGF, TrkA,. . . ) have been shown to lead to CIP. New analgesics can be generated from the discovery of these CIP causing genes. However, until now, clinical trials that have been initiated targeting these other CIP gene products have had limited success due to severe side effects (Drissi et al., British Medical Bulletin, 2020).
Despite the prevalence of chronic pain, currently available pain therapeutics often display limited efficacity and/or have serious side effects. The development of such innovative pain therapeutics requires a better understanding of the mechanisms leading to the development of pain as a pathological condition (Price & Gold, Pain medicine, 2018; Van Hecke et al., (2013) Chronic pain epidemiology and its clinical relevance. British Journal of Anaesthesia, 111(1), 13-18). There is therefore an unmet need to develop innovative strategies that are effective to modulate and steer pain sensation in a subject, which has not been addressed in the art thus far. While a considerable amount of research has been conducted on the subject of pain, no studies have investigated the role of the above-mentioned gene and gene product(s) in different pain contexts, and do not report on differences in pain type specific responses of said gene (products). SUMMARY OF THE INVENTION
The present invention identifies Prdml2 as an interesting pain target given its restricted expression in the nervous system suggesting that any compound targeting its action would have no side effect, its close link with NGF known to be involved in nociceptor sensitization, and the importance of epigenetic mechanisms in the induction and maintenance of chronic pain. Additionally, form the below, it can be concluded that in some pain disorders, it is the enhancement of Prdml2, rather than its blockade, that may have an analgesic effect.
The inventors obtained a detailed and thorough understanding of the molecular biology of Prdml2. Prdml2 functions as a transcriptional regulator in mature nociceptors of adult mice, controlling positively or negatively the expression of several receptors, ion channels and neuropeptides. Furthermore, loss of Prdml2 induced hypersensitivity in animals injected with formalin in the hindpaw during the second phase of the pain response, which is characterised by the involvement of inflammatory processes (Erami et al., (2017) Basic and Clinical neuroscience 8, 37-42).
The experimental data below provides evidence for the highly restricted expression of Prdml2 to sensory neurons in adult mice. In addition, we show that peripheral inflammation decreases its expression in mature nociceptors.
The bulk RNA-seq analysis of the transcriptome of DRG of both Advil and Rosa26 icKO models shows that the loss of Prdml2 slightly deregulates the expression of many genes in nociceptors. Comparing the list of DEGs in the two models, a core set of 71 DEGs was identified, mostly neuronal genes, about half of them being downregulated and the other half upregulated by the loss of Prdml2, suggesting that Prdml2 may act as an activator or a repressor in mature nociceptors. Landy and colleagues recently published the results of bulk RNA-seq analysis of DRGs of adult Prdml2 knockout mice, generated like us by crossing AvilCreERT2 mice to Prdml 2^ mice (Landy et al., 2021, Loss of Prdml2 during development, but not in mature nociceptors, causes defects in pain sensation. bioRxiv 2020:2020.09.07.286286. doi: 10.1101/2020.09.07.286286). Contrary to the results provided herein, they found that almost all DEGs were decreased in mutant mice. Comparing their transcriptomic data to our list of core 71 DEGs, surprinsingly, only 1 common gene was found, Chma6. One possible explanation for these discrepancies is that in the conditional knock-out mouse model they used, only the last exon of Prdml2 (exon V) is deleted. In this knockout, a transcript composed of exons I-IV is still detected. This transcript encodes a truncated protein with the conserved PR domain but lacking the zinc fingers. As it is thought that Prdml2 does not bind directly to DNA, this truncated protein may thus potentially retain some activity.
In the list of core Prdml2 DEGs genes, channel proteins and transmembrane receptors are overrepresented, providing first evidence that Prdml2 plays an essential role in the functional properties of adult nociceptors.
Despite the effect of Prdml2 deficiency on transcription, it was found that mice lacking Prdml2 exhibit normal response to thermal nociceptive stimuli under basal conditions. Unaltered responses to noxious cold is somehow surprising given the fact that we found that Trpm8 is reduced in Prdml2 icKO mice, as observed by Landy et al. (2021). This unaltered response to acute noxious cold may be due to the fact that Tprm8 expression is reduced, but not abolished in DRG neurons of Prdml2 icKO mice. It also likely reflects the complexity of the cold-sensing mechanisms in mouse DRG that involve other molecular candidates apart from Trpm8, such as TrpAl, that is not deregulated in Prdml2 icKO DRG. Mechanical nociception was also unaltered in icKO mice, a result in accordance with the absence of modification of the expression of TRPA1 and TRPV4, two channels suggested to be involved in mechanical pain (Helge Eilers and Mark A. Schumacher, Mechanosensitivity in Cells and Tissues. Moscow: Academia; 2005). We however found that in icKO mice spent less time licking their paw than controls after capsaicin injection. This reduced sensitivity is at first glance surprising as TRPV1 expression is not modified. This could however be due to differences between icKO and controls in the localization of TRPV1 receptors that is rapidly internalized after stimulation. Indeed, capsaicin can be used as analgesic and has been showed to reduce lesion or inflammation-induced pain by triggering receptor endocytosis or nerve retraction. It could also be due to post-transcriptional modifications of TRPV1, its activity being modulated by kinases (CaMKII, PKA, PKC) and phosphatases (calcineurin) or lipids such as PIP2, themselves regulated by G-coupled membrane receptors. More surprinsingly, it was also shown here that Prdml2 icKO mice develop in contrast increased sensitivity to formalin-induced inflammatory pain. Without wanting to be bound to any theory, the gene expression analysis of icKO mice revealed that the loss of Prdml2 dysregulates a set of genes known to contribute to formalin induced nociceptor sensitization. Among them Trpm8, Gfra3, Ntrkl, ASIC1 and Grikl that are downregulated, and Chrna6 and Cysltr2 that are upregulated in the absence of Prdml2 in basal conditions. The upregulation of Chrna6 is unexpected as its overexpression is protective against tactile allodynia associated with inflammatory injuries. The downregulation of Grikl and TrkA was also unpredicted since Grikl deletion reduces inflammatory pain and NGF acts as a mediator of inflammatory pain. However, the deregulation of other genes may play a role in the observed hypersensitivity of Prdml2 icKO mice. Indeed, previous studies have shown that formalin-evoked pain can be alleviated by cooling analgesia through Trpm8 -dependent receptor activation. Moreover, recent studies indicate that the activation of Trpm8 receptor primarily affects late, but not early phases of DRG neuron activity after formalin injection. The decreased expression of GFRa3 and ASIC1 may also contribute to the increased sensitivity to inflammatory pain in Prdml2 icKO mice. Indeed, artemin, a member of the GDNF family of ligand that signals through the GFRa3-RET complex has been shown to reduce formalin-induced pain behaviors and mice lacking ASIC1 display increased pain behavior in the formalin test. The upregulation of Cysltr2 may also contribute to the hyperalgesia. Indeed, Cysltr2 is a known mediator of inflammatory reactions. In their recent report, Landy and colleagues found that the knock out of Prdml2 exon V does not substantially modify pain sensitivity. A tendancy not reaching significance to a reduction of the licking time of exon V Prdml2 knockout was however apparent in their model after capscaicin injection. In the formalin test, they also observed an increase of licking but only 20 minutes after injection. We posit that the retention of exon II-IV in their knockout models, absent in our exon II Prdml2 knockout model, may be the cause of this milder phenotype.
The electrophysiological data provided herein further supports the idea that that Prdml2 plays a role in the functional properties of mature nociceptors. The increased excitability of cultured DRG neurons from icKO mice may provide some explanation to their hypersensitivity in the formalin test. The deregulation of some potassium channels such as the calcium-activated potassium channel Kcnn3 and the calcium-activated potassium channel subunit beta-1 Kcnmbl could be involved in this increased excitability and increased response to formalin, as it was shown for Kcnn4. It is to note that their downregulation alone would also have increased the response to capsaicin. The opposite observed effect could therefore result from other concomitant gene dysregulations.
Taken together, these results demonstrate that Prdml2 plays an important role in the transcriptional regulation of gene expression in mature nociceptors resulting in a complex modulation of pain-related behavior.
Thus, the inventors provide evidence that Prdml2, depending on the context, may have an analgesic effect, hereby providing the medical field with a new active pharmaceutical ingredient that is effective for managing pain responses, and thus pain sensation in subject. Indeed, in certain pain conditions, the enhancement of Prdml2, rather than Prdml2 blockade is beneficial for amelioration of the pain condition. Prdml2, or any modulator of Prdml2, may hence be used as a prophylactic or therapeutic component in pharmaceutical compositions. In particular, pain responses related to or caused by inflammatory processes such as but not limited to skin or joint inflammation and pain related to itch are envisaged to be particularly suited for treatment through Prdml2 modulation.
The present disclosure shows that Prdml2 function in pain responses is more complex than the role previously hypothesised in the art for this gene and related gene products. Surprisingly, Prdml2 function is dependent on previously disregarded parameters including different pain response phases and the specific pain condition that is induced. Undoubtedly, the findings, which are described in detail in and fully supported by the examples of the present disclosure profoundly change the traditional views on the role of Prdml2 in pain management.
The invention therefore relates to the following numbered aspects:
Aspect 1. A modulator ofPrdm!2 activity, foruse intreatment or prevention of a pain condition in a subject.
Aspect 2. The modulator for use according to aspect 1, wherein said modulator alters the expression level of Prdml2 in nociceptors and/or dorsal root ganglia of said subject.
Aspect 3. The modulator for use according to aspect 2, wherein said modulator increases Prdml2 expression in nociceptors and/or dorsal root ganglia of said subject, preferably wherein Prdml2 expression is increased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator.
Aspect 4. The modulator for use according to aspect 2, wherein said modulator decreases PRDM12 expression in nociceptors and/or dorsal root ganglia of said subject, preferably wherein PRDM12 expression is decreased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to PRDM12 expression in nociceptors and/or dorsal root ganglia of said subject before administration of said PRDM12 modulator.
Aspect 5. The modulator for use according to aspect 1, wherein the modulator alters the Prdml2-mediated G9a recruitment to histone H3, preferably wherein the modulator alters the methylation status of histone H3, preferably the methylation status of H3K9.
Aspect 6. The modulator for use according to aspect 5, wherein the modulator increases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to histone H3 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of said Prdml2 modulator. Aspect 7. The modulator for use according to aspects 5 or 6, wherein said modulator decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes comprising: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, by at least 25%, preferably at least 50%, more preferably at least 75%, most preferably 100% when compared to their transcription rate in nociceptors and/or dorsal root ganglia before administration of said Prdml2 modulator. Obviously modulators decreasing the activity of the gene product of any one of said genes, i.e. the protein is also envisaged in this aspect.
Aspect 8. The modulator for use according to aspect 5, wherein the modulator decreases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, most preferably inhibits methylation of histone H3, when compared to histone H3 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
Aspect 9. The modulator for use according to aspects 5 or 8, wherein said modulator increases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes comprising: PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, and NTS by at least 25%, preferably at least 50%, more preferably at least 75%, most preferably 100% when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator. Obviously modulators increasing the activity of the gene product of any one of said genes, i.e. the protein is also envisaged in this aspect.
Aspect 10. The modulator for use according to any one of aspects 1 to 9, preferably wherein the subject is suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or is a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, neuropathic itch, inflammatory itch such as atopic dermatitis, non-inflammatory itch such as pruritus-related pain, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
Aspect 11. The modulator for use according to any one of aspects 1 to 10, wherein the subject is a mammalian subject, preferably a human subject.
Aspect 12. The modulator for use according to any one of aspects 1 to 11, wherein said modulator is a Prdml2 inhibitor, a Prdml2 activator, mutant Prdml2, or Prdml2, preferably wherein the modulator is anull loss-of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutantcaps. Preferably said modulator is selected from the group comprising: Prdml2 binding molecules, Prdml2 protein or a polynucleotide encoding Prdml2, a functional fragment of Prdml2, or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene-expression or transcription targeting system, a transcription or expression inducer of the PRDM12 gene, a PRDM12 gene targeting system, inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof. Preferably, said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
Aspect 13. The modulator for use according to any one of aspects 1 to 12, wherein said modulator alleviates pain in a subject by at least 1, preferably at least 2, at least 3, at least 4, more preferably at least 5, at least 6, at least 7, at least 8 scale points as reported by the treated subject when using a self-reporting pain scale, more preferably wherein the self-reporting pain scale is the numeric rating scale (NRS-11), Stanford pain scale, or visual numeric scale.
Aspect 14. A pharmaceutical composition comprising Prdml2 and/or a Prdml2 modulator as described herein.
Aspect 15. A pharmaceutical composition according to aspect 14, for use in treating or preventing a pain condition in a subject, preferably wherein said subject is suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or is a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain (both inflammatory or noninflammatory), neuropathic itch, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
Aspect 16. The pharmaceutical composition according to aspect 14 or the pharmaceutical composition for use according to aspects 15, wherein said composition further comprises at least one additional analgesic, preferably wherein said analgesic is selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal -inflammatory drugs, opioids, muscle-relaxants, anti-anxiety agents, antidepressants, anticonvulsants and corticosteroids.
Aspect 17. A method for diagnosing a hypersensitivity to pain in a subject, the method comprising a step of determining the expression of Prdml2 in a biopsy of said subject, wherein reduced expression of Prdml2 in nociceptors and/or dorsal root ganglia is indicative of a hypersensitivity to pain. Aspect 18. The method according to aspect 17, wherein the hypersensitivity to pain is allodynia and/or hyperalgesia.
Aspect 19. An analgesic comprising a modulator of Prdml2 activity.
Aspect 20. The analgesic according to aspect 19, wherein modulator is PRDM12 or a functional fragment thereof, a Prdml2 inhibitor, a Prdml2 activator, mutant Prdml2, or Prdml2, preferably wherein the modulator is a null loss-of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutant. Preferably said modulator is selected from the group comprising: Prdml2 binding molecules, Prdml2 protein or a polynucleotide encoding Prdml2, a functional fragment of Prdml2, or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene-expression or transcription targeting system, a transcription or expression inducer of the PRDM12 gene, a PRDM12 gene targeting system, inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof. Preferably, said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
Aspect 21. A method of diagnosing a hypersensitivity to pain in a subject, the method comprising determining the level G9a recruitment to histone H3, preferably Prdml2-mediated recruitment to histone H3 in nociceptors and/or dorsal root ganglia in a biopsy of said subject. Aspect 22. A method for diagnosing a hypersensitivity to pain in a subject, the method comprising determining the methylation status or representative methylation status of H3K9 in nociceptors and/or dorsal root ganglia in a biopsy of said subject.
Aspect 23. The method according to aspects 21 to 22, wherein the hypersensitivity to pain is allodynia and/or hyperalgesia.
Aspect 24. A method of treating or preventing a pain condition in a subject, comprising administering a modulator of Prdml2 activity as described herein or a pharmaceutical composition comprising a Prdml2 modulator as described herein, or an analgesic according to aspect 19 or 20 to a subject. In some embodiments the subject is a subject suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, neuropathic itch, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch. Aspect 25. The method according to aspect 24, wherein the modulator of Prdml2 activity is an activator of Prdml2 activity. More preferably, when the modulator of Prdml2 activity is an activator, said pain condition is a pain condition induced by inflammation or itch, preferably inflammatory pain. More generally, said pain condition could be seen as a TRPV 1 -related pain condition, more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders (which include e.g. bladder pain syndrome (previously interstitial cystitis), irritable bowel syndrome (IBS) and fibromyalgia) and musculoskeletal pain. Many of these conditions are chronic pain conditions and many involve a burning sensation in the subject.
Aspect 26. The method according to aspect 25, wherein said modulator is a polynucleotide encoding Prdml2, or a functional fragment or activated mutant of Prdml2, or wherein said activator is a transcription or expression inducer of the PRDM12 gene. Preferably, saidPrdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
In one embodiment, said PRDM12 activator is a functional fragment of PRDM12 that retains a certain level of activity of the PRDM12 protein, such as 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the activity of the full-length PRDM12. In a particular embodiment, the PRDM12 activator is a fragment that comprises or consists of exons I-IV of the PRDM12 gene, resulting in a truncated protein with the conserved PR domain but lacking the zinc fingers. As it is thought that Prdml2 does not bind directly to DNA, this truncated protein may thus potentially retain some activity.
Aspect 27. The method according to any one of aspects 24 to 26, wherein the modulator is administered through gene therapy, or is administered systemically and/or topically.
Aspect 28. The method according to any one of aspects 24 to 27, wherein the modulator is comprised in a viral particle, preferably an adeno-associated viral particle. Well-known AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAVPHPS, AAVrh20 and AAV9. Gene expression in DRG neurons has been shown using AAV vectors based on serotypes 1, 2, 3, 4, 5, 6, 8, 8.2, 9 and rh20. Preferred vectors are AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self- complementary AAV9 vector (scAAV9). In a preferred embodiment, said vector system comprises the PRDM12 mRNA (SEQ ID NO:2), or encodes the Prdml2 protein or a functional fragment thereof.
Aspect 29. The method according to aspect 24, wherein the modulator of Prdml2 activity is an inhibitor of Prdml2 activity. More preferably, when the modulator of Prdml2 activity is an inhibitor, said pain condition is heat induced pain or chemically induced pain, allodynia and/or hyperalgesia, or a pain that can be treated by capsaicin such as back pain, joint pain or headaches.
Aspect 30. In a preferred embodiment of the method according to aspect 29, said inhibitor is selected from: inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof. Preferably, said Prdml2 protein is defined by SEQ ID NO:1 and/or said PRDM12 mRNA encoding Prdml2 is defined by SEQ ID NO:2.
When the modulator is an inhibitor of PRDM12 activity, the subject preferably is a subject suffering from heat-induced pain, operative pain, treatment-related pain, injury -related pain, or trauma-related pain.
Aspect 31. Use of a modulator of Prdml2 activity or PRDM12 expression as described herein for the manufacture of a medicament for the prevention or treatment of a pain condition as defined herein.
Aspect 32. Use of Prdml2, PRDM12, or a functional fragment thereof as described herein, or an activator of PRDM12 activity for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
Aspect 33. Use of an inhibitor of Prdml2 or PRDM12 activity, or a modulator thereof according to aspects 30 or 31, wherein said pain condition is heat induced pain.
Aspect 34. An in vitro method for identifying a molecule suitable as an analgesic, wherein said method comprises determining whether a candidate molecule modulates Prdml2 activity in nociceptors and/or dorsal root ganglia.
Aspect 35. The in vitro method according to aspect 34, wherein said method comprises contacting a nociceptor and/or a dorsal root ganglion with a candidate Prdml2 modulator, preferably wherein said nociceptor or dorsal root ganglion expresses Prdml2.
Aspect 36. The in vitro method according to aspects 34 or 35, wherein Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity. Aspect 37. The in vitro method according to aspect 34 or 35, wherein Prdml2 activity is assessed by measuring expression of one or more genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
Aspect 38. The in vitro method according to aspect 37, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 activator when the expression of one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
Aspect 39. The in vitro method according to aspect 37, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 inhibitor when the expression of one or more genes selected from the group consisting of: PRDM12, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
Aspect 40. The in vitro method according to any one of aspects 35 to 39, wherein the nociceptors and/or dorsal root ganglia are human nociceptors and/or dorsal root ganglia.
Aspect 41. In any one of aspects 1 to 33, said Prdml2 modulator can have been identified by the method according to any one of aspects 34 to 40.
More preferably, in any one of aspects 3, 8, 9, 10, 11, 12, 13, said Prdml2 activator can have been identified by the method according to any one of aspects 34 to 40; or alternatively, in any one of aspects 4, 5, 6, 7, 11, 12, 13, said Prdml2 inhibitor can have been identified by the method according to any one of aspects 34 to 40.
Aspect 42. A nucleotide sequence encoding Prdml2 or encoding a modulator of Prdml2 activity as described herein, wherein said nucleotide sequence encodes a molecule capable of binding to human Prdml2, or wherein said modulator is a nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2, preferably wherein Prdml2 is human Prdml2 as defined by SEQ ID NO: 1 or a functional fragment thereof as defined above. Aspect 43. The nucleotide sequence according to aspect 42, wherein the nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2 is a nucleotide sequence comprising a nucleotide sequence with a sequence identity of at least about 65%, preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 97%, at least about 99% to SEQ ID NO: 2 or a portion thereof.
Aspect 44. A viral vector encoding PRDM12 or encoding a modulator of Prdml2 activity as described herein, preferably wherein said modulator is a Prdml2 transgene, preferably wherein Prdml2 is human Prdml2 transgene, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or wherein said human PRDM12 transgene is defined by SEQ ID NO: 2. Alternatively, a functional fragment of PRDM12 as defined above can be used. Aspect 45. The viral vector according to aspect 44, for use in gene therapy, preferably human gene therapy.
Aspect 46. The viral vector according to aspect 44 or 45, wherein said viral vector is an AAV vector, such as an AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self-complementary AAV9 vector (scAAV9) viral vector. Aspect 47. A non-viral vector system encoding Prdml2 or encoding a modulator of Prdml2 activity as described herein, preferably wherein said modulator is a Prdml2 transgene, preferably wherein Prdml2 is human Prdml2 transgene, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or wherein the human PRDM12 transgene is defined by SEQ ID NO: 2. Alternatively, a functional fragment of PRDM12 as defined above can be used. In preferred embodiments, said non-viral vector is selected from the group comprising: cationic lipids, liposomes, nanoparticles, PEG, PEI; plasmid vectors (e.g. pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequencesO, a minicircle, an episomal vector, or a transposon-based vector, such as a PiggyBac-based vector or a Sleeping Beauty-based vector.
Aspect 48. In one embodiment of the above aspects, the Prdml2 modulator is a Prdml2 activator such as retinoic acid (RA) or zinc finger protein of the cerebellum 1 (Zicl).
Aspect 49. In one embodiments, said Prdml2 modulator is a Prdml2 inhibitor such as an inhibitor of alcohol and/or aldehyde dehydrogenases, for example citral.
Aspect 50. The method according to any one of aspects 24 to 29 comprising gene therapy using the viral or non viral vector according to any one of aspects 44 to 47.
Some interesting newly identified genes (i.e. to our knowledge not previously linked to pain management) are:
Ppefl: protein phosphatase with EF hand calcium-binding domain 1;
Ppmlj: protein phosphatase 1J;
Mettl7a3: methyltransferase like 7 A3;
Kcnmbl: potassium large conductance calcium-activated channel, subfam. M, beta member 1; Otoa: otoancorin;
Smr2: submaxillary gland androgen regulated protein 2; and
Ms4a3: membrane-spanning 4-domains, subfamily A, member 3.
Lrm4: leucine rich repeat neuronal 4;
A3galt2: alpha 1,3-galactosyltransferase 2; klk5: kallikrein related-petidase 5.
Hence the present application also provides for the following aspects:
Aspect 51. A method of identifying pain modulating agents comprising the step of screening candidate agents for their ability to modulate any one or more of the following genes:; Ppefl; Ppmlj; Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3, Lrm4, A2galt2 or Klk5. Aspect 52. A method of identify agents for use in treating a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch, comprising the step of screening candidate agents for their ability to restore or upregulate the expression, activity or function of any one or more of the following genes: Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3; Lrm4; A2galt2; or Klk5.
Aspect 53. A method of identify agents for use in treating a pain condition, more preferably wherein said pain condition is heat induced pain or chemically induced pain, comprising the step of screening candidate agents for their ability to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj. Aspect 54 The modulating agents identified in aspect 48 for use in treating a pain condition as defined herein.
Aspect 55. A method of treating a pain condition as defined herein comprising administering to a patient in need thereof a modulating agent identified in aspect 48.
Aspect 56. Use of a modulating agent identified in aspect 48 for the manufacture of a medicament for the prevention or treatment of a pain condition as defined herein.
Aspect 57. Use of an agent able to restore or upregulate the expression, activity or function of any one or more of the following genes: Ppefl; Ppmlj; Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3, Lrm4, A2galt2 or Klk5.2 for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
Aspect 58. A method of treating a pain condition comprising administering to a subject in need thereof a therapeutically effective amount of an agent able to restore or upregulate the expression, activity or function of any one or more of the following genes: Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3; Lrm4; A2galt2; or Klk5, more preferably wherein said pain condition is inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
Aspect 59. An agent able to restore or upregulate the expression, activity or function of any one or more of the following genes: Mettl7a3; Kcnmbl; Otoa; Smr2; Ms4a3; Lrm4; A2galt2; or Klk5 for use in the prevention or treatment of a pain condition, more preferably inflammatory pain or itch-induced pain, such as nerve injury preferably wherein said pain condition is caused by or suspected to be caused by skin or joint inflammation or itch.
Aspect 60. Use of an agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj for the manufacture of a medicament for the prevention or treatment of a pain condition, more preferably in heat induced pain or chemically induced pain, allodynia and/or hyperalgesia, or pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches.
Aspect 61. A method of preventing or treating of a pain condition, more preferably in heat induced pain or chemically induced pain, allodynia and/or hyperalgesia, or pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches, comprising administering to a subject in need thereof a therapeutically effective amount of an agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj or Ppefl.
Aspect 62. An agent able to downregulate the expression, activity or function of any one or more of the following genes: Ppmlj or Ppefl for use in the prevention or treatment of a pain condition, more preferably in heat induced pain or chemically induced pain, allodynia and/or hyperalgesia or pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Validation of the AdvillinCreERT2; Prdml2fl fl mice. Immunofluorescence with anti-Prdml2 antibodies on dorsal root ganglia (DRG) section of adult mice of the indicated genotype injected with Tamoxifen and sacrificed 4 days after injection for DRG dissection.
Figure 2. Loss of Prdml2 in DRG of adult mice does not lead to alteration of DRG neuronal subpopulations. A) DRG sections of Advillin-CreERT2; Prdml2fl/fl mice injected with Tamoxifen or with com oil as control were double labelled with peripherin (labelling small neurons) and IB4-biotin or anti-CGRP, or NF-200 (labelling large diameter neurons) and Navi.8. B) No significant difference in the number of Peripherin+ IB4 binding nonpeptidergic, peripherin+ CGRP expressing peptidergic, NF200+ proprio- and mechanoreceptive neurons and Navl.8+ nociceptors in DRG of icKO samples compared to controls. About 8 sections per animals were analysed (n=4 for each genotype). Data were analysed using the 2 tailed t-student test. Values are means ± SD.
Figure 3. Loss of Prdml2 in DRG of adult mice leads to altered expression of several nociceptive genes. Volcano plot showing the fold change and significance of the most misregulated genes in DRG between Advil icKO mutants (n=5) and controls (n=4), with nociceptor expressed genes decreased in the mutant (negative log2 fold change) indicated on the left-hand side of the dashed line and upregulated on the right-hand side of the dashed line. Figure 4. Validation of the differential expression of selected up- and down-regulated genes identified in the RNA-seq of TAM injected Advillin-CreERT2; Prdml 21111 mice (icKO) by RT-qPCR. As expected, Prdml2 is knocked-out. The data confirm the downregulation of Trpm8, Creg2 and Mrgprb5 and the upregulation of Cysltr2, Sst, Stk32a and Chrna6 in icKO (n= 7 for WT and 5 for mutants). *, **, ***, and **** indicate respective p values <0,05, <0,01, <0,001, and <0,0001 (Student's t-test, a = 0,05).
Figure 5. Validation of the differential expression of selected down-regulated genes identified in the RNA-seq of TAM injected Advillin-CreERT2; Prdml 2^ mice (icKO) by TSH on DRG sections. The data confirms the reduced expression of the indicated selected differential expressed genes. WT: wild type (Advillin-Cre-ERT2; Prdml 2^ mice injected with corn oil, or Prdml 2^ mice injected with tamoxifen).
Figure 6. Validation of the differential expression of selected up-regulated genes identified in the RNA-seq of TAM injected Advillin-CreERT2; Prdml 2M1 mice (Prdml2 icKO) by ISH on DRG sections. The data confirms the increased expression of Cysltr2. WT: wild type (Advillin-Cre-ERT2 ; Prdml 2^ mice injected with corn oil, or Prdml 2^ mice injected with tamoxifen).
Figure 7. Loss of Prdml2 increases sensitivity to inflammatory pain induced by subcutaneous injection of formalin in the hindpaw. (A) Durations of licking of TAM injected Advillin-CreERT2;Prdml2)l/fl mice (n= 11, closed square) and TAM injected Prdml 2^ control mice (n= 15, closed circle) of the formalin-injected paw is shown (characteristic movement shown in top graphic of the figure). Data are collected at 5 min intervals until 30 min after the formalin injection (0 min). There is no significant difference in licking duration between the TAM injected Adx’illin-CreERT2;Prdml2^ and control mice in the first phase of the response which results from direct sensitization of nociceptors. However, TAM injected Advillin-CreER12;Prdml2fl/-11 mice showed significantly enhanced pain behaviour in the second phase during which inflammatory phenomena take place. (B) Licking duration of individual mutant and control mice analysed at 30 min and 25 min after formalin injection.
Figure 8. Prdmll is expressed in nociceptors of adult mice and is downregulated upon CFA induced inflammation. (A) X-Gal staining of the brain (ventral view) with attached trigeminal ganglia (TG) of an adult Prdml 2 LacZ/+ mouse. Superior jugular ganglia (SJG) and spinal cord (SC) with attached dorsal root ganglia (DRG) are shown on the right. (B) Double
RECTIFIED SHEET (RULE 91) ISA/EP immunostainings on transverse sections of TG, SJG and DRG of adult mice with the indicated markers. (C) Representative images of the expression of Prdml2 and Trpvl in knee-innervating DRG neurons (L3-L4) of CFA injected mice and percentage of Prdml2 and TrpVl positive cells counted from the Ctrl side and CFA injected sides (n = 4, >300 neurons counted in each condition, scale bar = 50 pm). *indicates p < 0.05, **indicates p < 0.01, paired t-test.
Figure 9. Loss of Prdml2 in nociceptor of adult mice does not alter diversity of DRG neuronal subpopulations. (A) Representative images of DRG sections of Avil icKO and Rosa26 icKO injected with tamoxifen or with com oil as control immunolabelled for Peripherin, CGRP, Navi.8 and NF-200 or stained with IB4, with quantification showing that the number of cells positive for the different indicated markers is similar in DRG sections of Prdml2 icKO and control mice. (B) Proportion of CGRP+ neurons and of IB4+ neurons among Peripherin+ neurons in DRG are similar in both icKO and control mice. (C) Proportions of DRG neurons positive for Navi.8 and NF200 are unchanged in DRG sections of both icKO. (D) Tujl mmunostainings on skin tissue of Avil icKO and Rosa26 icKO injected with tamoxifen and controls. Nuclear are stained with DAPI. Arrows point skin epidermis innervation. E (epidermis), D (dermis), M (muscle). (E) Left panel, blll-Tubulin immunostainings on skin tissue of T AM-injected Rosa26 icKO and control mice. Arrows point skin epidermis innervation (E 5epidermis; D 5dermis). Scale bar: 50 mm. Right panel, Quantification showing the percentage of labelled sensory terminals invading the epidermis in TAM-injected Rosa26 icKO mice compared to controls. In all experiments, n > 3 for both genotypes. All quantifications were submitted to the 2 tailed t-student or two-way Anova tests. Values are means ± SD. **** p <0.001. DRG, dorsal root ganglia; icKO, inducible conditional knockout.
Figure 10. Transcriptomic analysis of the consequences of the loss of Prdml2 in DRG nociceptors of adult mice. (A) Volcano plots showing the -loglO adjusted p-value as a function of the log2 fold-changes of deregulated genes in DRG of Avil icKO (icKO n=5 versus control mice, n=4) and Rosa26 icKO (icKO n=5 versus control mice, n=4) one month after tamoxifen injection. The genes were identified by bulk RNA-seq and selected based on adjusted p-value <0.05. Genes with a log2FoldChange > 0,449 are indicated in red, genes below the threshold are in light grey. (B) Venn diagram showing the overlap between the DEGs identified in Avil icKO and Rosa26 icKO. (C) Proportion of neuronal genes among core Prdml2 DRGs. Neuronal genes up- and downregulated by the loss of Prdml2 are indicated in blue and red, respectively. (D) Classification of the common DEG’s based on their expression in the major different subtypes of DRG neurons, with downregulated genes in red and upregulated genes in blue. (E) Gene ontology classification of the DEGs. The graph shows the enriched biological molecular function associated with downregulated and/or upregulated genes.
Figure 11. Validation of some of the identified DEGs as Prdml2 targets in Advil icKO mice. (A) RT-qPCR analysis of the indicated genes. (B) ISH analysis of the expression of the indicated genes. *, **, ***, and **** indicate respective p values <0,05, <0,01, <0,001, and <0,0001 (Student's t-test, a = 0,05).
Figure 12. Knockout of Prdml2 in mature nociceptors does not alter thermal and mechanical nocociception but affects responses to formalin and capsaicin. (A) Avil icKO and control (TAM injected Prdml^M1 mice) spent similar time on the test side in a two- temperature choice assay. (B) Rosa26 icKO (n=l l) and control mice (n=9) show similar withdrawal latency in the tail flick test using a focus 30. (C) Rosa26 icKO (n=10) and control mice (n=12) show no difference in withdrawal latency in the cold plantar assay. (D) Rosa26 icKO and control mice take show the same behavior in the Mechanical conflict avoidance test. They spent the same time to cross half of the second chamber with nails to the floor. Results obtained with two different nail heights (2 and 5mm) are shown. (E) Rosa26 icKO mice (n=10) spent less time licking than control mice (TAM injected, n=12) after capsaicin injection. Values are means ± SEM. *p<0.05 (Student’s t-test, a = 0,05). (F) Time course of the nocifensive response (licking time in seconds) of tamoxifen injected Prdml2 icKO (n=l 1) and control mice (n=14) until 30 min after injection. Response of individual icKO and control mice in the first (0-5min post-formalin injection) and second phase (15-30min post-formalin injection) is shown on the right. Values are means ± SEM. **p<0.01 (Student’s t-test, a = 0,05).
(G) Time course of the nocifensive response (licking time in seconds) of Rosa26 icKO (n=15) and control mice (n=16) until 30 min after formalin injection. Response of individual Rosa26 icKO and control mice in the first (0-5min post-formalin injection) and second phase (15- 30min post-formalin injection) is shown on the right. Note that Rosa26 icKO mice spent more time licking their paw when compared to control mice during the second phase (p = 0.019). Values are means ± SEM. *p<0.05 (Student's t-test, a = 0.05).
Figure 13. Loss of Prdml2 leads to hypersensitivity in a model of inflammatory pain
(A) Representative traces from a control DRG neuron, showing subthreshold responses and subsequent action potential induced by injection of 1310 pA (top), and action potential frequency induced by a one-second 1500 pA step (bottom). The same protocol was applied to a Prdml2 KO DRG neuron (B). The current threshold was 750 pA for this neuron. Arrows indicate the current amplitude used to elicit the labeled response. (C) Comparison of current threshold in control (n = 19) and Prdml2 KO (n = 30) DRG neurons. (D) Maximal number of action potentials evoked in response to external current stimuli of 1 s, up to 1500 pA in control (n = 19) and Prdml2 KO (n = 30) DRG neurons. (E) Resting membrane potential in control (n=19) and Prdml2 KO (n = 30) DRG neurons. Each column represents mean ± SEM. *p<0.05, **p<0,01 (Student’s t-test).
Figure 14. Validation of the Avil icKO and Rosa26 icKO mice. (A) Prdml2 immunostaining performed one month after tamoxifen injection showing loss of Prdml2 in a DRG of a TAM- injected Avil icKO containing a Cre dependent tdTomato reporter gene (AvilCreERT2;Rosa26ill4;Prdml2fl/fl) compared to a control DRG (AvilCreERT2;Rosa26itl4;Prdml2fl/+) and quantification showing reduction of Prdml2+ ZtdTomato+ cells in DRG of Avil icKO mice.
(B) Double immunostaining for Prdml2 and Peripherin on a DRG of a Rosa26 icKO (TAM injected Rosa26CreERT2/Rosa26CreERT2; Prdml2fl/f1') and of a control mice (Prdml2+/+) and quantification showing reduction of Prdml2+ cells in DRG of mutant mice.
Figure 15. Gene ontology analysis and KEGG pathway analysis of the Prdml2 core DEGs. (A) Gene ontology analysis based in all categories of the 71 common DEGs indicating the 6 most enriched subclassifications. (B) KEGG pathway analysis indicating the 3 most enriched pathways. (C) Genes deregulated in the neuroactive ligand-receptor interaction pathway, in red the upregulated and in green the downregulated in the Prdml2 icKO’s.
Figure 16. Validation of some of the identified DEGs as Prdml2 targets in Rosa26 icKO mice by RT-qPCR. *, **, ***, and **** indicate respective p values <0,05, <0,01, <0,001, and <0,0001 (Student's t-test, a = 0,05).
Figure 17. Knockout of Prdml2 does not alter locomotor activity. (A) Rosa26 icKO (n=10) and control mice (n=12) mice travel the same distance and spent the same time moving in the open field device. (B) Rosa26 icKO (n=10) and control mice (n=12) spent the same time to cross the same distance in the beam walking test. Values are means ± SEM. Values are means ± SEM. **p<0.01 (Student’s t-test, a = 0,05)
Figure 18. Transcriptomic analysis of the consequences of the loss of Prdml2 in TG of adult mice. (A) Volcano plots showing the -loglO adjusted P-value as a function of the log2 fold-changes of deregulated genes in TG of Rosa26 icKO mice (icKO mice, n 56 vs control mice, n 56) 1 month after tamoxifen injection. The genes were identified by bulk RNA-seq and selected based on adjusted P-value ,0.05. Genes with a log2 fold-change .0.31 are indicated in red, genes below the threshold are in light grey. (B) Gene ontology classification of the DEGs. The graph shows the enriched biological molecular function associated with downregulated and/or upregulated genes. (C) Venn diagram showing the overlap between the DEGs identified in TG and DRG of Rosa26 icKO mice. The list of common DEGs in TG and DRG of Rosa26 icKO is shown in Table 5 of Example Y. DRG, dorsal root ganglia; TG, trigeminal ganglia; icKO, inducible conditional knockout.
Figure 19. Conditional knockout of Prdml2 in mature nociceptors does not alter thermal and mechanical nociception but affects responses to formalin and capsaicin. (A) Avil icKO and control mice spent similar time on the test side in a 2-temperature choice assay. (B) Rosa26 icKO (n 511) and control mice (n 59) show similar withdrawal latency in the tail flick test using a focus 30. (C) Rosa26 icKO (n 510) and control mice (n 512) show no difference in withdrawal latency in the cold plantar assay. (D) Rosa26 icKO and control mice show the same behavior in the mechanical conflict avoidance test. They spent the same time to cross half of the second chamber with nails on the floor. Results obtained with 2 different nail heights (2 and 5 mm) are shown. (E) Intradermal cheek injection of N-met LTC4 elicits scratching bouts that are not significantly different between Rosa26 icKO and control mice. (F) Rosa26 icKO mice (n 510) spent less time licking their paw than control mice (n 512) after capsaicin injection (P 50.011). (G) Time course of the nocifensive response (licking time in seconds) of Avil icKO (n 511) and control mice (n 514) until 30 minutes after formalin injection. Response of individual Avil icKO and control mice in the first (0-5 minutes after formalin injection) and second phase (15-30 minutes after formalin injection) is shown on the right. Note that Avil icKO mice spent more time licking their paw when compared with control mice during the second phase (P 50.001). Values are represented as mean 6SEM. *P ,0.05, **P ,0.01 (Student t test, a 50.05). icKO, inducible conditional knockout.
DETAILED DESCRIPTION
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of ± 10% or less, preferably ± 5% or less, more preferably ± 1% or less, and still more preferably ± 0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.
Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter aha a reference to any one of said members, or to any two or more of said members, such as, e.g. any 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more. Related hereto, numeric values indicated by “at least” as used herein are indicative for an interval between the lower value and an upper limit. For example, when describing a reduction of a parameter it is evident that “at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, respectively indicates intervals from about 10% to about 100%, from about 25% to about 100%, from about 35% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%, from about 99% to about 100%. Alternatively, when describing an increase of a parameter it is evident that the upper limit is not limited to 100%, and may be considerably higher than 100%, such as 150%, 200%, 250%, 500%, 1000%, or higher.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined.
In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
For general methods relating to the present disclosure, reference is made inter aha to well- known textbooks, including e.g. “Molecular Cloning: A Laboratory Manual, 4th Ed.” (Green and Sambrook et al., Cold Spring Harbor Laboratory Press, 2012). Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein.
Amino acids are referred to herein with their full name, their three-letter abbreviation or their one letter abbreviation. The term “amino acid” indicates naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D- and L-stereoisomers, provided their structure allows such stereoisomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A “naturally encoded amino acid” indicates an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy-lysine or selenocysteine. The 20 common amino acids are: alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Vai), tryptophan (W or Trp), and tyrosine (Y or Tyr). Also envisaged by said term are amino acid analogues, wherein at least one individual atom is replaced either with a different atom, an isotope of the same atom, or with a different functional group.
By “encoding” is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g. the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g. RNA or RNA analogue) relationship.
The inventors hypothesised that Prdml2 is an interesting potential pain target given its biological function, epigenetic transcriptional regulation, and restricted expression in pain sensing neurons. By extensive experimentation, the present inventors were able to identify new insights in the functioning of Prdml2, both on a molecular level and on the level of the subject as a whole. The findings expose numerous innovative strategies for interfering molecular pathways involved in pain sensation and/or pain signalling, and hereby provide a solution to the unmet need described above.
The following detailed description is not to be taken in a limiting sense. The scope of the present invention is defined by the appended claims. It is evident that disclosed embodiments may relate to both Prdml2 modulators and compositions comprising Prdml2 modulators. A skilled person will appreciate that when a certain isolated or purified modulator is administered to a subject wherein said administration causes an alleviation of a pain sensation or pain condition in the subject, a similar result is to be expected when a pharmaceutical composition comprising a therapeutically effective amount of the Prdml2 regulator is administered to the same subject. Terms such as “subject”, “patient”, “individual” may be used interchangeably herein and refer to animals, preferably warm-blooded animals, more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals, that have been the object of treatment, observation or experiment with a Prdml2 modulator. Preferred subjects are human subjects including all genders and all age categories thereof. Both adult subjects, newborn subjects, and fetuses are intended to be covered by the term “subject”. Preferred subject in the context of the invention are subjects that are experiencing pain, or suspected to experience pain either at a time of assessment or in an envisaged future point in time. In certain embodiments, the subject is diagnosed with a condition or disorder that is known in the art to be associated with physical pain or discomfort. In certain embodiments, the subject is an elderly subject. “Elderly subject” refers to a subject of old age, i.e. the age nearing or surpassing the life expectancy of a subject. An elderly subject is defined by an age of at least 65 years, preferably at least 70, at least 75, at least 80, at least 85, most preferably at least 85 years. In alternative embodiments, the subject is selected from the group consisting of: infants (i.e. juvenile subjects), adolescent subjects, and adult subjects. In certain embodiments described herein, the subject is diagnosed to be palliative or considered to be palliative.
When non-human subjects are intended, the term is further intended to include transgenic species. It is further envisaged that the present invention is suitable to suppress pain responses and/or pain sensation in test animals. Non-limiting examples of animals that may benefit from the present invention include but are not limited to warm-blooded animals, more preferably vertebrates, and even more preferably non-human mammals such as those selected from the non-exhaustive list of examples of domestic animals, commercial animals, farm animals, zoo animals, sport animals, pet and experimental animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Additionally, nonmammalian animals, particularly non-mammalian animals (e.g. fish and invertebrates) that are known to perceive and/or respond to noxious stimuli may act as a subject of the present invention.
In certain embodiments, subjects particularly suited for administering the Prdml2 modulator to are subjects that have a medical history of intolerance to traditional analgesics and/or may have experienced an analgesic addiction or susceptibility to analgesic addition, such as but not limited to opioid addiction, alcohol addiction, cannabis, any substance classified by authorities as “hard drug”, or any combination thereof. In one or more of the embodiments disclosed herein, a preferred subject may be a subject that is allergic to known analgesics. A skilled person is aware of the physical and physiological symptoms of opioid addiction and a healthcare practitioner is readily capable of establishing whether a subject is susceptible for, or is experiencing an opioid use disorder, all of which are described in detail in throughout the art (e.g. according to the DSM-5 criteria, Diagnostic and Statistical Manual of Mental Disorders Fifth Edition, 2013). In certain embodiments, the subject is a recovering opioid addict.
Pain, as defined by the International Association for the Study of Pain, is a submodality of somatic sensation and has been defined in the art as a complex constellation of unpleasant sensory, emotional and cognitive experiences provoked by real or perceived tissue damage and manifested by certain autonomic, psychological, and behavioral reactions (Terman and Bonica, Bonica’s management of pain, 2003).
The term “pain” as used within the context of the present disclosure refers to physical pain. However, a skilled person will readily appreciate that physical pain may additionally cause secondary unwanted emotional states that are associated with non-physical, i.e. mental pain. Hence, while the inventors envisage a wide applicability of the herein presented Prdml2 modulator for alleviating physical pain (sensation), said modulator may additionally exert an improvement in the emotional state of the subject. Particularly relevant in this context is the alleviation of chronic pain, which is known to be harmful and often detrimental for the mental well-being of a subject (described in a.o. Sheng et al., Neural Plasticity, 2017). Although by no means limiting, throughout the art, low levels of pain are commonly referred to as “discomfort” or synonyms thereof, while moderate to high levels of pain are referred to using the term “pain”. Both subjects experiencing discomfort or pain are suitable subjects in context of the present disclosure. Preferred subjects are subject that experience, or are considered to experience moderate to severe discomfort or pain, either at the time of diagnosing or in a future point in time. Evidently, the course of pain experienced by the subject over time is not-limiting for the applicability of the Prdml2 modulators disclosed herein. For example, the pain may be acute (i.e. temporary), chronic, intermittent, or increasing over time.
The term “pain” includes but is not limited to pain in subjects suffering from chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or wherein said is a palliative subject. More preferably said pain condition is nociceptive pain, inflammatory pain, non-inflammatory pain, neuropathic pain, itch-related pain, inflammatory itch-related pain caused by e.g. atopic dermatitis, non-inflammatory itch-related pain caused by e.g. pruritus, neuropathic itch, or any combination thereof, wherein in some embodiments said pain condition is induced by nerve injury or inflammation or is itch-induced (cf. Wong et al., (2017), Int J Mol Sci. 2017 Jul; 18(7): 1485).
Objective quantification of pain is a challenging area of research. Several scales for self- reporting of pain have been established, including the non-limiting examples of the numeric rating scale (NRS-11), Stanford pain scale, or visual numeric scale. The basic concepts of pain measurement instruments and techniques have been described in the art (e.g. in Younger et al., Current Pain And Headache Reports, 2010). More recently, technological advancements have driven the desire to quantify pain in a (near) objective manner, both in view of devices used to administer pain stimuli (Wagemakers et al., Pain Physician, 2019) and in view of pain measurement in a subject as such (Xu et al., FlOOOResearch, 2020). Emerging methods to assess pain include but are not limited to techniques such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). Any of these methods may be suitable for determining whether a person will, or is suspected to benefit from Prdml2 modulation. Hence, in certain embodiments the pain experienced by a subject is assessed by one or more methods selected from the group of methods consisting of: self-reporting scales, fMRI, EEG, or any combination thereof.
“Analgesic” as used herein is to be interpreted in its broadest interpretation and may therefore refer to any compound, substance, or composition that is able to reduce pain or suppress pain in a subject, and hence any product that is able to achieve analgesia in a subject. Throughout the art, any analgesic may interchangeably be referred to as “painkiller” or “pain reliever”. The analgesic may be used in the treatment of pain and/or in pain prevention (i.e. pain prophylaxis). An analgesic may act on the peripheral and/or central nervous system. In the art, analgesics are commonly classified according to the mechanism of action. Different classes of analgesics include but are not limited to acetaminophen (i.e. paracetamol), nonsteroidal -inflammatory drugs (NSAIDs), opioids, muscle-relaxants, anti-anxiety agents, antidepressants, anticonvulsants, and corticosteroids. All of these may be referred to herein as “traditional analgesics” As will be evident for a skilled person, any of these analgesics classes, subgroups of said analgesics classes, and individual analgesics may be used in combination with the Prdml2 modulator as described herein.
NSAID are classified by chemical structure, origin, and/or mechanism of action. Therefore, any NSAID referred to herein may be selected from an NSAID group consisting of salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, enolic acid derivatives (oxicam), anthranilic acid derivatives (fenamates), (selective) COX-2 inhibitors (coxibs), sulfonanilides, etc. Non limiting examples of NSAIDs include: aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid and salts thereof, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam, isoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, nimesulide, clonixin, licofelone,and h-harpagide.
“Opioids”, or alternatively opiates, may be classified according to the degree of chemical modification. Hence, an opioid referred to herein may be selected from the group consisting of natural opiates, morphine esters, semi-synthetic opioids, fully synthetic opioids, endogenous opioid peptides. The term “opioids” as referred to herein further encompasses substances or molecules classified in the art as “opioidergics”, said opioidergics are commonly referred to in the art to define groups of chemicals that modulate the opioid neuropeptide systems. Examples of natural opioids include alkaloids such as morphine, codeine, thebaine, Mitragyna speciosa (or leaves thereof), and salvorin A from Salvia dinorum. Examples of morphine esters include morphine prodrugs such as but not limited to diacetylmorphine, nicomorphine, dipropanoylmorphine, desomorphine, acetylpropionylmorphine, dibenzoylmorphine, and diacetyldihydromorphine. Examples of semi-synthetic opioids include but are not limited to hydromorphone, hydrocodone, oxycodone, oxymorphone, ethylmorphine, buprenorphine. Examples of fully synthetic opioids include fentanyl, pethidine, levorphanol, tramadol, tapentadol, and dextropropoxyphene. Endogenous opioid peptides include but are not limited to endorphins, enkephalins, dynorphins, and endomorphins. Alternative opioids or opioid-like compounds include tramadol, tapentadol, nefopam, orphenadrine, phenyltoloxamine, and antihistamines. Hence, the term opioid as referred to herein may optionally refers to one or more of the opioids of the group consisting of: codeine, morphine, thebaine, oripavine, diacetylmorphine (morphine diacetate; heroin), nicomorphine (morphine dinicotinate), dipropanoylmorphine (morphine dipropionate), diacetyldihydromorphine, acetylpropionylmorphine, desomorphine, methyldesorphine, dibenzoylmorphine, dihydrocodeine, ethylmorphine, heterocodeine, buprenorphine, etorphine, hydrocodone, hydromorphone, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl, alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl, pethidine (meperidine), ketobemidone, MPPP, allylprodine, prodine, PEPAP, promedol, propoxyphene, dextropropoxyphene, dextromoramide, bezitramide, piritramide, methadone, dipipanone, levomethadyl acetate (LAAM), difenoxin, diphenoxylate, loperamide, dezocine agonist/antagonist, pentazocine agonist/antagonist, phenazocine, buprenorphine partial agonist, dihydroetorphine, etorphine, butorphanol agonist/antagonist, nalbuphine agonist/antagonist, levorphanol, levomethorphan, racemethorphan, lefetamine, menthol, meptazinol, mitragynine, tilidine, tramadol, tapentadol, eluxadoline, AP-237, 7-Hydroxymitragynine, etc. Nono-limiting examples of allosteric modulators include BMS-986121 and BMS-986122 (both modulators of the p-opioid receptor, as described in Burford at al. PNAS USA, 2013), ignavine (modulator of the p-opioid receptor, Ohbuchi et al. Scientific Reports, 2016), Oxytocin (p-opioid receptor, Meguro et al., Journal of pharmacological sciences, 2018), 6-PAM (6-opioid receptor, Burford et al., Journal of Medicinal Chemistry, 2015), cannabidiol and tetrahydrocannabinol (6-opioid receptor, Kathmann et al., 2006), sodium (capable of modulating opioid receptors by sodium ions, Shang et al., 2014).
The term “corticosteroids” as referred to herein is representative for a class of steroid hormones, derivatives and related molecules that may have an effect on a plethora of processes in a subject such as stress responses, immune responses, inflammation regulation, metabolism, protein catabolism, electrolyte concentration in e.g. the blood, and behavior. Corticosteroids are commonly classified in the art in two classes, glucocorticoids (i.e. corticosteroids that bind to the glucocorticoid receptor) and mineralocorticoids, both of which are envisaged herein by the term corticosteroids. Alternatively, corticosteroids may be classified as natural or synthetic corticosteroids, said synthetic corticosteroids consisting of the group of corticosteroids selected from: progesterone-type, hydrocortisone-type, methasone-type, acetonides, cortivazol, and RU-28362, all of which are envisaged herein by the term “corticosteroids”. When referring to the term “corticosteroids” throughout the disclosure, it is envisaged that said corticosteroid may be selected from the group of corticosteroids consisting of: 11 -dehydrocorticosterone, 11- deoxy corticosterone, 11 -deoxy cortisol, 11 -ketoprogesterone, l ip-hydroxypregnenolone, l ip- hydroxyprogesterone, 1 ip,17a,21-trihydroxypregnenolone, 17a, 21 -dihydroxypregnenolone, 17a-hydroxypregnenolone, 17a-hydroxyprogesterone, 18-hydroxy-l 1 -deoxycorticosterone, 18-hydroxy corticosterone, 18-hydroxyprogesterone, 21 -deoxy cortisol, 21 -deoxy cortisone, 21- hydroxypregnenolone (prebediolone), aldosterone, corticosterone (17-deoxy cortisol), cortisol (hydrocortisone), cortisone, pregnenolone, progesterone, Flugestone (flurogestone) = 9a- fluoro-1 ip,17a-dihydroxypregn-4-ene-3, 20-dione, fluoromethoIone, medrysone
(hydroxymethylprogesterone), prebediolone acetate (21-acetoxypregnenolone), chloroprednisone, cloprednol, difluprednate, fludrocortisone, fluocinolone, fluperolone, fluprednisolone, loteprednol, methylprednisolone, prednicarbate, prednisolone, prednisone, tixocortol, triamcinolone, alclometasone, beclometasone, betamethasone, clobetasol, clobetasone, clocortolone, des oximetasone, dexamethasone, diflorasone, difluocortolone, fluclorolone, flumetasone, fluocortin, fluocortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, meprednisone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, ulobetasol (halobetasol), amcinonide, budesonide, ciclesonide, deflazacort, desonide, formocortal (fluoroformylone), fluclorolone acetonide (flucloronide), fludroxycortide (flurandrenolone, flurandrenolide), flunisolide, fluocinolone acetonide, fluocinonide, halcinonide, triamcinolone acetonide, cortivazol, and RU-28362.
“Muscle-relaxants” as referred to herein indicates a class of pharmaceutically active ingredients that are able to affect skeletal muscle function and decrease the muscle tone. Muscle-relaxants are commonly stratified into to categories: neuromuscular blockers and spasmolytics (the latter often being referred to as antispasmodics). Non-limiting examples of known muscle-relaxants include chlorzoxazone, carisoprodol, methocarbamol, cyclobenzaprine, orphenadrine, metocurine, botulinum toxin type A, botulinum toxin type B, baclofen, succinylcholine, cisatracurium, tizanidine, rocuronium, hexafluronium, doxacurium, chlormezanone, tubocurarine, dantrolene, mivacurium, pancuronium, pipecuronium, vecuronium, eperisone, thiocolchicoside, tolperisone, bromazepam, diazepam, clonazepam, flunitrazepam, lorazepam, nitrazepam, temazepam, eszopiclone, quinine, fludiazepam, etc.
“Anti-anxiety agents” are interchangeably annotated in the art as “antipanics” or “anxiolytic agents” and in addition to medication for preventing or reducing anxiety, several of them also display analgesic properties, and several anti-anxiety agents are therefore prescribed for both anxiety and pain conditions. Non-limiting exemplary classes of anti-anxiety agents include barbiturates, benzodiazepines, carbamates, antihistamines, opioids, antidepressants, sympatholytics (i.e. beta blockers, alpha blockers, and alpha-adrenergic agonists), phenibut, mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, pregabalin, menthyl isovalerate, propofol, and racetams.
The meaning of the term “antidepressant” is well known to a person in the art, and several antidepressants have been additionally used to treat pain, in particular chronic pain. Antidepressants have been categorised in several classes, including but not limited to selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors, and NMDA receptor antagonists. The term “anticonvulsant” as used herein indicates a class of antiepileptic and/or antiseizure drugs, which are increasingly used as pain treatment medication, particularly for treatment of neuropathic pain (Rogawski et al., Nature Reviews Neuroscience, 2004). Numerous anticonvulsant drugs have been developed and may be classified according to chemical structure. Anticonvulsants as envisaged herein include aldehydes, aromatic allylic alcohols, barbiturates, benzodiazepines, bromides, carbamates, fatty acids, fructose derivatives, hydantoins, oxazolidinediones, propionates, pyrimidinediones, pyrrolidines, succinimides, sulfonamides, triazines, valproylamides, and additionally anticonvulsants that are not generally classified under one of the former categories, including but not limited to perampanel, stiripentol, and pyridoxine.
In certain embodiments, the subject is insensitive or considered to be insensitive to traditional analgesics, preferably wherein the subject is insensitive or considered insensitive to one or more analgesics selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal-inflammatory drugs (NSAIDs), opioids, muscle-relaxants, anti-anxiety agents, antidepressants, and corticosteroids. The terms “insensitivity”, “unresponsiveness”, “insusceptibility” or “resistance” may be used interchangeably herein and refer to a subject having a pain condition or suspected to having a pain condition that is not relieved by traditional analgesics, i.e. where the pain conditions persists, optionally with a mild improvement despite treatment with traditional analgesics. In certain subjects, the insensitivity is the consequence of chronic pain and/or said subject being treated with traditional analgesics for a prolonged period of time.
Throughout this specification, the term “protein” is to be interpreted according to the commonly accepted meaning in the art, and therefore generally encompasses macromolecules comprising one or more polypeptide chains, i.e, polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. It is evident that the term “protein” is a collective name to indicate polypeptide chains as such but also encompasses proteins that carry one or more co- or post-expression-type modifications of at least one polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis corresponding canonical version of said protein, such as, e.g. amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and functional protein fragments, e.g. naturally- occurring protein parts or domain incorporated in the full-length protein which exerts or maintains a certain functionality when isolated from the full-length protein or used separately as part of a larger polypeptide chain distinct form the full-length protein.
Throughout the art, the terms “protein”, “polypeptide”, and even “peptide” are commonly used interchangeably, particularly in the context of relative short peptides. None of the terms is limited to any minimum length of the polypeptide chain. However, a consensus in the art is maintained that a peptide at least comprises two amino acids, which may be identical or different amino acids. A non-limiting production method of proteins, polypeptides and/or peptides is recombinant production, wherein a suitable host or host cell expresses by translation the envisaged protein, polypeptide, or peptide (i.e. wherein the host cell(s) function(s) as an expression system for an expression vector), and wherein said translated product is in a subsequent step isolated therefrom. A plethora of suitable expression systems have been described in the art and non-limiting examples thereof are bacterial, yeast, fungal, plant or animal cells or cell expression system). Alternatively, the proteins, polypeptides and/or peptides may be produced recombinantly by cell-free transcription and/or translation, or non- biological protein, polypeptide or peptide synthesis. By means of guidance and not limitation, a suitable peptide synthesis method known in the art that does not involve a live expression system is solid phase peptide synthesis (reviewed in detail in e.g. Jaradat, Amino Acids, 2018). The term “nucleic acid” as used herein typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Exemplary modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5- propynylcytosine. In particular, 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Sugar groups may include inter aha pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxy arabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-O-alkylated, e.g., 2'-O-methylated or 2'-0-ethylated sugars such as ribose; 2'-O-alkyloxy alkylated, e.g., 2’ -O-methoxy ethylated sugars such as ribose; or 2'-O,4'-C-alkylene-linked, e.g., 2'-O,4'-C-methylene-linked or 2'-O,4'-C-ethylene- linked sugars such as ribose; 2 ’-fluoro-arabinose, etc.). Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA. Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g., by a methyl, ethyl, alkyl, or alkyloxyalkyl). Preferably, ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units. Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA. Such deoxyribonucleoside unit(s) comprise 2'-H. Preferably, deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units. Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter aha phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate- based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.
The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used herein particularly encompasses lower hydrocarbon moi eties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2- propenyl, and isopropyl. Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.
These term “nucleic acid sequence” and the likes further encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids. RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). A nucleic acid can be naturally occurring, e.g. present in or isolated from nature, can be recombinant, i.e. produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised. A naturally occurring variant of a given sequence refers to all variants of the sequence which encode the same functional protein and that are present in or can be isolated from nature. Typically, this includes all variants of the sequence encountered in mammals, more particularly humans. It will be understood that variants from closely related species will have a higher sequence identity than variants from evolutionary more distant species. In particular embodiments, the natural variant of a given sequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 or 95% sequence identity with said given sequence. Without limitation, nucleic acids can be produced recombinantly by a suitable host or host cell expression system and isolated therefrom (e.g. a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription, or non-biological nucleic acid synthesis. Commercial nucleotide manufacturers that commonly rely on chemical synthesis are known to a person skilled in the art. A nucleic acid can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. The term “nucleic acid” further indicates any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (reviewed in e.g. Kurreck, European Journal of Biochemistry, 2003). Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof.
The term “Prdml2”, short for “PR domain zinc finger 12” is indicative for a protein that in humans is encoded by the PRDM12 gene which is located on chromosome 9 (9q34.12). Prdml2 is known to be a transcriptional regulator of sensory neuronal specification that plays a critical role in pain perception. Through this function, Prdml2 is involved in the development of nerve cells that assist in perception and sensation of pain. At least in vertebrates, Prdml2 has been shown to repress different genes, such as the non-limiting examples of the DBX1 and NKX6 genes, hereby acting as a general determinant of VI cell fate. VI interneurons in turn play an essential role in vertebrate locomotion (Thelie et al., development, 2015). Mutations in Prdml2 are associated in the art with a congenital insensitivity to pain (abbreviated herein and in the art as “CIP”). Further, Prdml2 mutations are associated with hereditary sensory and autonomic neuropathies (HSAN’s) affecting peripheral sensory and autonomic neurons (e.g. HSAN VIII), and anhidrosis. Additional consequences of Prdml2 mutations are an early loss of teeth, soft tissue injuries, dental carries, submucosal abscesses, and hypomineralisation of primary teeth, and mandibular osteomyelitis. Throughout the art, the PRDM12 gene and/or gene product may alternatively be annotated by one or more alternative names including but not limited to PFM9, HSAN8, PR domain 12, PR/SET domain 12, PR-containing protein 12, PR domain containing 12. The term Prdml2 denotes the Prdml2 peptide, polypeptide, protein, or nucleic acid as will be evident to a skilled person from the context of the specific disclosure. The full length human Prdml2 protein comprises an N-terminal PRDI-BF1 and RIZ homology (PR) domain, a SET domain, and three C-terminal C2H2 zinc finger DNA-binding domains (Chen et al. Nature Genetics, 2015). It has been described in the art that the human PRDM12 gene comprises 5 exons (Chen et al., Nature Genetics, 2015).
The human PRDM12 gene is annotated and publicly available under NCBI Genbank (www.ncbi.nlm.nih.gov) accession number (NCBI reference sequence NG_053081.1). The messenger RNA (mRNA) sequence of Homo sapiens PRDM12 is publicly accessible under NCBI reference sequence: NM_021619.3. The reference Human Prdml2 protein sequence is annotated and publicly available as NCBI reference sequence NP_067632.2 or alternatively retrievable on UniProt (www.uniprot.org) under accession number Q9H4Q1 (PRD12 HUMAN) and is by means of example reproduced below (SEQ ID NO: 1): MMGSVLPAEALVLKTGLKAPGLALAEVITSDILHS FLYGRWRNVLGEQLFEDKSHHAS PKTA FTAEVLAQS FSGEVQKLSSLVLPAEVI IAQSS I PGEGLGI FSKTWIKAGTEMGPFTGRVIAP EHVDICKNNNLMWEVFNEDGTVRYFIDASQEDHRSWMTYIKCARNEQEQNLEWQIGTS I FY KAIEMI PPDQELLVWYGNSHNTFLGI PGVPGLEEDQKKNKHEDFHPADSAAGPAGRMRCVIC HRGFNSRSNLRSHMRIHTLDKPFVCRFCNRRFSQSSTLRNHVRLHTGERPYKCQVCQSAYSQ L AGL RAH QKS ARH R P P S T AL QAH S PAL PA P H AH AP AL AAAAAAAAAAAAH H L P AMVL .
While SEQ ID NO:1 as depicted above is accepted as the general sequence, it is known in the art that the C-terminal poly-alanine stretch is polymorphic in the general human population and has a varying length (Chen et al., Nature Genetics, 2015). It is evident that these variations are also envisaged when reference is made to Prdml2 throughout this disclosure. Furthermore, a skilled person can, based on the above information for human Prdml2, readily identify and annotate PRDM12 genes and gene products in non-human subjects, which may also benefit from Prdml2 modulation. Furthermore, a skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of peptides, polypeptides, proteins, or nucleic acids and may include parts which are processed away from mature molecules commonly known under the respective designations in the art. The terms PRDM12 and Prdml2 encompass such peptides, polypeptides, proteins, or nucleic acids, of any subject where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
In view of the PRDM12 modulators described herein that are based on similarity to the DNA and/or mRNA nucleotide sequence of PRDM12 or hybridise to the DNA or mRNA sequence of PRDM12, the nucleotide sequence corresponding to the canonical mRNA sequence of PRDM12 which is annotated and publicly available as NCBI Reference Sequence NM_021619.3 (Homo sapiens PR/SET domain 12 (PRDM12), mRNA) is reproduced below (SEQ ID NO: 2): cccgcccacctcccccgtcggcccggccgtcccccggcgccggggagctccgggccgcccat gatgggctccgtgctcccggctgaggccctggtgctcaagaccgggctgaaggcgccgggac tggcgctggccgaggttatcacctccgacatcctgcacagcttcctgtacggccgctggcgc aacgtgctcggggagcagctcttcgaggacaagagccaccacgccagccccaagacagcctt caccgccgaggtgctggcgcagtccttctccggcgaagtgcagaagctgtccagcctggtgc tgcctgcggaggtgatcatcgctcagagctccatccctggcgagggcctcggcatcttctcc aagacgtggatcaaggcgggaaccgagatgggccccttcaccggccgcgtgatcgccccgga gcacgtggacatctgcaagaacaacaacctcatgtgggaggtgttcaatgaggatggcacgg tgcgctacttcatcgatgccagccaggaggaccaccggagctggatgacctacatcaagtgt gcacgtaacgaacaggagcagaacctggaggtggtccagatcggcaccagcatcttctacaa ggccattgagatgatcccacctgaccaggaactgctggtgtggtacggaaactcacacaaca ccttcctggggatcccaggtgtgcccgggctagaggaggaccagaaaaagaacaagcatgag gacttccacccggcggactcggcggctggccccgcgggccgcatgcgatgcgtcatctgcca ccgcggcttcaactcgcgcagcaacctgcgctcgcacatgcgcatccacacgctggacaagc ccttcgtgtgccgcttctgcaaccgccgcttcagccagtcgtccacgctgcgcaaccacgtg cgcctgcacacgggcgagcgcccctacaagtgccaggtgtgccagagcgcctactcgcagct ggccggcctgcgcgcccaccagaagagcgcgcggcaccggccgcccagcaccgcgctgcagg cacactcgcccgcgctgcccgccccgcacgcgcacgcgcccgcgctcgccgccgccgccgcc gccgccgccgccgccgccgcgcaccacctgccggccatggtgctgtgagcgcgcccgcgccc ccgccgggccccgcgcgctcctgggtccccggcaccccggccccgcagcgcgactcgccctc cagccccaacccccggcccggcgccgccgcggagccccgcgcgctggggttgcgccccggag gcggatctcaggcacccccgccttggcccgtgtcgcagatgaggacactgagggcggcgtcc ctcacccaggccacgcagctggtgcggctgttcggccgcctcctctgggagggggtccccct gcctggcctcgcccccgagtctccctgctgctgtagagccgggcgcccaggccggcatcccc ctcgtgggtggcaggagagcggacttagagcccccgagggcccctcagcagaggaaggggag tcccctcctccgccaccgagagtcctgtgcttcggaatgaagggagggcgacctccttgtcc gggtgtggtcgggcgcgggggttgtgggggcgccatctcctcttccggcccctgggactggt gtcctgaagtgtcgggagtccttacgcaaccttgacatgtgcgggctactggggtccatcgg cagcgggaagagacccggagagatgcatcttctgggcccttctctcctttaggccagcttac ccccaaacctggctcctggggacggatgaggaaggagctctttgcagtgcaccaggcacgtt gagagtggaggcacaatggaaatcctctgtgggaccccgagcaagaccctgcacctctttgg gcctcagtttccccatctgtaaaatgaaggagttggaccaatgccccctccccttcagctgt gacatcgtgcctgggaaggcgaaatatacccccagccccttccaacacacacactccagcga gaggagggcaagactggaaccgctgcccgagaggttaaggtggcttctgtggccacgaagcg cttccgagctgtgcccagtcccgggaattccgcccagtctgcccacacttccctcggtccct gcccgtttcctcaaattcgggccgtgcgcgccctctggtgtcgcctctcacactttgcagtc atttaccaggattcccgtagggcgttttgtaaaataaactataatattaatgaaaagtataa aatgtatagagttttcaagaaactgcgttctacttccagaagatggtcactttaaccttgta aatatttatctaaaatgatatttacaaaactgtaatatattattatttgattgtatatgtac aactgtaaatacatttgtacctctcttgtattctaaaataaaaattacttggaacattta .
Related hereto, a person skilled in the art is well aware of methods and tools to verify sequence homology, sequence similarity or sequence identity between different sequences of amino acids or nucleic acids. Non-limiting examples of such methods and tools are Protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/), SIM alignment tool (https://web.expasy.org/sim/), TranslatorX (http://translatorx.co.uk/) and T-COFFEE (https://www.ebi.ac.uk/Tools/msa/tcoffee/). The percentage of identity between two sequences may show minor differences depending on the algorithm choice and parameters.
The term “sequence identity” as used herein refers to the relationship between sequences at the nucleotide or amino acid level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the “% identity” is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence. A skilled person is aware of the related, yet different interpretations in the art of the terms “similarity”, “homology”, and “identity” (explain in detail in e.g. Pearson, Current protocols in bioinformatics, 2014).
The term “modulator” as used herein may be used interchangeably with synonymous terms such as “regulator” and indicate any moiety that may affect Prdml2 activity, either by affecting the function of a Prdml2 molecule, by affecting the expression level of Prdml2, or by both affecting the function and expression of Prdml2 or its gene PRDM12 in cells of a subject. Hence, certain modulator disclosed herein might exclusively alter expression levels of Prdml2 or PRDM12, while others might exclusively alter the intrinsic activity effected by Prdml2. Furthermore, certain modulators will alter both the function and the expression level of Prdml2, wherein both parameters may be upregulated or downregulated by said modulator, or wherein each parameter may be affected in a different direction. A skilled person is aware that modulation of the activity of a gene and/or its gene products may function by direct hybridisation or binding to the said target or to any of the molecules said target engages in an interaction with. Alternative, and optionally in addition to a direct physical interaction, the modulator may influence Prdml2 activity by binding to an element governing the expression of Prdml2, non-limiting examples hereof being certain genetic sequences including PRDM12 promoter or enhancer sequences. Nevertheless, a skilled person will appreciate that each of the above groups of modulators will affect the nett Prdml2 activity. By “affecting Prdml2 activity” it is clear that a moiety is only to be considered a modulator when a nett change in Prdml2 activity upon or after administration of said moiety is observed in a cell, tissue, and/or subject. Alternatively worded, a Prdml2 modulator is a moiety that by any mechanism causes a deviation or Prdml2 activity in a target cell, tissue, and/or subject from a baseline value, said baseline value being a predetermined Prdml2 activity level when said cell tissue, and/or subject is not subjected to, or contacted with, a Prdml2 modulator. Assays to determine protein expression levels and/or activity, and assays to determine RNA or DNA levels or concentrations are known in the art and suitable protocols are readily available. In certain embodiments referring to a modulation (i.e. increase or decrease) of the expression of a certain gene or gene product, the assay to quantify said expression, or change in expression is selected from the group consisting of: PCR (e.g. RT-PCR or qPCR), mass spectrometry analyses, spectrophotometric assays (e.g. UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays), ELISA, or a combination thereof. It is envisaged that the change in expression level may be expressed as a value relative to a suitable baseline or control expression value as discussed throughout the present disclosure.
By means of guidance and not limitation, a ubiquitously used technique to measure RNA expression levels is by means of a real-time polymerase chain reaction (RT-PCR or qPCR) experiment. RT-PCR has the ability to monitor the progress of the PCR as it occurs (i.e. in real time). Two main methodologies to perform quantitative PCR have been described: dye-based and probe-based detection. Each of these methods relies on calculating the initial (zero cycle) DNA concentration by extrapolating back from a reliable fluorescent signal. Protocols and related methods that rely on RT-PCR have been described in detail in the art (Ary a et al., Expert Review of Molecular Diagnostics, 2015). Commonly used techniques to measure protein expression levels include but are by no means limited to mass spectrometry analyses, spectrophotometric assays and enzyme -linked immunosorbent assays (ELISA). Non-limiting examples of targeted proteomics experiments include selective reaction monitoring and multiple reaction monitoring. Mass spectrometry approaches to measure protein expression levels have been extensively described in the art (Shi et al., Proteomics, 2016). Alternatively or complementary hereto, spectrophotometric assays have been described and include UV light absorption spectroscopy, dye-based protein assays, Coomassie blue (Bradford) assays, and Lowry alkaline copper reduction assays (Noble and Bailey, Methods in Enzymology, 2009). ELISA assays suitable for protein quantitation have also been described (Pamas and Linial, Brain research protocols, 1998).
The term “nociceptor” as used herein refers to any sensory neuron developed from neural-crest or placodal stem cells that respond to, or is considered to be able to respond to harmful (i.e. potentially damaging to the subject or tissue) stimuli. The term may interchangeably be used with the term “pain receptor”. Nociceptors constitute a key component of nociception (i.e. perceiving of pain). Nociception in turn may optionally induce one or more physiological and/or behavioural responses in a subject. In any of the embodiments and aspects described herein, the term may refer to the total amount of nociceptors in the subject, or alternatively to a certain subgroup of nociceptors. The subgroup may be defined by their localisation, molecular characteristics, innervation pattern or a combination thereof. The Prdml2 modulators as described herein may exert their function in peptidergic nociceptors and/or nonpeptidergic nociceptors. Alternatively, the Prdml2 modulators as described herein may exert their function in external nociceptors and/or internal nociceptors.
“Dorsal root ganglia”, and the singular form “dorsal root ganglion” may be used interchangeably with terms such as “spinal ganglion” and “posterior root ganglion”. The terms indicate a cluster of neurons (i.e. a ganglion) in a dorsal root of a spinal nerve that carry sensory signals to the central nervous system from the peripheral nervous system. Dorsal root ganglion axons are commonly considered “afferents” in the art. The right and left spinal nerve pairs in humans form out of afferent sensory dorsal axons (the dorsal root) and motor ventral efferent axons (the ventral root). As the dorsal root emerges from the intervertebral neural foramina, it forms the dorsal root ganglion, which is a group of cell bodies responsible for the transmission of sensory messages from receptors such as thermoreceptors, nociceptors, proprioceptors, and chemoreceptors, to the central nervous system for a response. The cell bodies of dorsal root ganglia are separated by layers of satellite glial cells that inhibit the interaction between somas. Dorsal root ganglion neurons are accepted in the art to be, or behave as pseudo-unipolar neurons, with one axon that bifurcates into two separate branches resulting in a distal process and proximal process. The present day understanding of dorsal root ganglia is that these somas are active participants in the signalling process; they sense specific molecules and produce molecules needed to regulate the process (Ahimsadasan et al., StatPearls, 2020). Dorsal root ganglia are relatively easily accessible from the exterior of the epidural space through the neuroforamina and from the epidural space to the outside. In view of the above, dorsal root ganglion stimulation is enjoying increased popularity as neuromodulation therapy, having an effectiveness comparable to spinal cord stimulation in reducing certain pain conditions, including but not limited to chronic postsurgical pain, regional pain syndromes, and pain from failed back surgery syndrome (Liem, Progress in neurological surgery, 2015).
A skilled person is aware that external nociceptors include nociceptors with endings localised in tissues such as skin, cornea, and mucosa. Internal nociceptors in contrast have projections that are localised in or in near proximity of organs including but not limited to: muscles, joints, bladder, visceral organs, and the digestive tract. The detection and transduction of noxious stimuli occurs in the peripheral terminal of the nociceptor. Noxious stimuli are commonly categorised in the art as: thermal, mechanical, and chemical (Loeser et al., Pain, 2008). Nociceptors may respond to a single category of noxious stimuli, more than one category of noxious stimuli (i.e. polymodal nociceptors), or even respond to none of the above noxious stimuli. The latter group of nociceptors are commonly referred to in the art as sleeping or silent nociceptors, and are often involved in the response to inflammation which is a preferred pain condition, cause of pain, or pain-associated condition in the context of the present invention. Hence, the Prdml2 modulators as described herein may selectively act on nociceptors consisting of the group of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, polymodal nociceptors, or any combination thereof. Alternatively, the Prdml2 modulator may act exclusively on sleeping nociceptors.
Without wishing to be bound by theory, the Prdml2 modulators as described herein may affect pain sensation in a subject by modulation of the peripheral sensitisation. Hence, Prdml2 modulator administration may induce a change in one or more groups of nociceptors as described herein from acting as a noxious stimulus detector to an inactive nociceptor, or alternatively change an inactive, silent, or defective nociceptor to a noxious stimulus detector. In one or more embodiments, the Prdml2 modulator may affect the function of A6 fiber axons, C fiber axons, or both. It is known in the art that A6 fiber axons conduct action potentials faster than C fiber axons (about 20 meter/second compared to about 2 meter/second), which is mainly due to light myelination of A6 fiber axons, while C fiber axons are not myelinated (Williams et al. Neuroscience, 2001). As a consequence, A6 fiber axons attribute mainly to a first pain phase (i.e. an initial sharp pain sensation), while C fiber axons contribute mainly to a second pain phase (i.e. a more prolonged and less intense pain sensation). Hence, the Prdml2 modulators as described herein may affect the first pain phase, the second pain phase, or both pain phases.
Instances of the present disclosure referring to “control” or “baseline” conditions, levels, and/or activity intend to specify one or more comparative values (i.e. reference values) which may be used as a benchmark (i.e. standard) to assess any influence of aPrdm!2 modulator, or candidate Prdml2 modulator on Prdml2 activity. Control conditions and baseline values may indicate a Prdml2 expression level or activity on the molecular level in absence of any Prdml2 modulator. Alternatively or in addition hereof, a control condition or baseline value may refer to a certain metric on the subject as a whole, a non-limiting example hereof being the quantification of hind paw licking by a rodent when not subjected to treatment comprising the administration of a Prdml2 modulator or candidate Prdml2 modulator. Evidently, a person skilled in the art readily appreciates that the above examples of baseline values and/or control conditions are non-limiting and is able to devise alternative yet equally appropriate control conditions or baseline values at any measurement level given a certain context. For example, a skilled person would understand how to obtain a reference value for Prdml2 activity on tissue level, or may in the context of pain on the level of the subject as a whole, for which numerous assays and quantification techniques have been described in the art (e.g. reviewed extensively in Deuis et al., Frontiers in Molecular Neuroscience, 2017).
By means of illustration and not limitation, behavioral methods to measure nociception in animals may be divided into stimulus-evoked (e.g. mechanical, heat, cold, etc.) and nonstimulus evoked methods. For example, a skilled person appreciates that to assess mechanical pain stimuli, methods including the manual Von Frey test, the electronic Van Frey test, the Randall-Selitto test and variations of each of these tests may be used. Likewise, suitable nonlimiting methods to assess heat stimuli are the tail flick test, the Hargreaves test, and the thermal probe test. Routinely used tests for assessing cold stimuli are the cold plate test, the acetone evaporation test, and the cold plantar test. By means of illustration and not limitation, nonstimulus evoked nociception can be quantified by parameters such as grimace scales, burrowing, wait bearing, gait analysis, or any combination thereof. Relevant parameters relating to gait analysis in freely walking test animals include paw intensity (a measure of paw pressure or weight bearing), paw print parameters (e.g. toe spread, print length, print width, print area), dynamic parameters (e.g. stance phase, swing phase, duty cycle, stride length, swing speed) and regularity index (a measure of interlimb coordination). Alternatively, automated behavioural analysis are used in the art to assess non-stimulus evoked pain Inflammatory responses are, in accordance with the examples of the present disclosure, routinely assessed by licking of the inflamed area, for example hind paw licking when said hind paw is inflamed.
Suitable control conditions differ depending on the experimental design of an assay. When a single subject is analysed before, after, and optionally during Prdml2 modulator administration, a suitable baseline value may be derived from a point in time before Prdml2 modulator administration, and/or in a point in time after Prdml2 administration if the pain condition is or considered to be temporary and reversible. Alternatively or additionally, the baseline value may be a measure of centre (non-limiting examples being a mean value or median value) in a control population of subjects, wherein the treatment population (i.e. the population subjected to Prdml2 modulator administration) may be the control population in a different point in time or a different subject population. As is commonly accepted in the art and also employed in the present disclosure, a Prdml2 modulator is a substance that induces a significant change vis-a-vis a suitable baseline value.
The Prdml2 modulator as described herein may alleviate pain in a subject by at least 1, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 as reported by the treated subject when using a self-reporting pain scale. In preferred embodiments, the self-reporting pain scale is the numeric rating scale (NRS 11), Stanford pain scale, or visual numeric scale. In further embodiments, the self-reported pain sensation experienced by a subject and expressed in a numeric value is halved by administration of the Prdml2 modulator. In embodiments wherein the subject is unconscious, optionally as a consequence of severe pain sensation, the Prdml2 modulator may significantly alleviate pain in said subject as measured by objective quantitation methods including but not limited to functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). In certain embodiments, the pain alleviation in a subject is assessed by at least two methods selected from the group consisting of: self-reporting scales, fMRI, and EEG, which are all techniques suitable to study or measure pain as indicated above.
The modulators as described herein may have an immediate or near immediate effect and/or a delayed effect on the pain sensation of the subject to which said modulator is administered. In view of the complex role of Prdml2 in pain responses which the inventors have discovered, the (near) immediate effect may markedly differ from the delayed effect of said modulator. Certain modulators as described herein may be characterised by a dosage-dependent effect, while others may display a consistent effect over time as long as a certain threshold concentration in the target cell, tissue, or subject is equalled and/or surpassed.
The terms “treatment” or “treat” encompass both the therapeutic treatment of an already developed pain condition, such as the therapy of an already developed inflammatory pain, nociceptive pain, or neuropathic pain, as well as prophylactic or preventive measures to prevent an envisaged pain condition in a future point in time, wherein the aim is to prevent or lessen the chances of incidence of pain, such as to prevent occurrence, development and progression of an inflammatory pain, nociceptive pain, or neuropathic pain. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of pain, stabilised (i.e. not worsening) pain, delay or slowing of the pain sensation, amelioration or palliation of the pain condition, and the like. “Treatment” can also indicate an increased quality of life when compared to the quality of life without Prdml2 modulator.
Related hereto, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the object, or goal, is to change a subjects body or a part of a subjects body from an undesired physiological state, disease or disorder, such as any pain condition described herein, to a desired state, such as a less severe state (e.g. amelioration or palliation), or even back to its normal, healthy state (e.g. restoring a pain-free or near pain-free status of a subject), to keep it (i.e. not worsening) at said undesired physiological status (e.g. stabilisation), or slow down progression to a more severe or worse pain condition or pain sensation compared to said undesired pain condition or pain sensation prior to treatment with a Prdml2 modulator as described herein. Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Generally, the terms encompass both curative pain treatments and pain treatments directed to reduce pain and/or slow progression and/or stabilise the pain condition over time.
“Prevention” or “prevent” as used in the context of the invention refers to an aversion of manifestation of a pain condition in a subject, i.e. the establishment of preventive measures or prophylactic measures. Preventive treatment refers to treatments wherein the object is to avoid a subject’s body showing a physiological pain response and/or avoid a subject’s perception of a pain sensation.
The terms "predicting" or "prediction" generally refer to a statement, declaration, indication, anticipation or foretelling of a pain condition in a subject not (yet) showing any, or a limited, clinical indication of said pain condition, or more relevant in the context of the present invention a pain-inducing condition. A prediction of a pain condition in a subject may indicate a probability, chance or risk that the subject will develop said condition, for example within a certain time period (expressed in minutes, hours, days, weeks, months, and/or years), or by a certain age. This probability, chance or risk may be formulated or phrased as any suitable qualitative or quantitative expression, optionally including both an expected or predicted time interval and additionally a measure of the predicted severity of the pain condition or pain sensation. Alternatively this probability, chance, or risk may be indicated relative to a suitable control subject or subject population (such as, e.g. relative to a general, normal or healthy subject or subject population). Hence, the probability, chance or risk that a subject will develop a pain condition may be advantageously indicated as increased or decreased, or as fold- increased or fold-decreased relative to a suitable control subject or subject population. The term “prediction” of the conditions or pain-inducing diseases as taught herein in a subject may also be expressed as the subject having a 'positive' prediction of such, i.e. that the subject is at risk of having such (e.g. the risk is significantly increased vis-a-vis a control subject, control subject population, or an earlier examination of said subject).
In a first aspect, the invention relates to a modulator of Prdml2 activity, for use in treatment or prevention of a pain condition in a subject.
In certain embodiments, the Prdml2 modulator is a Prdml2 activator. In further embodiments, the Prdml2 activator is a conditional Prdml2 activator.
In alternative embodiments, the Prdml2 modulator is a Prdml2 inhibitor. In further embodiments, the Prdml2 inhibitor is a conditional Prdml2 inhibitor.
In certain embodiments, the Prdml2 modulator may have an opposite effect on Prdml2 activity in distinct temporal pain responses. For example, the Prdml2 modulator may have an activating effect on Prdml2 activity in the primary or first pain response and an inhibiting effect on Prdml2 activity in a subsequent pain response.
Vice versa, the Prdml2 modulator may have an inhibiting effect on Prdml2 activity in the primary or first pain response and an activating effect on Prdml2 activity in a subsequent pain response.
The Prdml2 modulator as disclosed herein may be used for any type of pain. In certain embodiments, the Prdml2 modulator described herein is used for treatment of one or more occupational injuries. In certain embodiments, the Prdml2 modulator described herein is used for treatment of disease-related pain or age-associated pain, preferably chronic disease-related pain or chronic age-related pain. In certain embodiments, the Prdml2 modulator is used as a prophylactic, optionally in a subject, wherein said subject is envisaged to be or scheduled to be undergoing a medical procedure, such as but not limited to invasive medical procedures such as an operation.
The Prdml2 modulator as described herein may be used as a topical analgesic or systemic analgesic. In certain embodiments envisaged herein, the Prdml2 modulator for use in treatment or prevention of a pain condition in a subject, in support of a distinct analgesic known in the art is intended. In certain embodiments, the effect of the Prdml2 modulator is juxtaposed to the effect of the distinct analgesic. In alternative embodiments, administration of the Prdml2 modulator exerts a synergistic effect with said analgesic.
“Prdml2 activity” as used throughout the present disclosure is to be interpreted according to its broadest interpretation. Hence, Prdml2 activity may intent to denote any parameters of Prdml2 that may influence or alter the nett resulting activity of Prdml2 in a subject, preferably in a nociceptor of said subject and therefore includes but is not limited to parameters such as Prdml2 expression (subject to both transcription and translation rates), half-life, functionality, localisation, capacity to bind to other proteins (including but not limited to G9a and histones), or any combination thereof.
In the context of the invention, a Prdml2 modulator is considered a Prdml2 inhibitor when said modulator negatively effects the activity of Prdml2. Hence, a Prdml2 modulator as disclosed herein is considered a Prdml2 inhibitor when said modulator gives rise to a measured Prdml2 activity value that is lower than the baseline value. The term “inhibitor” is to be interpreted by its commonly accepted meaning in the art, and the associated verb “to inhibit” may be annotated by non-limiting synonyms such as to reduce, to diminish, to render void, to nullify, to negate, to mitigate, to downregulate, to turn off, to decrease, to reduce, to minimise, to lessen, to weaken, to attenuate, to constrain, to hinder, to impede, to deter, to stop, etc. the activity of a certain target molecule, here Prdml2. Prdml2 inhibitors as envisaged herein may inhibit Prdml2 activity either partially (i.e. to a certain degree) or completely. When referring to a complete inhibitor or the likes it is understood by a skilled person that the activity of Prdml2 and/or it gene product(s) is diminished from a control or baseline activity to 0%, or below an activity level that can be measured by methods available in the art.
Similarly, in the context of the present invention, a Prdml2 modulator is considered a Prdml2 activator when said modulator positively affects the activity of Prdml2. Hence, a Prdml2 modulator as described herein is considered a Prdml2 activator when said modulator leads to a Prdml2 activity value that is higher than the baseline value. The term “activator” is to be interpreted by its commonly accepted meaning in the art, and the associated verb “to activate” may be annotated by non-limiting synonyms such as to induce, to actuate, to exaggerate, to amplify, to elevate, to upregulate, to turn on, to increase, to ameliorate, to enhance, to improve, to stimulate, to effectuate, etc. the activity of a certain target molecule, here Prdml2.
Given the intricate and context-dependent role of Prdml2 in the pain response and consequently pain sensation in a subject, a Prdml2 inhibitor may have a positive or negative effect on the resulting pain sensation, optionally even differing in time within one pain response. Similarly, a Prdml2 activator may also have a positive or negative effect on the resulting pain sensation, optionally even differing in time within one pain response.
In certain embodiments, the Prdml2 modulator alters the expression of Prdml2 in nociceptors of a subject. In further embodiments, the alteration in expression level of Prdml2 may be limited in time e.g. while the Prdml2 modulator is present in said nociceptors or permanent, i.e. until a point in time wherein no detectable levels of Prdml2 modulator are present in said nociceptors). In certain embodiments, the Prdml2 modulator may alter Prdml2 expression by binding or modification of the Prdml2 promoter sequence. A “promoter” as defined herein is a region of DNA that initiates transcription of a particular gene and hence enables a gene to be transcribed. A promoter is recognised by RNA polymerase, which then initiates transcription. Thus, a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase. A promoter sequence may also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer(s), while typically at the 5’ end of a coding region, can also be separate from a promoter sequence, e.g. can be within an intronic region of a gene or 3’ to the coding region of the gene. Promoters may be located in close proximity of the start codon of genes, in preferred embodiments on the same strand and typically upstream (5’) of the gene. Promoters may vary in size and are preferably from about 100 to 1000 nucleotides long, although this length is not to be interpreted as a strict limitation.
In any of the described embodiments herein, the Prdml2 modulator may conditionally alter the expression of Prdml2 in nociceptors of a subject. A person skilled in the art is aware of inducible expression and/or inducible activation systems, the Tet-on and Tet-off systems being typical, yet non-limiting examples hereof (e.g. Das et al. Current Gene therapy, 2016). In yet alternative embodiments, the Prdml2 modulators as described may affect the Prdml2 expression in nociceptors by impacting the subcellular localisation of Prdml2 in nociceptors, optionally in a conditional manner. As a non-limiting example, the Prdml2 modulator may sequester Prdml2 away from the nucleus of a nociceptor (inducing an extranuclear or mainly extranuclear localisation), resulting in decreased expression of Prdml2 in the nucleus of said nociceptor.
In one or more preferred embodiments, the Prdml2 modulator increases the expression of Prdml2 in nociceptors of a subject, preferably by at least about 10%, at least about 25%, at least about 35%, more preferably at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator. In certain embodiments, the Prdml2 modulator increases the expression of Prdml2 in nociceptors in a subject by at least 1.5-fold, at least 2-fold, at least 5-fold, or even at least 10-fold when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator. Alternatively, the increased expression of Prdml2 as described above may be vis-a-vis a reference Prdml2 expression value of a group of subjects that are not administered said modulator, e.g. a mean or median value. A skilled person will appreciate that when the Prdml2 modulator is administered in response to an existing pain sensation or ongoing pain response in a subject, the reference value is the Prdml2 expression level during said pain sensation or pain response before Prdml2 administration. In further embodiments, the increased Prdml2 expression occurs selectively in sleeping nociceptors. Without any limitation hereto, the Prdml2 modulator may increase the half-life of Prdml2 by binding to Prdml2 or inhibiting proteasomal degradation of Prdml2 in said nociceptors. Alternatively, the Prdml2 modulator may increase the Prdml2 expression by upregulation of transcription and/or translation of Prdml2. Non-limiting examples of suitable Prdml2 modulators that increase Prdml2 expression are described further below. It is to be understood that Prdml2 modulators that increase Prdml2 expression may for examples also be exogenous oligonucleotide sequences that express additional Prdml2 protein in the nociceptors, or even functional mutants of Prdml2 with increased intrinsic potency, as discussed further below.
In certain embodiments, the Prdml2 modulator reduces the expression of Prdml2 in nociceptors of a subject, preferably by at least about 10%, at least about 25%, at least about 35%, more preferably at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably reduces the expression of Prdml2 in nociceptors of a subject with about 100%, i.e. to a level below the quantification and/or detection limit by suitable techniques for expression measurement available in the art.
The reduction in expression level is expressed in view of the Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator. In further embodiments, the reduction of Prdml2 expression occurs selectively in sleeping nociceptors. Without any limitation hereto, the mode of action of the Prdml2 modulator may rely on binding with the Prdml2 protein or Prdml2 RNA, or alternatively by binding and/or modification of the Prdml2 encoding genomic sequence. In these latter embodiments, the Prdml2 modulator may act as a so-called road block for Prdml2 expression or induce a change in the genomic Prdml2 sequence or Prdml2 promoter sequence which reduces or eliminates expression of a (fully) functional Prdml2 protein. Alternatively, the half-life of Prdml2 may be shortened by the Prdml2 modulator. Yet alternatively, non-limiting examples of suitable Prdml2 modulators that decrease Prdml2 expression are described further below.
Suitable techniques for expression measurement include but are not limited to (reverse transcription polymerase chain reactions (rt-PCR), qPCR, and/or enzyme-linked immunosorbent assays (ELISA). In another aspect the Prdml2 modulator for use in treatment or prevention of a pain condition in a subject, wherein the modulator alters the Prdml2-mediated G9a recruitment to histone H3. The human G9a protein has been described in detail in the art (e.g. in Rani Shankar et al., Epigenetics, 2013, accessible by UniprotKB identifier Q96KQ7 and also referred to in the art as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2)) and has as main function lysine methylation of both histone and non-histone substrates. By means of guidance, human G9a is annotated under NCBI Genbank Gene ID: 10919 and the canonical human G9a amino acid sequence is by means of example reproduced below (SEQ ID NO: 29):
MAAAAGAAAAAAAEGEAPAEMGALLLEKETRGATERVHGSLGDTPRSEETLPKATPDSLEPA GPSS PASVTVTVGDEGADTPVGAT PLIGDESENLEGDGDLRGGRILLGHATKS FPSS PSKGG SCPSRAKMSMTGAGKS PPSVQSLAMRLLSMPGAQGAAAAGSEPPPATTS PEGQPKVHRARKT MSKPGNGQPPVPEKRPPEIQHFRMSDDVHSLGKVTSDLAKRRKLNSGGGLSEELGSARRSGE VTLTKGDPGSLEEWETWGDDFSLYYDSYSVDERVDSDSKSEVEALTEQLSEEEEEEEEEEE EEEEEEEEEEEEEDEESGNQSDRSGSSGRRKAKKKWRKDS PWVKPSRKRRKREPPRAKEPRG VNGVGSSGPSEYMEVPLGSLELPSEGTLS PNHAGVSNDTSSLETERGFEELPLCSCRMEAPK IDRISERAGHKCMATESVDGELSGCNAAILKRETMRPS SRVALMVLCETHRARMVKHHCCPG CGYFCTAGTFLECHPDFRVAHRFHKACVSQLNGMVFCPHCGEDASEAQEVTI PRGDGVTPPA GTAAPAPPPLSQDVPGRADTSQPSARMRGHGEPRRPPCDPLADTIDSSGPSLTLPNGGCLSA VGLPLGPGREALEKALVIQESERRKKLRFHPRQLYLSVKQGELQKVILMLLDNLDPNFQSDQ QSKRT PLHAAAQKGSVEICHVLLQAGANINAVDKQQRT PLMEAWNNHLEVARYMVQRGGCV YSKEEDGSTCLHHAAKIGNLEMVSLLLSTGQVDVNAQDSGGWTPI IWAAEHKHIEVIRMLLT RGADVTLTDNEENICLHWAS FTGSAAIAEVLLNARCDLHAVNYHGDTPLHIAARESYHDCVL LFLSRGANPELRNKEGDTAWDLTPERSDVWFALQLNRKLRLGVGNRAIRTEKI ICRDVARGY ENVPI PCVNGVDGEPCPEDYKYISENCETSTMNIDRNITHLQHCTCVDDCSS SNCLCGQLS I RCWYDKDGRLLQEFNKIEPPLI FECNQACSCWRNCKNRWQSGIKVRLQLYRTAKMGWGVRA LQTI PQGTFICEYVGELIS DAEADVREDDSYLFDLDNKDGEVYCIDARYYGNISRFINHLCD PNI I PVRVFMLHQDLRFPRIAFFS SRDIRTGEELGFDYGDRFWDIKSKYFTCQCGSEKCKHS AEAIALEQSRLARLDPHPELLPELGSLPPVNT .
In certain embodiments, the Prdml2-mediated G9a recruitment to histone H3 is reduced by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 99% or even about 100%. In further embodiments the Prdml2-mediated G9a recruitment to histone H3 is eliminated or reduced to levels below quantification or even detection by methods known to a skilled person. In certain embodiments, the above reduction in Prdml2-mediated G9a recruitment occurs in one or more classes (i.e. groups) of nociceptors consisting of the group of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, and polymodal nociceptors. In preferred embodiments, the reduction in G9a recruitment is selectively, or even exclusively observable in sleeping nociceptors. In alternative embodiments, the Prdml2- mediated G9a recruitment to histone H3 is increased by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, most preferably at least about 100%. In accordance to the above, in certain embodiments the Prdml2-mediated G9a recruitment to histone H3 is increased by 1.5-fold, 2-fold, 2.5-fold, 5- fold, 10-fold, or higher. In certain embodiments, the above described increase in Prdml2- mediated G9a recruitment occurs in one or more classes (i.e. groups) of nociceptors consisting of the group of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, and polymodal nociceptors. In preferred embodiments, the increase in G9a recruitment is selectively, or even exclusively observable in sleeping nociceptors.
As acknowledged in the art, histone H3 methylation by G9a is mediated by recruitment of G9a to histone H3 by Prdml2, and it is established that this recruitment occurs by binding, i.e. physical interaction of Prdml2 with G9a (e.g. Yang and Shinkai, Cell Structure and Function, 2013). Hence, a skilled person can assess the level of G9a recruitment by known means to detect protein-protein interactions, which include non-limiting exemplary assays such as yeast- two-hybrid assays, complementation assays, immunofluorescence assays such as FiSH, etc (Rao, International Journal of Proteomics, 2014). However, when the Prdml2 modulator affects the expression level of the canonical Prdml2 protein in a subject, preferably the human Prdml2 protein as identified by SEQ ID NO: 1, then a skilled person may reasonable assume that G9a recruitment to H3 will be correlated to Prdml2 expression, optionally linearly correlated. Alternatively, when the recruitment of G9a to histone H3 is modified by modifying the physical interaction between Prdml2 and G9a, a skilled person may appreciate that the (changed) affinity of Prdml2 for G9a will determine the extent of, and impact on, Prdml2- mediated G9a recruitment.
In certain embodiments, the Prdml2 modulator alters the methylation status of histone H3, preferably at position Lysine 9 (K9). In preferred embodiments the Prdml2 modulator alters the methylation status of H3K9. Methods to evaluate methylation at specific locations are readily available in the art (reviewed in Kurdyukov and Bullock, Biology, 2016). “Methylation of H3K9” as referred to herein may indicate a change in monomethylation status (H3K9mel), dimethylation (H3K9me2) or a combination thereof. A general method suitable to assess methylation routinely used in the art is bisulfite sequencing. In certain embodiments at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even about 100% of H3K9 is methylated in nociceptors of a subject in the effective temporal time window after administration of the Prdml2 modulator. In certain embodiments the at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, most preferably at least about 95%, or even about 100% of H3K9 is methylated in nociceptors and/or dorsal root ganglia of a subject in the effective temporal time window after administration of the Prdml2 modulator.
In further embodiments, the Prdml2 modulator increases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, more preferably at least about 100%, when compared to histone H3 methylation, preferably H3K9 methylation in respectively nociceptors or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
In yet further embodiments, the Prdml2 modulator decreases the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 by at least about 10%, at least about 25%, preferably at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even reduces said transcription rate by 100% (i.e. achieves a complete transcriptional halt) when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator. In yet further embodiments, the transcription rate of all the genes CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, and optionally PRDM12 is reduced by at least about 10%, preferably at least about 25%, preferably at least about 35%, preferably at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even most preferably reduces said transcription rate by 100%, when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator.
In alternative further embodiments, the Prdml2 modulator decreases methylation of histone H3 in nociceptors and/or dorsal root ganglia of said subject by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, more preferably at least about 100%, when compared to histone H3 methylation, preferably H3K9 methylation in respectively nociceptors or dorsal root ganglia of said subject before administration of said Prdml2 modulator.
In yet further alternative embodiments, the Prdml2 modulator increases the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, NTS by at least about 10%, preferably at least 25%, at least 35%, preferably at least about 50%, at least about 60%, more preferably at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%, when compared to their expression levels in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator. Alternatively worded, the transcription rate in nociceptors or dorsal root ganglia cells of at least one gene, preferably at least two genes, at least three genes selected from the group of genes consisting of PRDM12, CHRNA6, STK32A, STEAP3, CALCB, CYSLTR2, SKOR2, AGTR1A, ISM1, and KCNV1, SST, NTS is at least about 1.5-fold, at least about 2- fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold increased when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator.
In certain embodiments, the Prdml2 modulator decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or most preferably even completely inhibits said transcription rate (i.e. a reduction by about 100%); and additionally decreases the transcription rate in nociceptors and/or dorsal root ganglia of at least one gene selected from the group of genes consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 by at least about 10%, at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, more preferably at least about 95%, or most preferably even completely inhibits said transcription rate (i.e. a reduction by about 100%), when compared to their transcription rate in nociceptors and/or dorsal root ganglia cells before administration of said Prdml2 modulator. It is readily appreciated by the skilled person that one or more of the above embodiments relating to H3 (i.e. H3K9) methylation status and transcription rates, the change in methylation status and related hereto the transcription rate may be specific for one or more classes of nociceptors selected from the group consisting of: thermal nociceptors, mechanical nociceptors, chemical nociceptors, sleeping (silent) nociceptors, and polymodal nociceptors. Preferably, the change in H3K9 methylation induced by the Prdml2 modulator occurs at least in, or even occurs exclusively in sleeping nociceptors. Furthermore, the meaning of “transcription rate” as used herein is to be interpreted in line with the generally accepted meaning thereof in the art. Hence, any of the above embodiments concerning the increase or decrease of one or more genes is associated with an increase or decrease of their expression level in the modulated nociceptors or dorsal root ganglia. The transcription rate and expression level may be positively correlated, preferably linearly positively correlated or are alternatively characterised by an exponential correlation. The term “transcription rate” may relate to the nascent transcription rate and/or the rate of synthesis of mature mRNA (Perez-Ortin, BioEssays, 2013).
As will become apparent from the Examples section, the role of Prdml2 in pain model systems seems to be dual. On the one hand, knocking out Prdml2 in mouse model systems makes them hypersensitive to pain caused by formalin, while it seems to make them less sensitive to pain caused by capsaicin. This implies that Prdml2 activators as disclosed herein or as identified by the methods disclosed herein will generally be of use for treating pain caused or aggravated by reduced or lost Prdml2 expression. Preferred examples of such pain conditions are pain condition induced by inflammation or itch, preferably inflammatory pain. More generally, said pain condition could be seen as a TRPV1 -related pain condition, more preferably, but not limited to: pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders (which include e.g. bladder pain syndrome (previously interstitial cystitis), irritable bowel syndrome (IBS) and fibromyalgia) and musculoskeletal pain.
This further implies that Prdml2 inhibitors as disclosed herein or as identified by the methods disclosed herein will generally be of use for treating pain caused or aggravated by increased Prdml2 expression. Preferred examples of such pain conditions are pain conditions that can be treated by capsaicin such as back pain, joint pain or headaches. In the most rudimentary classification, pain may be classified in two categories, i.e. acute and chronic pain. The Prdml2 modulators may be used for both acute and chronic pain, as the Prdml2 modulators described herein may be used to treat any pain condition. However, different classifications of pain and pain perceptions have been defined in the art, and these pain responses and pain conditions are well known to a person skilled in the art and when referred to herein. Therefore, the pain sensations and pain responses disclosed herein should be interpreted according to their commonly accepted meaning in the art, and a skilled person would appreciate that certain terms may overlap. For example, nociceptive pain may be an acute pain and a somatic pain. Hence, in certain embodiments the pain condition is selected from the group consisting of: acute pain, chronic pain, somatic pain, visceral pain, neuropathic pain, nociceptive pain, inflammatory pain, neuropathic pain, radicular pain, or any combination thereof. In certain preferred embodiments, the pain condition is a nociceptive pain condition, an inflammatory pain condition, a neuropathic pain condition, or any combination thereof. In further preferred embodiments said pain condition is nerve injury, skin inflammation, joint inflammation, or allergy-related inflammation. In certain embodiments, the pain condition is an inflammation-induced pain condition wherein said pain is related to, or caused by an inflammatory disease having a main inflammatory localisation selected from the group consisting of: the nervous system, the cardiovascular system, the respiratory system, the digestion system, the integumentary system, the musculoskeletal system, the urinary system, the reproductive system, the reproductive system, the endocrine system, the lymphatic system, or any combination thereof. In certain embodiments, the Prdml2 modulator is used for treating or preventing allodynia and hyperalgesia in a subject.
Preferred inflammatory diseases related to or causative of pain conditions that benefit from any of the aspects of the invention described herein are selected from the group consisting of: encephalitis, myelitis, meningitis, arachnoiditis, neuritis, dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, otitis externa, otitis media, labyrinthitis, mastoiditis, carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, capillaritis, sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis, mediastinitis, stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis (i.e. parotitis), cheilitis, pulpitis, gnathitis, esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis, proctitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, dermatitis, folliculitis, cellulitis, hi dradenitis, arthritis, dermatomyositis, myositis, synovitis (i.e. tenosynovitis), bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis (i.e. osteitis, osteomyelitis), spondylitis, periostitis, chondritis, nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis, urethritis, oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, posthitis, balanoposthitis, chorioamnionitis, funisitis, omphalitis, insulitis, hypophysitis, thyroiditis, parathyroiditis, adrenalitis, lymphangitis, and lymphadenitis.
“Acute pain” is defined in the art by a sudden onset which is caused by a specific noxious stimulus, of which non limiting examples are given below when defining “nociceptive pain”. The arbitrary cut-off to define “acute” in the field of pain management is 6 months. In contrast, “chronic pain” is commonly described as an ongoing pain that continues after 6 months, wherein the pain sensation perceived by the subject may be constant, increasing, or intermittent in nature. Non-limiting examples hereof are headache, arthritis, cancer, nerve pain, back pain, and fibromyalgia pain. As illustrated by these non-limiting examples, certain chronic pain conditions are representative for a disease state or may be considered a disease state (Grichnik and Ferrante, The Mount Sinai Journal of Medicine, 1991).
The term “nociceptive pain” is used herein and in the art as a collective name to indicate pain that arises, or is activated in response to underlying tissue damage, injury or a stimulus that is capable of causing an injury (Dubin and Patapoutian, The Journal of Clinical Investigation, 2010). Subject experiencing nociceptive pain typically describe the pain as sharp, achy, throbbing, or a combination thereof. However, the final pain sensation or pain sensation may depend on the local injury site environment that may alter the specific characteristics of nociceptors, central connections and the autonomous nervous system. Typically, in nociceptive pain, the peripheral nociceptors are directly activated by a stimulus and transmit a signal (i.e. action potential) throughout the nervous system over the A6 fiber axons and C fiber axons. After transmission through the primary afferent neurons and second-order neurons, the pain stimulus is received and perceived by the brain. In terms of clinical classification, nociceptive pain has a defined cause, wherein removal of said cause or recovery from a causative injury resolves the pain. Acute nociceptive pain lasts for less 6 months. Non-limiting examples of nociceptive pain are sprains, strains, contusions, post-operative pain, tissue trauma, bum pain, vascular pain, and ischemic pain. While nociceptive pain is by definition a distinct pain from inflammatory pain, the two pain conditions may often co-occur by inflammation of the injury site.
“Neuropathic pain” is a pathological state of pain that is triggered by changes of the somatosensory nervous system. Where nociceptive pain and inflammatory pain may be considered protective responses, neuropathic pain is perceived throughout the art as a disease state wherein pain occurs spontaneously or apparent spontaneously in response to innocuous stimuli. Neuropathic pain is indicative of a dysfunction of the nervous system which may include defect in circuitry, signal transmission and/or altered action potentials. Neuropathic pain is commonly described by patients as a burning or shooting sensation that optionally may fluctuate in intensity over time (Costigan et al., Annual Review of Neuroscience, 2009). Neuropathic pain may arise as a consequence of, or have an increased chance to develop following a series of diverse (disease) conditions. Non-limiting conditions associated with (the onset of) neuropathic pain include: addictions (e.g. alcoholism), limb removal (e.g. amputation), treatment-induced pain (e.g. chemotherapy), metabolic disorders (e.g. diabetes), infection (e.g. herpes infection, or HIV infection), neurodegenerative diseases (e.g. multiple sclerosis), damage to the nervous system (e.g. nerve compression such as carpal tunnel syndrome or herniated disc, nerve trauma), sexual transmitted infections (e.g. syphilis), hormonal disturbance (e.g. hypothyroidism, hyperthyroidism), post-herpetic neuralgia (e.g. reactivation of Varicella zoster virus, (often referred to in the art as shingles)), chronic physical pain (e.g. back pain), cancer neuropathy (e.g. multiple myeloma), phantom limb pain, and channel opathies. Channelopathies have been described in detail in the art and a skilled person is therefore capable of identifying this heterogenous group of disorders that are the result of ion channel dysfunction in the membranes and cellular organelles (Kim, Korean Journal of Pediatrics, 2014). Neuropathic pain is associated with a decreased quality of life, including amongst others sleep impairment, lost workdays, and psychological distress. “Allodynia” and “hyperalgesia” are two specific pain conditions that are associated with, or considered related to neuropathic pain and have a generally accepted meaning within the technical field (Jensen and Finnerup, The Lancet Neurology, 2014). A skilled person readily appreciates that allodynia refers to pain due to a stimulus that does not usually provoke pain, and hyperalgesia refers to increased pain (sensation) from a stimulus that usually provokes pain.
The term “inflammatory pain” as used herein indicates both inflammatory pain as such and pain arising from inflammation in a subject. Inflammation is associated with the release of several immune mediators including but not limited to bradykinin, histamine, 5- hydroxytryptamine, adenosine triphosphate, and nitric oxide. Further downstream activation by these mediators leads to the recruitment of immune cells that release additional molecules such as cytokines, chemokines that are considered algogenic. During inflammation, the ongoing exposure to these ligands will cause hypersensitivity of sensory neurons at or near the site of initial insult (known in the art as peripheral sensitisation). Continued peripheral sensitisation may in turn lead to sensitisation of the (near) complete nociceptive pathway (i.e. central sensitisation).
“Radicular pain” as used herein is caused by irritation of the sensory root or dorsal root ganglion of a spinal nerve and generally is used in the art to indicate back pain that is caused by a combination of compression sensitising the nerve root to mechanical stimulation, stretching, and a chemically mediated noncellular inflammatory reaction (often as a consequence of a herniated disc in the subject). Radicular pain is typically perceived as sharp, shooting, piercing, or stabbing pain down the length of the leg. Depending on the severity, loss of sensation and/or loss of motor function may be observed (Dydyk and Das, StatPearls, 2020; Govind, Australian Family Physician, 2004). The Prdml2 modulators as described herein may be used to treat or prevent cervical radicular pain, lumbar radicular pain, spinal radicular pain, or a combination thereof.
“Somatic pain” as used herein indicates pain as the result of activation of pain receptors in tissues and may be further stratified into superficial somatic pain and deep somatic pain, which both can be constant or intermittent in nature. Superficial pain occurs by activation of nociceptors in for examples skin, mucus, and mucous membranes. Common injuries such as skin cuts may be considered a non-limiting example of superficial somatic pain. In contrast, deep somatic pain is typically described as a localised pain which is considered as aching, gnawing, throbbing, or cramping by the subject. Deep somatic pain occurs when stimuli active pain receptors deeper in the body of a subject such as those part of tendons, joints, bones, and muscles. Typical deep somatic pain is often experienced by but not limited to subjects having cancer and bone metastasis (Carver and Foley, Holland-Frei Cancer Medicine, 6th edition).
The term “visceral pain” is caused by activation of nociceptors in for example the cardiovascular, respiratory, gastrointestinal, and genitourinary systems and is typically described by subjects as a dull, deep, squeezing pressure or colicky pain. Visceral pain is often difficult to localise and is often referred to a distinct and typically superficial structure (i.e. referred pain) due to the dual innervation of somatic and visceral structures by common afferent fibers that converge in the dorsal horn in the spinal cord. There is no pathology or cause for experiencing the pain at the referred somatic localisation. The referred pain is sharper in nature and less likely to be co-occurring with autonomic or emotional distress. For example, shoulder pain, resulting from diaphragmatic irritation from a pleural disease, is an example of a cutaneous referral of a visceral pain (Carver and Foley, Holland-Frei Cancer Medicine, 6th edition). The Prdml2 modulator as described herein may broadly refer to any chemical (e.g. inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g. biological macromolecule), a combination or mixture thereof, a sample of undetermined composition, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Preferred, though non-limiting modulators include nucleic acids, oligonucleotides, ribozymes, peptides, polypeptides, proteins, peptidomimetics, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, preferably organic molecules, more preferably small organic molecules, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof. The term “modulator” may denote a “therapeutic agent”, “drug”, or “active pharmaceutical ingredient”, useful for or used in the treatment, cure, prevention, or diagnosis of a pain condition as taught herein.
Examples of groups of molecules or systems that can be considered to increase or reduce the expression and/or activity of a protein are known in the art. Therefore, in certain embodiments the Prdml2 modulator is selected from the group consisting of: Prdml2 binding molecules, Prdml2 or a polynucleotide encoding Prdml2, a functional fragment of Prdml2 or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene targeting system, or any combination thereof. In preferred embodiments, the PRDM12 gene targeting system is a PRDM12 antisense agent or a PRDM12 gene editing system.
“Prdml2 binding molecule” as used herein refers to any protein or non-protein chemical molecule that binds to Prdml2 (either the PRDM12 gene, PRDM12 RNA, or Prdml2 protein), preferably human Prdml2. Therefore, as envisaged herein, the Prdml2 modulator, is a Prdml2 or PRDM12 binding molecule, selected from the group consisting of a chemical substance, an antibody, an antibody fragment, an antibody-like protein scaffold, a protein or polypeptide, a peptide, a peptidomimetic, an aptamer, a photoaptamer, a spiegelmer, a soluble receptor, an antibody mimetic (non-limiting examples hereof include alphabodies, designed ankyrin repeat proteins (DARPins), monobodies, affibodies, anticalins, avimers, versabodies, and duocalins), a PRDM12 gene targeting system, or any combination thereof. In further embodiments the Prdml2 modulator is selected from the group consisting of a chemical substance, a Prdml2- binding antibody, a PRDM12 RNA antisense agent, and a PRDM12 gene editing system. In certain embodiments, the Prdml2 modulator specifically recognises and/or binds to one or more specific Prdml2 C-terminal polyalanine stretch variants, Prdml2 gene isoforms, or nucleotides encoding said variants and/or isoforms. A skilled person is aware that the terms “recognizing” and “targeting” can be interchangeably used in this context. As used throughout this disclosure and in accordance with the art, the term “binding” preferably indicates the binding of a first moiety to a second moiety, for example the binding of a transcription factor to a DNA sequence, the binding of a protein to another protein, the binding of a chemical substance to Prdml2 protein, etc. In contrast, the more specific term “hybridizing” as used herein is indicative for a specific interaction between two nucleotide sequences based on sequence complementarity. A suitable synonym for “hybridizing” is “annealing”, as appreciated by a person skilled in the art. A specific yet non-limiting example of hybridizing in the context of the invention is the hybridisation of an antisense agent to PRDM12 RNA. The nucleic acid sequences that are suitable for hybridisation are not limiting, and therefore encompass DNA-DNA hybridisation, DNA-RNA hybridisation, and RNA-RNA hybridisation. The term “specifically” in the context of “binding”, “hybridizing to” or “targeting” implies that the modulator binds to, influences, or targets the Prdml2 protein sequence or PRDM12 DNA/RNA sequence, without substantially binding or hybridizing to the sequence of another protein or DNA/RNA. Hence, by “specifically binding” or “specifically interacting” is meant that a modulator of Prdml2 activity binds to, influences, or targets Prdml2 or a nucleotide sequence encoding Prdml2 or associated sequences (e.g. the Prdml2 promoter sequence) substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g. at least about 1000-fold or more greater, at least about lxl04-fold or more greater, or at least about lxl05-fold or more greater, than its affinity for a non-target molecule.
The binding or interaction between the modulator of Prdml2 activity and Prdml2 may be covalent (i.e. mediated by one or more chemical bonds that involve the sharing of electron pairs between atoms) or, more typically, non-covalent (i.e. mediated by non-covalent forces, such as for example, hydrogen bridges, dipolar interactions, van der Waals interactions, etc.). Preferably, the agent may bind to or interact with its intended target(s) with an affinity constant (KA) of such binding of at least about KA > IxlO6 M’1, more preferably at least about KA > IxlO7 M4, at least KA > 1 x 108 M4, at least about KA > 1 x 109 M4, and most preferably at least about KA > IxlO10 M4 or KA > IxlO11 M4, wherein KA = [A_T]/[A][T], A denotes the modulator of Prdml2 activity (i.e. the agent) and T denotes Prdml2 (i.e. the target). Determination of KA can be carried out by methods known in the art, such as for example, using equilibrium dialysis and Scatchard plot analysis (Attie and Raines, Journal of Chemical Education, 1995). The binding of a modulator of Prdml2 activity as described herein to Prdml2 and the affinity and specificity of said binding may be determined by any methods known in the art. Non-limiting examples thereof include co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, label transfer, phage display, proximity ligation assay (PLA), Tandem affinity purification (TAP), in silico docking and calculation of the predicted Gibbs binding energy and competition binding assays (e.g. review in Hunter and Cochran, Methods in Enzymology, 2016).
The term “chemical substance” as used herein in its broadest sense and generally refers to any substantially pure substance that has a constant chemical composition and characteristic properties. Preferably, the chemical substance is an organic molecule, preferably a small organic molecule. The term “small molecule” refers to organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g. proteins, peptides, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, e.g. up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g. up to about 900, 800, 700, 600 or up to about 500 Da.
The term “antibody” is used herein in its broadest sense and hence generally refers to any immunologic binding agent, such as a whole antibody or antibody fragments, including without limitation a chimeric, humanised, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest. The term antibody thereby includes a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanised antibody, a human antibody, or an immunologically effective fragment of any of these. The term thus specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g. 2-, 3- or more-valent) and/or multi-specific antibodies (e.g. bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments. The term “antibody” is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g. a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo.
The term antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g. birds and mammals. Without limitation, the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant. Also without limitation, the antibodies may be human, murine (e.g. mouse, rat, etc.), porcine, donkey, rabbit, goat, sheep, guinea pig, monkey (e.g. cynomolus monkeys), camel (e.g. Camelus bactrianus and Camelus dromaderius) also including camel heavy-chain antibodies, llama (e.g. Lama paccos, Lama glama or Lama vicugna) also including llama heavy-chain antibodies, or horse.
Furthermore, the term antibody as used herein also encompasses “chimeric antibodies” which originate from at least two animal species. More specifically, the term “chimeric antibody” or “chimeric antibodies” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as for example antibodies having murine heavy and light chain variable regions linked to human, non-human primate, canine, equine, or feline constant regions. Chimeric antibodies comprise a portion of the heavy and/or light chain that is identical to or homologous with corresponding sequences from antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, exhibiting the desired biological activity (Morrison et al., PNAS USA, 1984). Chimeric antibodies are made through merging DNA encoding a portion, such as the Fv region, of a monoclonal antibody from one species, e.g. mouse or monkey, with the antibody-producing DNA from another species, e.g. human.
In certain embodiments wherein the modulator of Prdml2 activity is a Prdml2 affinity ligand such as an antibody, it is understood by a skilled person that this indicates that said affinity ligand recognises and binds to at least one epitope of Prdml2, preferably an epitope unique for Prdml2 within the relevant proteomic background, a preferred proteomic background being the human proteome. The term “epitope” includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. An antibody is said to specifically bind an antigen when it preferentially recognises its target antigen in a complex mixture of proteins and/or macromolecules. In accordance with their generally accepted meaning in the art, the terms “binding region”, “binding site” or “interaction site” indicates a certain site, part, domain or stretch of amino acid residues that is responsible for binding to an antigen of interest. Such binding region essentially consists of specific amino acid residues of the affinity ligands such as antibodies described herein, which residues are in contact with Prdml2.
Related hereto, the term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an antibody) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.
Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an antibody) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigenbinding proteins (such as antibodies) will bind with a dissociation constant (KD) of 10'5 to 10’ 12 moles/liter (M) or less, and preferably 10'7 to IO42 moles/liter (M) or less and more preferably 10'8 to IO42 moles/liter, and/or with an association constant(KA) of at least 107 M’ \ preferably at least 108 M4, more preferably at least 109 M4, such as at least 1012 M4. Any KD value greater than 104 M is generally considered to indicate non-specific binding. Preferably, an antibody will bind to the desired antigen with an KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.
A skilled person appreciates that a full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds. The amino terminal portion of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy -terminal portion (i.e. C-terminal portion) of each chain defines a constant region primarily responsible for effector function. The CDRs are interspersed with regions that are more conserved, termed framework regions (FR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred to as “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions is in accordance with the well- known Kabat numbering convention, which refers to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain regions of an antibody (Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, 1991 ). The positioning of CDRs in the variable region of an antibody follows Kabat numbering or simply, “Kabat.”
Light chains are classified as kappa or lambda, and are characterised by a particular constant region as known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g. IgGl, IgG2, IgG3, IgG4. Each heavy chain type is characterised by a particular constant region with a sequence well known in the art. In certain embodiments, the Prdml2 binding molecule is an antibody selected from the group consisting of: IgA, IgD, IgE, IgG and IgM antibodies, preferably an IgG class antibody. In certain embodiments, the antibody may be a polyclonal antibody, e.g. an antiserum or immunoglobulins purified there from. In other embodiments, the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility.
As used herein, the term “monoclonal antibody” refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies preferably exist in a homogeneous or substantially homogeneous population. Monoclonal antibodies and antigenbinding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g. CDR- grafting, or combinations of such technologies, or other technologies known in the art. By means of example and not limitation, monoclonal antibodies may be made by the hybridoma method (initially described by Kohler et al. Nature, 1975), or may be made by recombinant DNA methods. Monoclonal antibodies may also be made using phage antibody libraries (Clackson et al., Nature, 1991).
The term “antibody fragment” or “antigen -binding moiety” comprises a portion or region of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, Fv , scFv fragments, single domain (sd)Fv, such as VH domains , VL domains and VHH domains, diabodies, linear antibodies, single-chain antibody molecules, in particular heavy-chain antibodies; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g. dibodies, tribodies, and multibodies. The above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning. The term “antigen-binding portion” or “antigen-binding region” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. These may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 1989); which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) (Bird et al., Science, 1988). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are known to a person skilled in the art and are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger et al., PNAS, 1993).
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas, 1995) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., Molecular Immunology, 1994). Antibody portions, such as Fab and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules may be obtained using standard recombinant DNA techniques.
In certain embodiments, the Prdml2 antibody fragment may be a Nanobody®. The terms “Nanobody®” and “Nanobodies®” are trademarks of Ablynx NV (Belgium). The term “Nanobody” is well-known in the art and as used herein in its broadest sense encompasses an immunological binding agent obtained (1) by isolating the VHH domain of a naturally occurring heavy-chain antibody, preferably a heavy-chain antibody derived from camelids; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanisation" of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanised VHH domain; (4) by "camelisation" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelised VH domain; (5) by "camelisation" of a "domain antibody" or "dAb" as described in the art, or by expression of a nucleic acid encoding such a camelised dAb; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.
The amino acid sequence and structure of a Nanobody can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region For "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. The total number of amino acid residues in a Nanobody can be in the region of 110-120, and preferably 112-115. It should however be noted that parts, fragments, analogs or derivatives of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are preferably suitable for the purposes described herein.
In accordance with the terminology commonly employed in the art, the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to herein as “VL domains”). As mentioned in the prior art referred to above, VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4- chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a VH domain covalently linked to a VL domain).
In certain embodiments, the anti-Prdm!2 antibody fragment is an domain antibody (dAb) (described in e.g. Ward et al., Nature, 1989). Single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", as described in Streltsov et al., Protein Science, 2005). In further embodiments, the antibody or antibody fragment may be multispecific (such as a bispecific, trispecific, etc. antibody) comprising at least two (such as two, three, etc.) binding sites, each directed against a different antigen or antigenic determinant. In some embodiments, the therapeutic agent may be a dual variable domain immunoglobulin (DVD-Ig™) (described in e.g. DiGammario et al., Methods in Molecular Biology, 2012).
In certain embodiments, the anti-Prdm!2 antibody may be a “fully human antibody”. As used herein, the term “fully human antibody” refers to an antibody of which the encoding genetic information is of human origin. Accordingly, the term “fully human antibody” refers to antibodies having variable and constant regions derived only from human germline immunoglobulin sequences. The term “fully human antibody” is thus not to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. Fully human antibodies may be derived from phage human antibody libraries as described above, or they may be obtained through immunisation of transgenic mice which have been engineered to replace the murine immunoglobulin encoding region as described in Lonberg and Husznar 1995 (Int. Rev. Immunol. 13 (1): 65-93). Fully human antibodies that are made using phage display are preferably produced by recombinant expression in a human cell line resulting in antibodies with a human glycosylation pattern. Non-limiting examples of fully human antibodies are HuCAL® antibodies (Morphosys). The genetic information for constructing a HuCAL® antibody is extracted from the HuCAL® antibody library (Morphosys) and introduced into human PER.C6® cells in the form of a vector (i.e. transfection). The transfected cells translate the genetic information into protein. The protein is further modified by glycosylation and the resulting antibody molecule is finally secreted by the cells into the culture medium. The term antibody as used further encompasses “humanised antibodies”, which are antibodies derived from non-human species whose protein sequence have been modified so as to increase their similarity to antibodies produced naturally in humans. More particularly, the term “humanised antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non -human species (e.g. a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e. more similar to human germline variable sequences. One type of humanised antibody is a CDR-grafted antibody, in which non-human CDR sequences are introduced into human VH and VL sequences to replace the corresponding human CDR sequences.
The humanised antibody is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanised antibody comprises substantially all, or at least one, and typically two, variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e. donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanised antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. A humanised antibody may contain both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively, a humanised antibody may only contain a humanised light chain, or a humanised heavy chain. An exemplary humanised antibody contains a humanised variable domain of a light chain and a humanised variable domain of a heavy chain. By means of illustration and not limitation, humanised antibodies may be derived from conventional antibodies (i.e. an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds) from the family Camelidae, in particular from the llama (e.g. Lama paccos, Lama glama or Lama vicugna), whose variable domains exhibit a high degree of amino acid sequence identity with the variable domains of human antibodies. A suitable platform for the production of such humanised antibodies is the SIMPLE Antibody™ platform (ArGEN-X, WO 2011080350).
Methods for immunising animals, e.g. non-human animals such as laboratory or farm animals, using immunising antigens optionally fused to or covalently or non-covalently linked, bound or adsorbed to a presenting carrier, and preparation of antibody or cell reagents from immune sera is well-known per se and described in documents referred to elsewhere in this specification. The animals to be immunised may include any animal species, preferably warmblooded species, more preferably vertebrate species, including birds and mammals. In certain embodiments, the antibody capable of modulating Prdml2 activity is a bird antibody, preferably wherein said antibody is selected from the group consisting of chicken, turkey, goose, duck, guinea fowl, quail, or pheasant antibody. Alternative embodiments, the antibody capable of modulating Prdml2 activity is a mammalian antibody, preferably wherein said antibody is selected from the group consisting of: human, murine (e.g. mouse, rat), porcine, donkey, rabbit, goat, sheep, guinea pig, camel, llama or horse antibody.
The skilled person is further aware that production methods for producing recombinant antibodies have been described in the art. Such methods rely on a host organism or cell. The terms “host cell” and “host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host organisms or cells for the production of antibodies include inter alia unicellular organisms, such as bacteria (e.g. E. coli), and (cultured) animal cells (e.g. mammalian cells or human cells). The advantages of producing antibodies in bacteria are amongst other the relatively safe and straightforward handling of bacterial cells and the rapid replication cycles of microorganisms. Bacteria are particularly suitable for the production of antibody fragments with a simple structure. For the production of full-length immunoglobulins or more complex antibody fragments in prokaryotic cells, the bacterial cells may be transformed with at least two nucleic acids each encoding a different portion of the antibody fragment or the immunoglobulin, e.g. the heavy chain or the light chain, as described in WO 2009021548 for full-length immunoglobulins. In the bacterial cell, the genetic information encoding the antibody is read and translated into a protein. The resulting antibodies accumulate in the periplasmic space and can be harvested upon lysis of the bacterial cells. A further separation step may be performed to purify the antibodies. WO 2009021548 describes an E. Coli-based secretion system wherein the bacteria release the antibodies in the surrounding culture medium due to the introduction of a signal sequence into the antibody encoding construct. This enables the easy and convenient purification of the antibodies from the cell culture medium. An exemplary mammalian cell line that can be used for the production of antibodies is the Chinese hamster ovary (CHO) cell line. An exemplary human cell line suitable for the production of antibodies includes the PER.C6® cell line as deposited under EC AC no. 96022940. Particularly preferred for the production of fully human antibodies are human cell lines due to their capacity to produce antibodies that contain a human glycosylation pattern.
The term “PRDM12 gene targeting system” as used herein encompasses both PRDM12 antisense agents and PRDM12 gene editing systems. In certain embodiments wherein the Prdml2 modulator is an antisense agent or a gene editing system, the modulator is directed to a portion of a nucleotide sequence encoding Prdml2 in a genomic sequence of a subject. In alternative embodiments wherein the modulator of Prdml2 is an antisense agent or a gene editing system, the inhibitor is directed to a portion of the PRDM12 promoter sequence. In further embodiments wherein the Prdml2 modulator is an antisense agent or a gene editing system, the inhibitor comprises a nucleotide sequence that has a sequence identity of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99% compared to a portion of a naturally occurring genomic sequence of PRDM12, SEQ ID NO: 2, or PRDM12 promoter sequence in a subject, preferably a human subject. In alternative embodiments, the modulator of Prdml2 inhibits Prdml2 activity by direct hybridisation to PRDM12 RNA. In alternative embodiments, the inhibitor of Prdml2 inhibits Prdml2 activity by direct hybridisation to PRDM12 RNA and additionally targeting it for degradation, wherein said degradation is optionally mediated by an RNA-induced silencing complex. In yet further embodiments, the inhibitor specifically targets and hybridises to a sequence comprised within SEQ ID NO: 2, or a sequence encoding SEQ ID NO: 1 or a naturally occurring variant thereof. In particular embodiments, the antisense agent is a sequence of between 8 and 50 nucleotides in length which is a sequence which is complementary to a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% sequence identity to an mRNA encoding SEQ ID NO: 1 or a portion thereof, or wherein the antisense agent is complementary to a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% sequence identity to a portion of SEQ ID NO: 2 and is able to hybridise to said sequence.
The term “antisense agents” indicates an oligonucleotide configured to specifically anneal with (i.e. hybridise to) a given sequence in a target nucleic acid, typically an mRNA and in the context of the present invention in particular PRDM12 mRNA. An PRDM12 antisense agent in the context of the present invention comprises, consists essentially of or consists of a nucleic acid sequence that is complementary or essentially complementary to said target PRDM12 nucleic acid sequence. Antisense agents suitable for use as a PRDM12 modulator are able to hybridise to the PRDM12 target nucleic acid sequences at high stringency conditions, and are able of hybridising specifically to PRDM12 mRNA under physiological conditions. Synthesis, manipulation and introduction into cells of antisense agents have been described in the art and are therefore known to a skilled person (for example Dias and Stein, Molecular Cancer Therapeutics, 2002).
The sequence of an antisense agent does not need to be a perfect “match” (i.e. 100% complementary to the PRDM12 nucleic acid sequence) to that of its target sequence to anneal or hybridise specifically with the latter. An antisense agent may be said to be specifically hybridisable when interaction of the agent to a target nucleic acid molecule interferes with the normal function of the target nucleic acid such as to attain an intended outcome (e.g. loss of utility), and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense agent to non-target sequences under conditions in which specific hybridisation is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. In certain preferred embodiments, the PRDM12 antisense agent is an inhibitor of Prdml2 activity, wherein said antisense agent reduces the Prdml2 activity by at least about 10%, preferably at least about 25%, at least about 35%, at least about 50%, at least about 60%, preferably at least about 75%,; more preferably at least about 80%, at least about 85%, at least about 90%, at least about 95% when compared to the Prdml2 activity in absence of said antisense agent. In further preferred embodiments, the antisense agent reduces the Prdml2 activity by about 100%, i.e. below a detection threshold. The reduction in Prdml2 activity may be limited to nociceptors and/or dorsal root ganglia, to one or more tissues or organs, or on the level of the subject as a whole. In particular embodiments, the Prdml2 inhibitor is selected from the group of antisense agents consisting of: RNAi (RNA interference), shRNA (short hairpin RNA), siRNA (silencer RNA), and miRNA (micro RNA).
The terms “complementary” or “complementarity” as used herein with reference to nucleic acids, refer to the normal binding (i.e. hybridisation) of single-stranded nucleic acids under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson- Crick base pairing. By means of example, complementary Watson-Crick base pairing occurs between the bases A and T, between A and U, and between G and C. In one or more embodiments disclosed herein wherein the Prdml2 modulator is an antisense agent, the nucleotide sequence of the antisense agent may comprise at least one modification, preferably a terminal modification that may optionally inhibit or reduce degradation of the PRDM12 antisense nucleotide sequence by cellular machinery of a host cell. A non-limiting example of a modification that inhibits or reduces degradation of the antisense nucleotide sequence is a phosphorothioate bond in the phosphate backbone of an oligo wherein a sulfur atom is substituted for a non-bridging oxygen. In certain embodiments, the at least one phosphorothioate bond is introduced at the 5’ or 3’ terminus. In certain embodiments, the at least one phosphorothioate bond is present internally.
Alternatively or in addition to modifications that alter cellular degradation rates of the antisense nucleotide sequence, said nucleotide sequence may be physically coupled (i.e. conjugated) to other moi eties or conjugates that alter other characteristics of said nucleotide sequence, or even impart additional functions to said nucleotide sequence. Non-limiting examples of such characteristics are altered activity, cellular distribution, immunogenicity and/or cellular uptake. Non-limiting examples of modifications include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g. dodecandiol or undecyl residues, a phospholipid, e.g. di-hexadecyl-rac-glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecyl amine or hexylamino-carbonyl- oxy cholesterol moiety.
“Gene editing system” as used herein indicates any molecular tool or system that is able to induce one or more targeted nucleic acid modifications in the sequence of PRDM12, PRDM12 promoter sequence and/or optionally PRDM12 enhancer sequence(s) within an intact and living cell. The PRDM12 sequence may be the genomic PRDM12 sequence or PRDM12 (m)RNA sequence. In view hereof, in certain embodiments modulator of Prdml2 activity is a (endo)nuclease or a variant thereof having altered or modified activity. (endo)Nucl eases known in the art comprise programmable, sequence-specific DNA- or RNA-binding modules linked to a nonspecific DNA or RNA cleavage domain (review in e.g. Gaj et al., Cold Spring Harbor Perspectives in Biology, 2016). When targeting DNA, these nucleases create site-specific double-strand breaks at desired locations in the genome. The induced double-stranded breaks are repaired through non-homologous end-joining or homologous recombination, resulting in targeted mutations in the PRDM12 sequence. Alternatively modified, i.e. mutated forms of the endonucleases may be used to generate DNA “nicks” instead of double stranded breaks. By DNA “nick” is intended herein a double stranded DNA sequence wherein only one of the two strands contains a breaks, while the second strand remains intact, or may be separately nicked by a complementary nicking endonuclease optionally at a location proximal to the location of the nick location on the first strand. Non-limiting examples of endonucleases are restriction enzymes, meganucleases, zinc-finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and CRISPR-associated (Cas)-based nucleases. In preferred embodiments, the Prdml2 modulator, is selected from the group of endonucleases consisting of restriction enzymes, meganucleases, ZFNs, TALENs, CRISPR-Cas. In preferred embodiments, the Prdml2 modulator is selected from the group of endonucleases consisting of ZFNs, TALENs, CRISPR-Cas. In further preferred embodiments, the Prdml2 modulator is a CRISPR-Cas endonuclease, preferably a CRISPR-Cas9 endonuclease. In one or more embodiments described herein wherein the Prdml2 modulator is an endonuclease, said endonuclease specifically binds, or has a preference of binding to the human promoter sequence of Prdml2 or at least one exon selected from one of the 5 human PRDM12 exons. In yet further embodiments, the endonuclease specifically binds, or has a preference of binding to exon 1, 2, or 3 of human PRDM12.
The term “restriction enzyme” as used herein can be used interchangeably with “restriction endonuclease” or “restrictase” and refers to a class of endonucleases that cleave DNA at or in close proximity to specific recognition sites, which are commonly referred to as “restriction sites” in the art. Numerous restriction enzymes have been identified in the art. Methods, tools, and databases have been described in the art and are freely available to find information on both restriction enzyme activity and restriction sites (Roberts et al., Nucleic Acids Research, 2007). “Meganucleases” are a class of nucleases that are characterised by a larger recognition site than traditional (i.e. standard) restriction enzymes, wherein said recognition site typically has a length of between about 12 and 40 base pairs. Meganucleases have been identified in a considerable number of organisms, including but not limited to Archaea, bacteria, phages, fungi, yeast, algae, and plants. Additionaly, methodologies and tools are available in the art that allow the design and production of artificial meganucleases (Bartsevich, et al., Molecular Therapy, 2016). In one or more embodiments wherein the Prdml 2 modulator is a meganuclease targeting a PRDM12 sequence, the meganuclease is selected from the group consisting of one of the following families (based on sequence and structure motil): LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)XK.
“Zinc-finger nucleases”, commonly abbreviated as “ZFNs”, are artificial restriction enzymes that comprise a zinc finger DNA binding domain fused to a DNA cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences which allows for a skilled person to design zinc finger nucleases that are able to target unique sequences within a given genomic sequence, such as but not limited to the PRDM12 genomic sequence or PRDM12 promoter sequence. Traditional zinc finger DNA binding domains contain between about three and six zinc finger repeats which will each recognise 9 to 18 nucleotides. Methods to design and produce zinc finger arrays are known to a skilled person (Wu et al., Custom-designed zinc finger nucleases: What is Next?, Cellular and molecular life sciences, 2007). By means of guidance and not limitation, an example of a suitable non-specific cleavage domain is the obligate dimeric endonuclease FokI domain, and engineered FokI domains with enhanced cleavage activity such as Sharkey (Guo et al., Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases, Journal of molecular biology, 2010). Obligate heterodimeric ZFNs containing FokI domains wherein the FokI domains comprise modified dimerisation interfaces whereby only the heterodimeric FokI reconstituted species display catalytic activity (Szczepek et al., Structure-based redesign of the dimerisation interface reduces the toxicity of zinc-finger nucleases, Nature Biotechnology, 2007) are preferred embodiments when the Prdml2 modulator is a ZFN.
“Transcription-activator like effector nucleases”, or short “TALENs” are another class of artificial restriction enzymes that comprise a Transcription Activator Like (TAL) effector DNA binding domain which is fused to a DNA cleavage domain. Similar to ZFNs, it is possible to engineer TAL effector domains to specifically bind any given DNA sequence present in a genome, such as but not limited to (a portion ol) the PRDM12 genomic sequence or PRDM12 promoter sequence. TAL effector domains comprise a repeated conserved of about 33 or 34 amino acid sequence with variable amino acids at the 12th and 13th position, commonly annotated as repeat variable diresidues (RVDs). Said RVDs determine the specific nucleotide recognition pattern of the TALEN. Tools and protocols to generate TAL effector domains specific for a desired sequence are publicly available (Heigwer et al., Nucleic acids research, 2013; Neff et al., BioMedCentral Bioinformatics, 2013). In accordance to the above-described ZFNs, TALENs may be based on the use of a (modified) FokI domain as DNA cleavage domain, but can in theory include any DNA cleavage domain.
The term “CRISPR-associated (Cas)-based nucleases”, which may be used interchangeably with “CRISPR/Cas nuclease”, “CRISPR-Cas” is indicative for a genome engineering system comprising an endonuclease that relies on the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences to recognise and cleave specific strands of nucleotide sequences complementary to the CRISPR sequence. Both single stranded, double stranded, RNA and DNA cleaving CRISPR/Cas systems have been described in the art and are therefore known to a person skilled in the art (Makarova and Koonin, Annotation and classification of CRISPR-Cas systems, Methods in molecular biology, 2015, and Makarova et al., Classification and nomenclature of CRISPR-Cas systems: where from here?, The CRISPR journal, 2018). By means of guidance, examples of Cas proteins include Cas3, Cas 8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, CaslO, Csm2, Cmr5, CaslO, Csxll, CsxlO, Csfl, Cas9, Csn2, Cas4, C2cl, C2c3, Casl2 (i.e. Cpfl), Casl3a, Casl3b, Casl3c, and Casl3d. in accordance to what is described in the art, a skilled person appreciated that different Cas proteins require different CRISPR sequences. A particularly preferred CRISPR-Cas system is the CRISPR-Cas9 system. Cas9 comprises two nuclease domains, a RuvC domain and a HNH nuclease domain, which are responsible for cleavage of the nontarget DNA strand and the target strand respectively. CRISPR-Cas9 can be specifically or preferentially to virtually any DNA sequence that comprises a protospacer adjacent motif (PAM), with the PAM being the nucleotide sequence NGG, wherein N may be any nucleotide. It is established in the art that a functional Cas9 relies on complex formation of the protein with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), which may be presented as a single functional guide RNA (gRNA) sequence (e.g. Wang et al., Annual Review of Biochemistry, 2016). In one or more embodiments wherein the modulator of Prdml2 activity is based on targeted activity of a CRISPR-Cas9 system, the Cas9 protein is a modified Cas9 protein comprising a mutagenised HNH and/or RuvC catalytic domain. In further embodiments, said Cas9 protein is a nickase Cas9 (nCas9). In yet alternative embodiments, said Cas9 protein is a catalytically inactive Cas9 (i.e. dead Cas9, or dCas9) (cf. also https://www.origene.com/products/gene-expression/crispr-cas9/crispra-crispri). In further embodiments, double stranded breaks in a genomic PRDM12 sequence or PRDM12 promoter sequence may be achieved by fusing the catalytically inactive Cas9 protein to a DNA cleavage domain such as the non-limiting example Fokl. Hence in certain embodiments described herein, the crRNA sequence or target specific portion of the gRNA sequence has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In certain embodiments, the single guide RNA or crRNA comprises a portion of the nucleotide-encoded Prdml2 sequence. In further embodiments, the single guide RNA or crRNA comprises a portion of a sequence contained in exon 1, 2, or 3 of the PRDM12 sequence, preferably the human PRDM12 sequence. In alternative embodiments, the single guide RNA or crRNA comprises a portion of the endogenous PRDM12 promoter sequence, preferably the human PRDM12 sequence. Tools for designing gRNA sequences have been reported in the art (review for example in Cui et al., Interdisciplinary Sciences, Computational Life Sciences, 2018). In particular embodiments, the gene-editing system comprises a guide RNA that is complementary to, and hence able to hybridise to, a sequence within the human PRDM12 genomic sequence or PRDM12 promoter sequence. More particularly, the Cas9-based PRDM12 modulator comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and preferably 100% complementary to a nucleotide sequence encoding SEQ ID NO: 1 or a nucleotide sequence comprised in the sequence of SEQ ID NO: 2.
While the above-mentioned gene editing systems are suitable for inhibiting or deleting the PRDM12 target sequence, a skilled person aware of the state of the art appreciates that these gene editing systems may be further mutagenised or modified to act as a transcriptional repressor or transcriptional activator of PRDM12 embodiment. For examples, a catalytically inactive endonuclease specifically targeting the promoter sequence of Prdml2 may be coupled or genetically fused to a transcriptional activator or repressor domain. Non-limiting examples of transcriptional activators suitable for coupling include VP16, VP64, MyoD, FoxA, VPR, TET, p300, SMYD3, DOT1L, and PRDM9. Non limiting examples of transcriptional repressors suitable for coupling include the KRAB domain, DNMTs, LSD1, HDAC3, and EZH2 (Hirai et al., The International Journal of Developmental Biology, 2012; and Xu et al., International Journal of Molecular Sciences, 2020).
Alternatively, in one or more embodiments described herein Prdml2 as such or a mutant Prdml2 protein is used as a modulator of Prdml2 activity. In certain embodiments, the Prdml2 has a sequence having at least about 65%, preferably at least about 75%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, more preferably 100% identity to the Prdml2 amino acid sequence of SEQ ID NO: 1. It is appreciated to a skilled person that when using a Prdml2 protein that mimics the activity of naturally occurring Prdml2, the Prdml2 modulator will exaggerate or increase the function of the naturally occurring Prdml2. Hence, addition of exogenous Prdml2 or a nucleotide sequence wherefrom Prdml2 is translated and/or transcribed from will increase Prdml2 activity in the host cell or host tissue. Depending on the exact pain conditions, the additional Prdml2 may act therefore act as an inhibitor or enhancer of the pain response and/or pain sensation. In pain conditions as described herein wherein Prdml2 expression leads to an inhibition of the pain response and/or pain sensation, increasing the Prdml2 expression or activity will hence have an analgesic effect, which is unexpected based on what has been described in the art. In alternative embodiments, the modulator of Prdml2 activity is a mutant Prdml2 protein or nucleotide sequence encoding a mutant Prdml2 protein that exerts a higher intrinsic activity than the endogenous Prdml2 protein. In yet alternative embodiments, the modulator of Prdml2 activity is a mutant Prdml2 protein or nucleotide sequence encoding a mutant Prdml2 protein that exerts a lower intrinsic activity than the endogenous Prdml2 protein, or is an inactive Prdml2 protein. Considering the above, in one or more embodiments the mutant Prdml2 protein is selected from the group of Prdml2 mutants consisting of: null loss-of-function Prdml2 mutants, leaky loss-of function Prdml2 mutants, and gain-of-function Prdml2 mutants. In preferred embodiments, the Prdml2 mutant is a gain-of-function mutant. A skilled person is capable to identify different classes of mutant proteins in accordance to information readily available in the art (Griffiths et al., An Introduction to Genetic Analysis, 7th edition, 2000). In embodiments where a mutant Prdml2 is described, said mutant Prdml2 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations when compared to the canonical sequence of Prdml2, preferably the human Prdml2 sequence of SEQ ID NO: 1, which may optionally be encoded by a nucleotide sequence as described by SEQ ID NO: 2 or a portion thereof.
In certain embodiments, the modulator of Prdml2 activity may be a fusion protein of at least two distinct proteins, at least two functional fragments of proteins, at least two concatenated proteins, or any combination thereof. The terms “fusion protein” or “fusion polypeptide” and “protein conjugate” or “polypeptide conjugate” as used interchangeably in the art denote hybrid or chimeric molecules comprising at least two proteins or polypeptides linked, connected or joined together in a manner not normally found in nature. The molecules may be suitably denoted as amino acid-based compounds, i.e. as substances or molecules as including primarily but not necessarily exclusively amino acid residues. Any recombinantly, semi-synthetically or synthetically produced fusion proteins or proteins conjugates are encompassed. The fusion proteins as envisaged herein may be modified by glycosylation, phosphorylation, sulfonation, methylation, acetylation, lipidation, pegylation or the like. In further embodiments, the terms “fusion protein” or “fusion polypeptide” indicate genetic fusions, whereby two or more proteins, polypeptides or variants or fragments thereof are joined by a co-linear and/or covalent linkage via their individual polypeptide backbones, through genetic expression of a single contiguous oligonucleotide molecule encoding the fusion product. Typically, to produce the contiguous oligonucleotide molecule encoding the fusion product, two or more open reading frames (ORFs) each encoding a given polypeptide segment are joined to form a continuous longer ORF in a manner that maintains the correct reading frame for each original ORF. In the resulting recombinant fusion polypeptide the two or more polypeptide segments encoded by the original ORFs are joined in the same polypeptide molecule, whereas they are not normally so joined in nature. While the reading frame is thus made continuous throughout the fused genetic segments, the so fused polypeptide segments may be physically or spatially separated by, for example, an in-frame polypeptide or peptide linker. In alternative embodiments, the terms “protein conjugate” or “polypeptide conjugate” denote substances or molecules in which two or more proteins, polypeptides or variants or fragments thereof are joined by non-genetic means, whereas they are not normally so joined in nature. The polypeptide segments may be joined via their individual polypeptide backbones or via one or more of their respective amino acid side chains, or one protein segment may be joined via its polypeptide backbone to an amino acid side chain of another polypeptide segment.
In certain embodiments, the Prdml2-binding molecule is a mutant G9a protein, preferably a mutant G9a protein that has either reduced or increased activity when compared to the native G9a protein. For examples, when downregulation of the transcription rate of Prdml2 effector genes is envisaged, a hyperactive G9a protein can be introduced or expressed in nociceptors or dorsal root ganglia to induce increased methylation of H3K9, and alternatively an inactive or attenuated G9a protein can be introduced or expressed in nociceptors or dorsal root ganglia to induce a decrease in H3K9 methylation. In preferred embodiments, the mutant G9a protein is a mutant human G9a protein, more preferably a mutant human G9a protein characterised by a sequence having at least about 65%, preferably at least about 75%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 99%, identity to the G9a amino acid sequence of SEQ ID NO: 3.
A further aspect of the invention is directed to a nucleotide sequence encoding Prdml2 or encoding a modulator of Prdml2 activity as described herein, wherein said nucleotide sequence encodes Prdml2 or a molecule capable of binding to human Prdml2, or wherein said modulator is a nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2, preferably wherein Prdml2 is human Prdml2 as defined by SEQ ID NO: 1 and/or wherein said nucleotide sequence encoding the Prdml2 protein is the PRDM12 gene (SEQ ID NO: 2). In certain embodiments, the nucleotide sequence is a DNA sequence. In further embodiments, the DNA sequence is a single stranded or double stranded DNA sequence. In alternative embodiments, the nucleotide sequence is an RNA sequence. The nucleotide sequence may contain one or more modifications as described herein. In certain embodiments, the nucleotide sequence consists of a modulator of Prdml2 activity. In certain embodiments, the nucleotide sequence comprises, consists or, or essentially consists of a nucleotide sequence capable of hybridising to a target nucleotide sequence encoding Prdml2 (i.e. a PRDM12 antisense agent), preferably the human nucleotide sequence encoding Prdml2 as identified by SEQ ID NO: 2, or a nucleotide sequence having at least about 65%, preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, at least about 97%, at least about 99% to SEQ ID NO: 2 or a portion thereof. Also envisaged herein are viral or non-viral vectors encoding Prdml2 or a modulator of Prdml2 activity as described herein, preferably wherein Prdml2 is human Prdml2, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or encoded by SEQ ID NO: 2. In preferred embodiments, the viral or non-viral vector is designed for and/or used for gene therapy, preferably human gene therapy. In one or more embodiments, the viral or non-viral vector comprises at least one additional encoded protein or at least one nucleotide sequence encoding capable of influencing a pain condition. In certain embodiments, the viral or non-viral vector is designed to insert one or more nucleotide sequences comprised in said vector into the genome of the host cell, host tissue, or host organism. In certain embodiments, the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 further comprises a human promoter sequence. In alternative embodiments, the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 further comprises an inducible promoter sequence. In yet alternative embodiments, the nucleotide sequence comprised in the viral or non-viral vector encoding Prdml2 or a modulator of Prdml2 comprises a tissue-specific promoter sequence, preferably a human tissue-specific promoter sequence. Tissue-specific promoter sequences have been described in the art (e.g. in Zheng and Baum, Methods in Molecular Biology, 2009). In further embodiments, the tissue specific promoter sequence is a native promoter sequence. In alternative embodiments, the tissue specific promoter sequence is a composite promoter sequence. As is known in the art, native promoter sequences comprise a single fragment of a 5’ region of a gene, preferably wherein said gene is naturally expressed in a specific group of tissues or even one single tissue or cell type, while composite promoter sequences originate from the combination of promoter elements of different origins. Without limitation, a native promoter particularly suited in the context of neurons and thus in the context of the present invention is the neuron-specific enolase (NSE) promoter sequence. In certain embodiments, the viral vector comprises at least two distinct modulators or Prdml2 activity.
The term 'vector' as used in the application refers to nucleic acid molecules which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. The vectors can be episomal vectors (i.e. , that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. The term 'vector' may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors. Non- viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI, plasmid vectors (e.g. pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a nonlimiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.
In preferred embodiments, the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector, more preferably an AAV vector. AAV vectors are preferably used as self-complementary, double-stranded AAV vectors (scAAV) in order to overcome one of the limiting steps in AAV transduction (i.e. single-stranded to doublestranded AAV conversion) (McCarty, 2001, 2003; Nathwani et al, 2002, 2006, 2011; Wu et al., 2008), although the use of single-stranded AAV vectors (ssAAV) are also encompassed herein. Well-known AAV vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAVrh20 and AAV9. Gene expression in DRG neurons has been achieved using AAV vectors based on serotypes 1, 2, 3, 4, 5, 6, 8, 8.2, 9 and rh20. Preferred vectors are AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector more particularly a self- complementary AAV9 vector (scAAV9). In yet other embodiments, the vector comprises viral and non-viral elements. Especially for DRG expression, AAV5 and AAV1 are preferred as well as AAV6, AAV8 and AAV9.lt is understood by the skilled person that expression systems as described herein encoding for Prdml2 or a functional fragment thereof are typically considered as activators of Prdml2, i.e. of modulators that restore or increase the expression of Prdml2 in the subject. Such activators can typically be used for treating pain conditions caused or aggravated by reduced or lost expression of Prdml2 (as compared to the expression level in subjects not suffering from said pain condition) in DRG or TG neuronal cells as defined herein elsewhere.
In contrast, antisense agents and Prdml2 binding molecules as disclosed herein are understood to typically be negative modulators of Prdml2, or inhibitors of Prdml2. Such inhibitors can typically be used for treating pain conditions caused or aggravated by increased expression of Prdml2 (as compared to the expression level in subjects not suffering from said pain condition) in DRG or TG neuronal cells as defined herein elsewhere.
A further aspect of the invention relates to a pharmaceutical composition comprising Prdml2, a modulator of Prdml2 activity as described herein, or a combination thereof. In a preferred embodiment of the invention, said pharmaceutical composition is used for treatment or prevention of a pain condition in a subject. The terms “pharmaceutical formulation”, pharmaceutical composition”, “formulation” or “composition” may be used interchangeably herein. In any of the embodiments concerning one or more of the pharmaceutical composition described herein, it is evident that said composition may comprise one or more pharmaceutically acceptable carriers (i.e. excipients). The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
It is evident that pharmaceutical compositions in the context of the invention are indicative for those compositions that comprise a therapeutically or prophylactically effective amount of Prdml2 and/or Prdml2 modulator. In the context of the invention, the Prdml2 of Prdml2 modulator present in the pharmaceutical composition is considered as at least one of the pharmaceutical active ingredients of the pharmaceutical composition. Wherein the modulator of Prdm activity may be administered as such, as a nucleotide sequence encoding said modulator, as an nucleotide expression vector encoding said modulator, or as a viral vector encoding said modulator. “Pharmaceutical active ingredient” or “API” as referred to herein is to be interpreted according to the definition of the term by the World Health organisation: “a substance used in a finished pharmaceutical product (FPP), intended to display pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings”. The term “therapeutically effective dose” or “therapeutically effective amount” as used herein refers to an amount of Prdml2, or an amount of Prdml2 modulator (i.e. Prdml2 inhibitor or Prdml2 activator) as described herein, that when administered brings about a clinical positive response with respect to treatment of a subject afflicted by a pain condition, e.g. a patient having been selected (e.g. diagnosed) to have or is expected to have or be exposed to a certain pain condition. In certain embodiments, the patient is diagnosed with a nociceptive, neuropathic or inflammatory pain condition, wherein said pain condition may be chronic in nature.
The term “prophylactically effective dose” or “prophylactically effective amount” refers to an amount of Prdml2 modulator that inhibits or delays in a subject the onset of a pain conditions as being sought by a researcher, veterinarian, medical doctor or other clinician. While a skilled person appreciates that occupational injury cannot be predicted, the prediction of pain in the context of the present invention relates to the prediction of a pain-inducing disease or condition. By means of example and not limitation, a subject diagnosed with cancer, such as multiple myeloma is expected to perceive pain in a future point in time (i.e. treatment-related pain, pain due to disease progression, or a combination thereol). Alternatively, a person scheduled for surgery can be expected to perceive pain in a future point in time, i.e. when recovering from the surgery. A skilled person understands that the required dosage or amount of Prdml2 modulator that is needed to arrive at a therapeutically effective or prophylactically effective dose needs to be assessed on a case-by-case and subject-to-subject basis. It is standard practice to adapt a dosage to a certain individual to obtain an optimal, i.e. ideal effect or response. Hence, a skilled person is aware that a plethora of parameters may or depending on the situation are even required to be assessed when determining an optimal dosage, or dosage schedule and that said parameters include but are by no means limited to the nature and degree of the pain condition or pain-inducing condition to be treated, gender of the subject, subject age, body weight, other medical indications, nutrition, mode of administration, metabolic state, interference or influence by or efficacy of other pharmaceutically active ingredients, etc. Furthermore each individual may have a certain intrinsic degree of responsiveness to the Prdml2 modulator that is used.
The term “excipient” as used interchangeably herein and in the art with “carrier” may be indicative for any solvent, diluent, buffer (including but not limited to neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubiliser (including but not limited to Tween 80 or Polysorbate 80), colloid, dispersion medium, vehicle, filler, chelating agent (including but not limited to EDTA or glutathione), amino acid, protein, disintegrant, binder, lubricant, wetting agent, stabiliser, emulsifier, sweetener, colorant, flavoring, aromatiser, thickener, any agent suitable to achieve a depot effect, coating, antifungal agent, any preservative (including but not limited to Thimerosal™, benzalkonium chloride, or benzyl alcohol), antioxidant (including but not limited to ascorbic acid, sodium metabisulfite), tonicity controlling agent, absorption delaying agent, adjuvant, bulking agent (including but not limited to lactose, mannitol) and any other ingredient that may influence any parameter or characteristic of the pharmaceutical formulation, or of the Prdml2 modulator comprised in the pharmaceutical composition described herein. A skilled person understands that one or more excipients may be used in the pharmaceutical formulation on condition that the one or more excipient is compatible with the one or more pharmaceutical ingredient (i.e. in the context of the present invention at least the modulator of Prdml2 activity) and that a pharmaceutically acceptable formulation is obtained. “Pharmaceutically acceptable” as used herein is indicative for the ingredients to be compatible with each other and that the combination of ingredients in the pharmaceutical formulation does not lead to deleterious effects (including but not limited to toxicity) to the subject receiving the formulation.
In certain embodiments, the excipient may be an active pharmaceutical ingredient excipient, binder excipient, carrier excipient, co-processed excipient, coating system excipient, controlled release excipient, diluent excipient, disintegrant excipient, dry powder inhalation excipient, effervescent system excipient, emulsifier excipient, lipid excipient, lubricant excipient, modified release excipient, penetration enhancer excipient, permeation enhancer excipient, pH modifier excipient, plasticiser excipient, preservative excipient, preservative excipient, solubiliser excipient, solvent excipient, sustained release excipient, sweetener excipient, taste making excipient, thickener excipient, viscosity modifier excipient, filler excipient, compaction excipient, dry granulation excipient, hot melt extrusion excipient, wet granulation excipient, rapid release agent excipient, increased bioavailability excipient, dispersion excipient, solubility enhancement excipient, stabiliser excipient, capsule filling excipient, or any combination hereof. A skilled person is aware that use of such media and agents for pharmaceutical active substances is common practice and incorporation of these excipients is hence well known in the art. It is evident that all of the used ingredients should be non-toxic in the concentration contained in the final pharmaceutical composition and should not negatively interfere with the activity of the one or more pharmaceutically active ingredients, in this context at least the Prdml2 activity modulator. In certain embodiments, more than one excipient which a skilled person would classify as belonging to the same group of excipients is added to the pharmaceutical composition. In further embodiments, more than one excipient wherein the different excipients belong to different groups is added to the pharmaceutical composition. In certain embodiments, the excipients may fulfill more than one function and/or be classified by a skilled person as belonging to different groups or classes of excipients.
Furthermore, the formulation may comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, preservatives, complexing agents, tonicity adjusting agents, wetting agents and the like, nonlimiting examples include sodium acetate, sodium lactate, sodium phosphate, sodium hydroxide, hydrogen chloride, benzyl alcohol, parabens, EDTA, sodium oleate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. In certain embodiments, at least one additional component is combined with the pharmaceutical formulation prior to administration. In further embodiments, the additional component is combined with the pharmaceutical formulation immediately prior to administration. In alternative embodiments where the pharmaceutical formulation is stored under a lyophilised condition, the additional component may be part of the solvent used to reconstitute the pharmaceutical composition. Aqueous solutions suitable for reconstitution of pharmaceutical compositions are known to a person skilled in the art. A non-limiting example of a suitable aqueous solution is water for injection. In certain embodiments, the amount of the additional component added to the pharmaceutical formulation is calculated based on certain patient parameters including but not limited to age, weight, gender, severity of the disease condition, and other known disease conditions of the patient or disease conditions the patient is suspected to be afflicted with.
Any of the pharmaceutical compositions described to herein may be formulated into a unit dosage form, including but not limited to hard capsules, soft capsules, tablets, coated tablets such as lacquered tablets or sugar-coated tablets, granules, aqueous or oily solutions, syrups, emulsions, suspensions, ointments, pastes, lotions, gels, inhalants or suppositories, which may be provided in any suitable packaging means known in the art, non-limiting examples being troches, sachets, pouches, bottles, films, sprays, microcapsules, implants, rods or blister packs. In certain embodiments, the pharmaceutical composition is suitable for direct administration to a subject. Preferred administration routes include oral, rectal, bronchial, nasal, topical, buccal, sublingual, transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intra-arterial, intracerebral, intracerebroventricular intraocular injection or intravenous infusion) administration, or in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration. In embodiments where the pharmaceutical composition is administered parentally, the composition is comprised in an aqueous solution which is preferably pyrogen- free and additionally has a suitable pH, isotonicity and stability. In those embodiments, the aqueous solution optionally contains at least one of the following: sugars, alcohols, antioxidants, buffers, bacteriostatics (bacteriostats), solutes, suspending agents or thickening agents. In certain embodiments, the pH of the pharmaceutical composition to be administered parentally is adjusted to a physiologic pH in the region of 7 to 9. In certain embodiments, the pH of the pharmaceutical composition to be administered parentally is from about 7.35 to about 7.45.
In certain embodiments, the pharmaceutical composition may be formulated as an immediate release formulation dosage form. In alternative embodiments, the pharmaceutical formulation may be comprised in a delayed release dosage form. In yet alternative embodiments, the pharmaceutical composition may be comprised in a controlled release formulation dosage form. A skilled person understands the meaning of the terms “immediate release”, “delayed release”, and “sustained-release” or “controlled release” and is aware that these terms are indicative for the particular release profile of a pharmaceutical composition. In immediate release the pharmaceutical composition is about immediately, or near immediately released from a dosage form to a body of a subject or patient. In delayed release dosage forms, the pharmaceutical composition is delivered in the body, or its contents contacted or exposed to the body or part thereof with a delay after administration. In sustained release or controlled release dosage forms, the dosage form is designed to release a pharmaceutical composition at a predetermined rate in order to maintain a constant drug concentration for a specific period of time.
The release profile of a dosage form can be assessed as described in the major pharmacopeias published by government authorities or medical or pharmaceutical societies. For example, immediate release is defined by the European Medicines Agency as dissolution of at least 75% of the active substance within 45 minutes (European Pharmacopeia (Ph. Eur.) 9th edition). A skilled person appreciates that suitable tests and time windows may vary depending on therapeutic ranges, solubility and permeability factors of the drug substance. Techniques regarding the formulation and administration of pharmaceutical compositions are known to a skilled person and have been described in the art (e.g. the reference book: Remington: The Science and Practice of Pharmacy, periodically revised). By means of illustration and not limitation, solid dosage forms may be manufactured by mixing the one or more active ingredients with a portion of the excipients or all excipients followed by wet or dry granulation, or direct compression. In one or more embodiments described herein, the pharmaceutical composition comprises in addition to Prdml2 and/or one or more Prdml2 modulator a further pharmaceutically active ingredient, preferably wherein said further pharmaceutically active ingredient is an analgesic. In preferred embodiments the further analgesic pharmaceutical active ingredient is an analgesic selected from the group consisting of: acetaminophen (i.e. paracetamol), nonsteroidal- inflammatory drugs (NSAIDs), opioids, muscle-relaxants, anti-anxiety agents, antidepressants, anticonvulsants, corticosteroids, and any combination thereof. In certain embodiments, the further analgesic pharmaceutical active ingredient is an NS AID, preferably an NS AID disclosed in the present disclosure, more preferably an NSAID selected from the group consisting of: salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, enolic acid derivatives (oxicam), anthranilic acid derivatives (fenamates), (selective) COX-2 inhibitors (coxibs), sulfonanilides, or any combination thereof.
In alternative embodiments, the further analgesic pharmaceutical active ingredient is an opioid, preferably an opioid disclosed in the present disclosure, more preferably an opioid selected from the group consisting of natural opiates, morphine esters, semi-synthetic opioids, fully synthetic opioids, endogenous opioid peptides, or any combination thereof. In yet alternative embodiments, the further analgesic pharmaceutical active ingredient is corticosteroid as disclosed in the present disclosure, more preferably a glucocorticoid and/or a mineralocorticoid. In yet alternative embodiments, the further analgesic pharmaceutical active ingredient is an anticonvulsant, preferably an anticonvulsant selected from the group consisting of: aldehydes, aromatic allylic alcohols, barbiturates, benzodiazepines, bromides, carbamates, fatty acids, fructose derivatives, hydantoins, oxazolidinediones, propionates, pyrimidinediones, pyrrolidines, succinimides, sulfonamides, triazines, valproylamides, perampanel, stiripentol, pyridoxine, or any combination thereof. In yet alternative embodiments, the further analgesic pharmaceutical active ingredient is an muscle-relaxant as disclosed herein, more preferably a neuromuscular blocker or a spasmolytic. In yet alternative embodiments, the further analgesic pharmaceutical active ingredient is an anti-anxiety agent, preferably an anti-anxiety agent selected from the group consisting of: barbiturates, benzodiazepines, carbamates, antihistamines, opioids, antidepressants, sympatholytics (i.e. beta blockers, alpha blockers, and alpha-adrenergic agonists), phenibut, mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, pregabalin, menthyl isovalerate, propofol, racetams, alcohol, inhalants, and combinations thereof. In yet alternative embodiments, the further analgesic pharmaceutical active ingredient is an antidepressant, preferably an antidepressant selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, serotonin modulators and stimulators, serotonin antagonists and reuptake inhibitors, norepinephrine reuptake inhibitors, norepinephrinedopamine reuptake inhibitors, tricyclic antidepressants, tetracyclic antidepressants, monoamine oxidase inhibitors, NMDA receptor antagonists, and combinations thereof.
In certain embodiments wherein a pharmaceutical composition is used that comprises or consists essentially of a Prdml2 modulator (e.g. Prdml2, a Prdml2 activator, or Prdml2 inhibitor) and one of the above mentioned further analgesics, a therapeutic effect may be achieved by incorporating doses or concentrations of said Prdml2 modulator and/or the further analgesic which are not effective in isolation. As a non-limiting example, a modulator of Prdml2 activity as described herein may be combined in a pharmaceutical composition with an opioid, wherein both the Prdml2 modulator and the opioid are present in amounts or concentrations that are below the therapeutically effective dose when used in isolation. Yet, is envisaged by the inventors that such a pharmaceutical composition would nevertheless result in a favorable clinical outcome, i.e. a diminishment or cessation of the pain condition and/or pain sensation.
Certain aspects of the invention relate to an analgesic comprising of, or essentially consisting of a modulator of Prdml2 activity. In further embodiments, the analgesic comprises or essentially consists of Prdml2 and/or a mutant Prdml2, preferably human Prdml2 and/or human mutant Prdml2. In certain embodiments, the analgesic comprises or essentially consists of a Prdml2 activator. In alternative embodiments, the analgesic comprises or essentially consists of a Prdml2 inhibitor. Where the analgesic comprises Prdml2 or mutant Prdml2, the analgesic may comprise or essentially consist of a nucleotide sequence encoding Prdml2 and/or mutant Prdml2. In such embodiments, the analgesic may comprise a DNA sequence encoding Prdml2 and/or mutant Prdml2. In certain embodiments, the analgesic is a null loss- of-function Prdml2 mutant, leaky loss-of function Prdml2 mutant, or gain-of-function Prdml2 mutant. In yet alternative embodiments, the analgesic may be administered to a subject by use of a normally exogenous living delivery vehicle. In yet further embodiments, the living delivery vehicle is a prokaryotic or eukaryotic cell. In even further embodiments, the living delivery vehicle is a human cell, such as a stem cell. Alternatively, the living delivery vehicle is a bacterial cell, for example a bacterial cell capable of surviving and/or propagating within the gut of the subject.
An alternative aspect is the use of a modulator of Prdml2 activity as additive to pharmaceutical compositions comprising a traditional analgesic as described herein. For example, the modulator of Prdml2 activity is added to, and optionally suspended and/or dissolved in a liquid pharmaceutical analgesic composition suitable for administration by injection.
A further aspect of the invention relates to a method of diagnosing a hypersensitivity to pain in a subject, wherein the method comprises determining the expression of Prdml2 in a biopsy of said subject, wherein reduced expression of Prdml2 in dorsal root ganglia cells and/or nociceptors is indicative of a hypersensitivity to pain. In certain embodiments, the method is directed to diagnosing allodynia and/or hyperalgesia in a subject. In certain embodiments, the Prdml2 expression in the biopsy of the subject is determined by a method selected from the group consisting of: PCR (preferably RT-PCR or qPCR), mass spectrometry analyses, spectrophotometric assays (preferably UV light absorption spectroscopy assays, dye-based protein assays, Coomassie blue (Bradford) assays, or Lowry alkaline copper reduction assays), ELISA, or a combination thereof. In certain embodiments, a Prdml2 expression level of at least about 75%, at least about 60%, preferably at least about 50%, preferably at least about 25%, preferably at least about 10% when compared to a suitable control or baseline value as defined herein is indicative of allodynia and/or hyperalgesia.
“Biopsy” as used herein and in the art refers to a medical method comprising a step of extracting sample cells or tissues from a subject by a procedure that allows further examination of said cells or tissues. The nature of the further examination is not limited, and may be for example chemical analysis or histological analysis. In preferred embodiments of the invention, the biopsy is performed on inflamed tissue or tissue suspected or expected to be inflamed. Different non-limiting examples of biopsies include excisional biopsies, incisional biopsies (also referred to in the art as core biopsy), and needle aspiration biopsy. In certain embodiments, the biopsied site is selected from the group consisting of: bone tissue, bone marrow tissue, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, nervous system tissue, urogenital tissue, lymph node tissue, muscle tissue, skin tissue.
Alternatively, a method of diagnosing a hypersensitivity to pain (such as allodynia and/or hyperalgesia) in a subject comprises determining the level of G9a recruitment to histone H3 in dorsal root ganglia cells and/or nociceptors, preferably Prdml2-mediated recruitment to histone H3 in dorsal root ganglia cells and/or nociceptors, wherein a reduction in G9a recruitment indicates decreased Prdml2 activity and indicates hypersensitivity to pain of the subject. Yet alternatively, a method of diagnosing a hypersensitivity to pain (such as allodynia and/or hyperalgesia) in a subject comprises determining the methylation status, or representative methylation status of H3K9 in dorsal root ganglia cells and/or nociceptors in a biopsy of a subject, wherein reduced methylation indicates reduced Prdml2 activity and a hypersensitivity to pain of the subject. In further embodiments, the method of diagnosing provides information for prognosticating the severity of pain that may be experienced by a subject, and/or pain that is expected to be experienced by a subject.
The term "diagnosing" as used herein is indicative for a process of recognizing, deciding on or concluding on a pain-inducing disease or pain condition in a subject on the basis of symptoms and signs and/or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and/or quantity of one or more biomarkers of or clinical symptoms characteristic for the diagnosed disease or condition). Particularly relevant tools for diagnosing a pain condition in the context of pain are self-reporting pain scales. “Diagnosis of’ the diseases or conditions as taught herein in a subject may particularly mean that the subject has such disease or (pain) condition. A subject may be diagnosed as not having such despite displaying one or more conventional symptoms or signs reminiscent of such. "Prognosticating" in the context of the invention is indicative for anticipation on the progression of a paininducing disease or pain condition condition and the prospect (e.g. the probability, duration, and/or extent) of recovery, and/or the severity of experiencing or amelioration of pain. The term "a good prognosis of generally encompass anticipation of a satisfactory partial or complete recovery from a diagnosed pain-inducing disease or pain condition, optionally within an acceptable time period. Alternatively, the term may encompass anticipation of not further worsening or aggravating of such, preferably within a given time period. The term "a poor prognosis of the disease or condition typically encompass an anticipation of a substandard recovery and/or unsatisfactorily slow recovery, or no recovery at all, or further worsening of said pain-inducing disease or pain condition, or any clinical manifestation associated with said disease or condition.
In certain embodiments of the above described methods, the pain is selected from nociceptive pain, neuropathic pain, inflammatory pain, or any combination thereof.
A further aspect of the invention relates to a method of treating or preventing a pain condition as described herein, comprising administering a modulator of Prdml2 activity as described herein or an analgesic comprising a modulator of Prdml2 activity as described herein, or a pharmaceutical composition comprising a modulator of Prdml2 activity as described herein to a subject. An alternative method of treating or preventing a pain condition as described herein comprises administering Prdml2, a functional fragment thereof, or mutant Prdml2 to a subject. In certain preferred embodiments, the administration is intravenous administration.
Optionally, the Prdml2 modulator may be administered in combination with a further analgesic to the subject. Preferably, the further analgesic that is administered to the subject is administered before, after, simultaneously, or intermittently to the subject. The administration may be topical and/or systemic. In certain embodiments, the modulator of Prdml2 activity is administered daily during a treatment period. In further embodiments, the modulator of Prdml2 activity is administered at least once a day during the treatment. In further embodiments, the modulator of Prdml2 activity is administered at least twice a day during the treatment. In further embodiments, the modulator of Prdml2 activity is administered at least three times a day during the treatment. In certain embodiments, the modulator of Prdml2 activity may be administered continuously during the treatment for instance in an aqueous drinking solution or in an intravenous solution. In certain embodiments, different administrations of the modulator of Prdml2 activity each contain an about equal amount and/or concentration of said modulator. In certain embodiments, including but not limited to embodiments wherein the modulator of Prdml2 activity is a gene editing system the methods of treatment comprise a single administration moment. In certain embodiments of the described methods of treatment, the Prdml2, functional fragment thereof, or mutant Prdml2 is expressed in the subject after administration of a nucleotide sequence as described herein encoding said protein. In further embodiments, the nucleotide sequence is a DNA sequence, which is optionally part of an expression vector. Said expression vector may comprise the endogenous Prdml2 promoter sequence, a constitutive heterogenous promoter sequence, an inducible heterogenous promoter sequence, or a combination thereof. In alternative further embodiments, the nucleotide sequence is an RNA sequence. The Prdml2, functional fragment of Prdml2, mutant Prdml2, Prdml2 inhibitor, or Prdml2 modulator may be packaged in a delivery vector, preferably a viral delivery particle prior to administration to the subject. By means of illustration and not limitation, a suitable viral delivery particle is an adeno-associated viral particle.
In a further aspect of the invention, the use of a modulator of Prdml2 activity as described herein is intended for the manufacture of a medicament for the prevention or treatment of a pain condition. Also intended as substantiated by the above described embodiments is the use of Prdml2, functional fragment of Prdml2, mutant Prdml2, or a nucleotide sequence as described herein encoding one or more of the preceding moieties, is intended for the manufacture of a medicament for the prevention or treatment of a pain condition. In certain embodiments, said use is intended for the manufacture of a medicament for the prevention or treatment of nociceptive pain, neuropathic pain, inflammatory pain, or any combination thereof. In further preferred embodiments, said use is intended for the manufacture of a medicament for the prevention or treatment of inflammatory pain or nerve injury pain, preferably wherein said inflammatory pain condition is selected from the group consisting of: skin inflammation, joint inflammation, and allergy-related inflammation.
Another aspect of the invention is directed to kit of parts comprising means to assess Prdml2 expression and/or activity levels in a subject, or in a biopsy of a subject. The terms “kit of parts” and “kit” as used herein refer to a product containing components necessary for carrying out the methods (e.g. the diagnosis methods), packed so as to allow their transport and storage. Materials suitable for packing the components comprised in a kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, flasks, vials, ampules, paper, envelopes, or other types of containers, carriers or supports. Where a kit comprises a plurality of components, at least a subset of the components (e.g., two or more of the plurality of components) or all of the components may be physically separated, e.g., comprised in or on separate containers, carriers or supports.
The components comprised in a kit may be sufficient or may not be sufficient for carrying out the specified methods, such that external reagents or substances may not be necessary or may be necessary for performing the methods, respectively. Typically, kits are employed in conjunction with standard laboratory equipment, such as liquid handling equipment, environment (e.g., temperature) controlling equipment, analytical instruments, etc. In addition to the recited set of isolated oligonucleotides as taught herein, optionally provided on arrays or microarrays, the present kits may also include some or all of solvents, buffers (such as for example but without limitation histidine-buffers, citrate-buffers, succinate-buffers, acetate- buffers, phosphate-buffers, formate buffers, benzoate buffers, TRIS (Tris(hydroxymethyl)- aminomethan) buffers or maleate buffers, or mixtures thereof), enzymes (such as for example but without limitation thermostable DNA polymerase), detectable labels, detection reagents, and control formulations (positive and/or negative), useful in the specified methods. In certain embodiments, the kits include instructions for use thereof, non-limiting examples hereof being a printed insert and/or a computer readable medium. The terms may be used interchangeably with the term “article of manufacture”, which encompasses any man-made tangible structural product, when used in the present context.
Another aspect of the invention is directed to a nucleotide sequence encoding a modulator of Prdml2 activity as described herein. As described in the art, said nucleotide sequence may be introduced into a host cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle. In preferred embodiments, said nucleotide sequence encoding the modulator of Prdml2 activity is incorporated or part of an expression vector. A skilled person is aware that expression vectors may act as autonomous expression vectors (i.e. the expression vector is maintained extrachromosomally in the target cell) or as integrative expression vectors (i.e. the expression vector is integrated into the target. Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g. URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and/or selection of those cells transformed with the desired nucleic acids. Expression vectors can also include an autonomous replication sequence (ARS). The ARS may comprise a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS 18 or ARS68.
Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. The first and second insertable DNA fragments are each about 200 (e.g. about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed. A nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene. Integrative vectors can be linearised prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome.
Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g. amplified) in bacterial cells such as Escherichia coli (E. coli). The vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu. The purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells. In the context of the present invention, the host cell may be a any cell deemed suitable by a skilled person.
The term “host cell” may be used interchangeably with the term “host organism” and is indicative for cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter aha unicellular organisms, such as bacteria (e.g. E. coli, Salmonella tymphimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g. Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g. from Arabidopsis thaliana or Nicotiana tobaccum) and (cultured) animal cells (e.g. vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter aha multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are nonhuman.
The Prdml2 modulator may be isolated from a host cell or natural environment. The term “isolated” with reference to a particular component (such as for instance a nucleic acid, protein, polypeptide or peptide) generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment. For instance, an isolated human or animal protein or complex may exist in separation from a human or animal body where it naturally occurs. The term “isolated” as used herein may preferably also encompass the qualifier “purified”. As used herein, the term “purified” with reference to peptides, polypeptides, proteins, or nucleic acids does not require absolute purity. Instead, it denotes that such peptides, polypeptides, proteins, or nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in the starting composition or sample.
“A discrete environment” as referred to in the above paragraph denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc. Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc. Purified peptides, polypeptides or proteins may preferably constitute by weight > 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment. Protein content may be determined, e.g. by the Lowry method (Lowry et al. Journal of Biological Chemistry, 1951). Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.
Further, there are several other well-known methods of introducing nucleic acids into animal cells, any of which may be used herein. At the simplest, the nucleic acid can be directly injected into the target cell or target tissue. Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors (i.e. derived from lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like. Other techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilised) NA molecule(s) into micropumps delivering the product. Another possibility is the use of implantable drugreleasing biodegradable microspheres. Also envisaged is encapsulation of NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; and co-inj ection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used. In a further aspect, the invention is directed to an in vitro method for identifying a molecule suitable as an analgesic, wherein said method comprises determining whether a candidate molecule modulates Prdml2 activity in nociceptors and/or dorsal root ganglia. In further embodiments, the in vitro method comprises a step of contacting a nociceptor or dorsal root ganglion with one or more candidate Prdml2 modulators and a step of measuring Prdml2 activity in the nociceptor or dorsal root ganglion. In yet further embodiments the Prdml2 activity is measured by assessing the Prdml2 expression level before and after contacting the nociceptor and/or dorsal root ganglion with a candidate molecule. In alternative embodiments the Prdml2 activity is measured by assessing H3K9 methylation. In alternative embodiments, the in vitro method is a computer-implemented method. In one or more embodiments of the in vitro method, the Prdml2 is human Prdml2, preferably human Prdml2 as defined by SEQ ID NO: 1. In certain embodiments, the method is an in vitro method for identifying a molecule suitable as an analgesic in pain-inducing inflammatory conditions and/or pain conditions induced by nerve injury.
In certain embodiments of the in vitro method for identifying a molecule suitable as an analgesic, the Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity. Exemplary assays can be tailor made sequencing kits, PCR-based assays, or ChIP assays. As explained in detail throughout the present disclosure, the suitability of a molecule as an analgesic will depend on the influence on Prdml2 activity combined with the specific pain condition. Hence in certain embodiments the molecule is considered an analgesic when G9a recruitment to histone H3 and/or H3K9 methylation is increased. However, in alternative embodiments the molecule is considered an analgesic when G9a recruitment to histone H3 and/or H3K9 methylation is decreased. In certain embodiments, the molecule is considered prima facie a molecule suitable as an analgesic when the G9a recruitment to histone H3 and/or the H3K9 methylation is altered by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, more preferably at least about 90%, at least about 95%, most preferably at least about 100% when compared respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
Additionally or alternatively to G9a recruitment to histone H3 and/or the H3K9 methylation assessment, a suitable means to assess Prdml2 activity is by measuring expression of one or more genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, MRGPRB5, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity. In certain embodiments, the expression is assessed at the RNA level. In alternative embodiments, the expression is assessed at the protein level. In yet alternative embodiments, the expression is assessed at the RNA and protein level. In one or more embodiments, said genes are the human PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, MRGPRB5, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 genes and/or the nociceptors and/or dorsal root ganglia are human nociceptors and/or dorsal root ganglia.
In further embodiments, the candidate modulator of Prdml2 activity is considered a Prdml2 activator when the expression of one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity. In alternative further embodiments, the candidate modulator of Prdml2 activity is considered a Prdml2 inhibitor when the expression of one or more genes selected from the group consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity. In one or more of these embodiments the gene expression is assess by PCR, preferably qPCR or RT-PCR. In one or more of these embodiments, the gene expression is expressed as a value normalised to the expression level of one or more housekeeping genes, preferably one or more housekeeping genes included in the HRT Atlas Database (https://www. housekeeping.unicamp.br, Hounkpe et al., Nucleic Acids Research, 2020).
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in the light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples. EXAMPLES
Previous research by the inventors and others have shown that Prdml2 is selectively expressed in sensory ganglia in developing pain sensing neurons, termed nociceptors, and that it is required for their development (Desiderio et al., 2019; Bartesaghi et al., 2019). Interestingly, earlier published data of the inventors also indicated that Prdml2 remains expressed in mature nociceptors in adult animals (Desiderio et al., 2019; Fig. 1, bottom panels), suggesting it could modulate the function of mature nociceptors.
Materials and Methods
Mice
All mice were maintained on a C57BL/6J background and mice of either sex were used. Mice were provided ad libitum with standard mouse lab pellet food and water and housed at room temperature with a 12h light/dark cycle. The experimental protocols were approved by the CEBEA (Comite d’e thique et du bien etre animal) of the IBMM-ULB and conformed to the European guidelines on the ethical care and use of animals. The following mouse strains were used: Prdmn^ (Desiderio et al, 2019), Advillin-CreERT2 (Lau et al., 2011) and RosaCreERT2 (JAX# 008463)(Ventura et al., Nature 445, 661-665, 2007). Females carrying Prdml2 conditional alleles were crossed to Advillin-CreERT2 males to generate tamoxifen (TAM)-inducible Prdml2 conditional knockout offspring in which Prdml2 is specifically deleted in somatosensory neurons following treatment with tamoxifen. Tamoxifen has been administrated (1,9 mg/25g body weight) intraperitoneally 2 times in a week in more than 8 week old Advillin-Cre-ERT2; Prdml2fl;fl mice and, as control, Advillin-Cre-ERT2; Prdml2fl;A mice injected with com oil, or Prdml2fl;fl mice injected with tamoxifen. Tamoxifen dissolved in com oil has been administrated (1,9 mg/25g body weight) intraperitoneally in more than 56-days old ^VilCreERT2-Prdml2fl:fl mice two times in a week, and four times in a week in the case of RosaCreERT2;Prdml2fl:fl mice. Injected animals were sacrificed 28-35 days after the second tamoxifen injection for analysis. For tissue recovery, mice were anesthetized with Domitor (Img/Kg) and Ketamine (75mg/Kg) or with Ketamine (75mg/kg) and Rompun (Img/kg). Whole-mount X-gal staining of dissected brain and cranial ganglia of Prdml^ mice was performed as described [13], For retrograde labelling studies, intra-articular injections of Fast Blue (1.5 ul, 2% in 0.9% saline; Polysciences) and Complete Freund’s adjuvant (7.5 ul, 10 mg/ml; Chondrex) were conducted in mice under anesthesia (ketamine, 100 mg/kg and xylazine, 10 mg/kg, i.p.).
PCR genotyping was used to detect the transgenic alleles. Prdml2 floxed allele was detected using primers forward 5’- GCTGATCGAGTCCAGGAGAC-3 (SEQ ID NO: 25) and reverse 5’ CCAAACATCCACAACCTTCA-3. (SEQ ID NO: 26) Cre allele was detected using primers forward 5 -GACAGATTATCTGCAATCTCTCTAAG-3 (SEQ ID NO: 27) and reverse 5 - GTTCCTGATGTTCCTGGCATCTGTC-3’ (SEQ ID NO: 28), for R26LSL-tdTomato using primers forward 5’-AAGGGAGCTGCAGTGGAGTA-3' and reverse 5'-CCGAAAATCTGTGGGAAGTC-3' and for RosaCreERT2 using primers forward 5' - CGTGATCTGCAACTCCAGTC - 3' and reverse 5' - AGG CAA ATT TTG GTG TAC GG - 3' to detect the Cre and forward 5' - CTGGCTTCTGAGGACCG - 3' and reverse 5' - CCGAAAATCTGTGGGAAGTC - 3' to detect the wild type allele.
Pain behavior assay
For all behavior tests, non-inducible mice injected with tamoxifen were used as controls. Formalin behavior assays were performed on 11 conditional mutants and 15 WT mice, both male and female, between 5-7 months of age. Experimenter was blind to the genotype of the test subjects. Animals were habituated the day before the test for 5 minutes in a plexiglass cage with mirrors on the walls and the floor covered with litter. On the day of the test, the right hind paw of the mouse was injected with 25ul of a 3% formalin solution. The mouse was observed for 30 minutes after the injection. The duration of the licking time of the injected paw was recorded at 5 min intervals until 30 minutes. The subsequent statistical analysis (Student’s T- test) was done on all data point collected. After the test, mice were euthanized with Domitor (Img/Kg) and Ketalar (75mg/kg. Intracardiac perfusion with PBS was then carried out, and their DRG collected for validation of their genotype by IF and/or RT-qPCR. Plantar incision in the hindpaw is currently be used as model to test whether Prdml2 also affect neuropathic pain (Brennan et al., 2004).
For the thermal place preference test assay, we used the BIOSEB BIO-TC2T device that is composed of two joined thermal plates and is equipped with a camera to follow the transitions of the mouse from one plate to another and monitor the time spent on each plate. The day before the test, mice were habituated for 180 seconds on the device and both plates were set at 30°C, the reference temperature for all tests. Mice were recorded for 180 seconds for each temperature tested. The temperatures of 22°C, 10°C, 43°C and 52°C have been tested sequentially, with a day off in between hot and cold temperatures. Each day, the reference and the test plates were reversed.
Regarding the capsaicin test, mice were habituated for 5 minutes the day before the test in a plexiglass cage such as for the formalin test. The day of the test, the mouse was injected with 10 pl of a 0,35pg/pl capsaicin solution (0,9% saline, 10% ethanol, 10% Tween-20). Mice were recorded for 15 minutes after injection and the time licking the injected paw was measured. Tail flick was also tested in mice using the Bioseb device BX-TF. The mouse was restrained in a cylinder with the tail outside of the cylinder in a notch and the tail was subjected to a radiant heat source. The focus 30 was selected for this test meaning that the temperature applied on the tail was at 50°C after 5 seconds and at 60°C after 10 seconds. The withdrawal latency from the heat stimulus applied to the tail was therefore measured. In total, 3 measurements were carried out with a rest time of 15 seconds between measurement.
Cold plantar assay was used to evaluate the cold sensitivity in mice. Mice were habituated in small cages on a 3mm thick glass plate for 20 minutes. A 5ml syringe cut at the end and filled with powdered dry ice was applied under the hind paw against the glass plate and the withdrawal latency was measured. Three measurements were taken per paw and both paws were tested with a time interval of 5 minutes between measurements. The mechanical conflict avoidance test was used to evaluate the response to mechanical stimuli. For the habituation, the mouse was placed on the device for 5 minutes and was free to move around the different parts of the box. For the test, the mouse was placed in the first part which had a separation with the second part, after 10 seconds the lamp was turned on and after 15 seconds the separation was removed. The mouse went through the second part of this box with nails to get to the last part which was dark. The time it took for the mouse to cross half of the part with nails was measured. Two heights were tested: 2mm and 5mm on two different days and 3 measurements were taken per height with a rest time of 30 minutes between each measurement. Open field was used to analyse the activity of mice and their exploratory behaviour. Mice were placed for 10 minutes in a plexiglass cage (44x44cm) with a camera placed above the cage and the behaviour was analysed with a Noldus software. Several parameters have been analysed such as the distance travelled, the velocity, the time moving and the time spent in the center and on the borders. Statistical analysis was conducted with the student’s t-test. All graphs were processed with GraphPad.
RNA-sequencing and data processing Dissected lumbar DRG (8 DRG from one animal) from 5 mutants (2 males and 3 females) and 4 WT mice where immediately frozen at -80°C and the frozen pellet placed after in 1 ml Trizol (Thermo Fisher, ref 15596026) for RNA extraction. RNA-seq analysis and data processing has been done at the NGS Integrative Genomics platform of the University of Gotingen as described previously described (Desiderio et al, 2019).
Real-time RT-qPCR
DRGs were dissected individually in cold RF PBS (lx) and kept at -80°C until RNA extraction. RNA extraction and purification were performed using the Monarch Total RNA Miniprep kit (Ref: T2010S) following the manufacturer’s indications. RNA was transcribed to cDNA using the iScript cDNA Synthesis kit (Cat #1708891; BioRad) obtaining a final concentration of 20ng/pL. The cDNA was used as a template for RTqPCR and amplified using the GoTaq qPCR Master Mix (Cat #A6001; Promega). Each reaction consists of the reaction mix prepared following manufacturer’s indication and 40-5 Ong of cDNA template. The sequence of the primers used are listed in Table 4 below:
The following cycling parameters were used: 95°C for 2min (GoTaq Polymerase activation) and 40 cycles of denaturation 95°C for 15 seconds and annealing/extension 60°C for Imin. All qPCR lectures were performed using the CFX96 Dx In Vitro Diagnostics (IVD) Real-Time PCR Systems (Bio Rad). Gene expression was normalized using GADPH as housekeeping gene. Data analysis was performed comparing WT as control and icKO using the 2'AACT method previously described by Livak and Schmitgen (2001). Finally, one tailed student’s T-test (a=0,05) statistical analysis was applied in order to define statistical significance.
ISH and immunostaining
Tissue dissection for ISH and immunostaining was performed as previously described (Thelie et al., 2015; Desiderio et al., 2019). Plasmids used for generating probes for Prdml2, Ntrkl and Trpm8 were as described (Desiderio et al., 2019). Grikl,Mrgprb5 and Cysltr2 cDNA used for probes generation were obtained by PCR from mouse embryonic dorsal root ganglia using the following primers:
Chrna6, forward 5 -TTCCAGGTCGAAGGCAAGAC -3’ and reverse 5’- TTGGCAGGCCTCTTGGTATG -3’; Grikl forward 5 -GAATGACAAAGGGGAGTGGA- 3’ (SEQ ID NO: 19) and reverse 5’-AAGGTCATTGTCGAGCCA-3’ (SEQ ID NO: 20); Mrgprb5 forward 5’- CCATCAGTGTTGAGCGCTCT-3 ’ (SEQ ID NO: 21) and reverse 5’- GTCCTGCATGGCTCTCTGAA-3’ (SEQ ID NO: 22); Cysltr2 forward 5’- GATATTTGGGGACTTGGCCTG-3’ (SEQ ID NO: 23) and reverse 5’- GCTTTGAAATTCTCCCCAGCA-3’ (SEQ ID NO: 24). PCR products were cloned into the pCR4-Topo vector and the riboprobe synthetized (Notl, T3 for Grikl and Mrgprb5, Spel, T7 for Cysltr2).
ISH experiments were performed as previously described using antisense digoxy genin-labeled riboprobes (Thelie et al., 2015; Desiderio et al., 2019).
For immunostainings, after intracardiac perfusion with ice cold PBS, dissected DRG were fixed in 4% paraformaldehyde for 15 min and then cryopreserved and frozen as described above for ISH. For hind paw skin tissue, quenching was performed using 50mM Glycine dissolved in PBS to minimize background.
Primary antibodies used were: homemade rabbit and guinea pig anti-PRDM12 (respectively 1:5000 and 1:2000), anti-peripherin (1:1000, Abeam abl06276), goat anti-CGRP (1/200, Abeam ab36001), rabbit anti- Navi.8 (1/400, Abeam ab63331). Isolectin B4 conjugated to Al exafluor 594 (Invitrogen, 121413) was used in an 1 : 1000 dilution. Secondary antibodies used were: goat Anti-rabbit Alexa 594 (1:800, Invitrogen A11012), donkey anti-goat Alexa 594 (1:2000, Invitrogen A 11058), goat anti-guinea pig Alexa 488 (1:2000, Invitrogen A11073), donkey anti-guinea pig Alexa 488 (1:800, Bio connect) and donkey a-chicken Alexa 488 (1:1000, Bio connect - Jackson).
Immunostainings were performed as described (Thelie et al., 2015). Images collection and analysis were performed using a wide-field fluorescence microscope Zeiss Axio Observer Zl, a laser-scanning confocal microscope Zeiss LSM 710 using the Zeiss Zen microscopy software or a ste- reo microscope Olympus SZX16 and the softwares CellAF or CellSens Entry V2.1. Image analysis was performed using ImageJ/Fiji and Microsoft Excel 2020 and GraphPad version 9. Cell counts were conducted with at least 8 DRG sections analyzed per animals with an N >= 3.
Electrophysiology
Patch-clamp recordings were conducted at room temperature using an amplifier A-M system, INC Model 2400 controlled by WIN WCP software V5.52. Small DRG neurons (diameter<30 pm) were selected for current-clamp recording. In TAM injected AvilCreERT2 ;Rosa26A'14 ;Prdml 2fl/fl mice, Prdml2 icKO neurons were identified by the presence of red Tomato signal (Olympus 1X70 Inverted microscope equipped with Led fluorescence system). Electrodes were fabricated from 1.6 mm outer diameter borosilicate glass micropipettes (G86150T-4; Warner Instruments) using a Sutter Instruments P-97. Piezoelectric Burleigh micromanipulator was used to lower the electrode on cells. Liquid junction potential was not corrected. Cells were excluded from analysis if the resting membrane potentials was more positive than -40 mV. Neurons were recorded within 24 hours of culture to prevent neurite outgrowth that degrades space clamp.
For current-clamp recordings, electrodes had a resistance of 2.5-3.5 M.Q when filled with the pipette solution of the following composition (in mM): 140 K-Aspartate, 10 NaCl, 10 EGTA, 1 MgC12, 10 HEPES pH 7.20, with KOH (290±5 mOsm). The extracellular solution was composed of (in mM): 150 NaCl, 5 KC1, 1 MgC12, 2 CaC12, 10 HEPES, 10 Glucose, pH 7.40 with NaOH (290±5 mOsm). Whole-cell configuration was obtained in voltage-clamp mode before proceeding to the current-clamp recording mode.
Three variables were assessed in each neuron: resting membrane potential, threshold current and Action Potential frequency. Threshold current was determined by the first action potential elicited by a series of depolarizing current injections (300 ms) that increased in 20 pA increments. Action potential frequency was determined by quantifying the number of action potentials elicited in response to depolarizing current injections (2 s, 1500pA). Any modification in basal excitability will be evidenced by change in the threshold current and/or action potential frequency.
RNA-seq and NGS data analysis
Before tissue collection, mice were deeply anesthetized with Ketamin (75mg/kg) and Rompun (Img/kg) and intracardiacally perfused with ice-cold PBS. Whole lumbar DRG from inducible knockout animals were harvested 4 weeks after the last tamoxifen injection. Controls were either Cre negative TAM injected Prdml2Fl/Fl or Prdml2Fl/Fl mice or com oil injected Cre expressing Prdml2Fl/Fl mice.
Tissues were microdissected in ice-cold PBS and stored at -80°C in 1ml TRIZOL (Thermofisher, ref 15596026). RNA was extracted using the illustra RNAspin Mini RNA isolation kit from GE Healthcare (25-0500-70). Quality and integrity of RNA was assessed with the Fragment Analyzer from Advanced Analytical by using the standard sensitivity RNA Analysis Kit (DNF-471). RNA-seq libraries were performed using 100 ng total RNA of a non stranded RNA Seq, massively -parallel mRNA sequencing approach from Illumina (TruSeq RNA Library Preparation Kit v2, Set A; 48 samples, 12 indexes, Cat. N°RS-122- 2001). Libraries were prepared on the automation (Beckman Coulter’s Biomek FXP workstation). For accurate quantitation of cDNA libraries a fluorometric based system, the QuantiFluor™dsDNA System from Promega was used. The size of final cDNA libraries was determined by using the dsDNA 905 Reagent Kit (Fragment Analyzer from Advanced Bioanalytical) exhibiting a sizing of 280 bp in average. Libraries were pooled and sequenced on the Illumina HiSeq 4000 (SE; 50 bp; 30 Mio reads/sample). Sequence images were transformed to BCL files with the Illumina software BaseCaller software which were demultiplexed to fastq files using bcl2fastq v2.20.0.422. Sequencing quality was asserted using FastQC software (http://www. bioinformatics, babraham. ac.uk/proj ects/fastqc/)(version
0.11.5). Sequences were aligned to the reference genome Mus musculus (mmlO version 96, https://www.ensembl.org/Homo_sapiens/Info/Index) using the STAR aligner software version 2.5.2a allowing for 2 mismatches within 50 bases. Subsequently, read counting was performed using featureCounts version 1.5.0-pl. Read counts were analyzed in the R/Bioconductor environment version 3.6.1 (www.bioconductor.org) using the DESeq2 package version 1.24.0. Candidate genes were filtered using an FDR-corrected p-value threshold of 0.1. Genes were annotated using the Mus musculus GTF file mmlO version 96 used to quantify the reads within genes. Gene ontology and pathway analysis was performed in RStudio using Bioconductor version 3.12 selecting all DEG with an FDR- adjusted? value <0.05 and log2FoldChange>0.449. ClusterProfiler and enrichGO were used for gene ontology analysis based on molecular function, biological process and cellular component using a pvalueCutOff for the enriched categories of 0.03. KEGG pathways analysis was performed using enrichKEGG. Analysis and sensory neuron classification of deregulated genes was performed using the gene expression visualization tools developed by Usoskin et al., 2015
(linnarssonlab org) and Zeisel et al, 2018 (mousebrain.org) Deregulated genes were classified as ‘’Neuron enriched” if they had significant expression in at least one neuronal cell type. Deregulated genes were manually classified as ‘’aspecific” if they had unknown expression pattern, no significant expression at least in one neuronal cell type or significant expression in at least in one non-neuronal cell type.
DRG neuron culture
Mice (3 to 7-month-old) were anesthetized with a mixture of Domitor (Img/kg) and Ketamine (75mg/kg) and an intracardiac perfusion with PBS was performed. DRG from thoracic and lumbar parts were collected and then digested for 35 minutes at 37°C in an enzyme mixture containing Collagenase (12.5mg/mL) and Dispase (3mg/mL). DRG were quickly centrifuged at 150G after digestion and then the enzyme mix was removed and replaced by DMEM/F12. Dissociation using a P 1000 was carried out and the cell suspension was then filtered through a 0,2pm filter followed by a 7 minute centrifugation at 150G. Cells were then resuspended in complete medium containing DMEM/F12, 10% of fetal bovine serum and 1% of Penicillin- Streptomycin. Cells were plated in 4-well plates on glass coverslips coated with 0,lmg/mL of Poly-L-lysine and lOpg/mL of laminin and kept in complete medium at 37°C.
Results
Prdml2 is a conserved epigenetic transcriptional regulator whose mutation in human leads to congenital insensitivity to pain and that is required for the development of the nociceptors. Here we show that Prdml2 continues to display restricted expression in nociceptors in the peripheral nervous system of adult mice (Fig. 8). To address whether Prdml2 is required at adulthood in mature nociceptors for pain perception, the inventors crossed Prdml^fl mice with Advillin-Cre-ERT2 BAC transgenic mice to generate Advillin-CreERT2; Prdml^M1 (Prdml2 icKO) in which Prdml2 is specifically deleted in somatosensory neurons following treatment with tamoxifen or with Rosa26-CreERT2 mice that express Cre ubiquitously after treatment with tamoxifen, to generate Rosa26-CreERT2,' PrdmnAA (Rosa26 icKO). Controls were Prdml2fl/fl or Prdm^^ mice with no Cre transgene injected with tamoxifen; Cre expressing Prdml2fl/fl mice injected with com oil,; or Cre-expressing Prdml2+/+ mice injected with tamoxyfen. After having verified by immunofluorescence (IF) on dorsal root ganglia (DRG) sections the efficiency of the tamoxifen induced loss of Prdml2 in icKO mice and observing that the loss of Prdml2 in mature DRG of adult mice is dispensable for their survival (Figures 1, 2 and 9, 14), the inventors performed a transcriptomic analysis by bulk RNA-seq on dissected lumbar DRG and of TG of icKO models and control mice 1 month after TAM injection. The results obtained, validated for some of the identified differentially expressed genes by RT-qPCR and or in situ, revealed that the loss of Prdml2 alters the expression of a battery of genes essential for their functional properties (Figures 3-6 and 10, 11, 15, 16, 18).
To determine whether the molecular defects observed in sensory neurons of icKO lead to changes in neuronal excitability, current-clamp recordings were performed on cultured small diameter DRG neurons (i.e., putative nociceptors) from Avil icKO (marked by tdTomato fluorescence) and control mice. Measurements done revealed that the loss of Prdml2 alters DRG neuron excitability (Figure 13). To determine whether loss of Prdml2 in mature nociceptors of adult mice alters pain sensation, pain behavior assays were performed on icKO and control mice 1 month after TAM injection. It was observed that mice lacking Prdml2 exhibit normal responses to thermal and mechanical nociceptive stimuli but a reduced response to capsaicin and hypersensitivity to formalin-induced inflammatory pain (Figure 7 and 12,17,19). Together, this data indicate that Prdml2 regulates pain-related behavior in a complex way by modulating gene expression in adult nociceptors and controlling their excitability. They suggest that, depending on the type of pain condition, increasing or reducing its expression and/or activity, may provide analgesic effect.
TABLE 1: List of deregulated genes identified by RNA-seq in DRG of Advillin CreERT2;Prdml2fI/fl mice. Genes with negative fold changes (indicated with a”-“) are downregulated. Those with positive values are upregulated.
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
As a novel pain target, Prdml2 appears interesting given its restricted expression in the nervous system suggesting that any compound targeting its action would have no side effect, its close link with NGF known to be involved in nociceptor sensitization, and the importance of epigenetic mechanisms in the induction and maintenance of chronic pain. Additionally, it can be concluded from these data that in some pain disorders, it is the enhancement of Prdml2, rather than its blockade, that may have an analgesic effect. Overexpression of Prdml2 could be obtained using recombinant viruses encoding Prdml2 or encoding a modified version of Prdml2 that functions as a stronger transcriptional regulator.
Example 1: Prdml2 expression in mature nociceptors is dispensable for DRG nociceptor survival
To visualize Prdml2 expression in adult mice, the inventors performed X-gal staining on dissected tissues £wmPrdml2LacZ/+ mice. Fig. 8A shows that /Wm/2 remains expressed in all trunk and cranial ganglia containing nociceptors. Double immunostaining on transverse sections of trigeminal (TG), superior-jugular (SJG) and dorsal root (DRG) ganglia showed that in these distinct ganglia, Prdml2 is absent from mechanoreceptors (TrkB+) and proprioceptors (TrkC+), but is expressed in both peptidergic (TrkA+, CGRP+) and non-peptidergic (Ret+) nociceptors (Navl.8+, TrpVl+), as well as in some itch-mediating Somatostatin (Sst+) neurons and c-fiber low-threshold mechanoreceptors (C-LTMRs) expressing tyrosine hydroxylase (TH+) (Fig. 8B). Altered epigenetic mechanisms are known to contribute to inflammation- induced pain hypersensitivity.
Injection of Complete Freund’s adjuvant (CFA) into the knee joint produces acute and inflammatory pain in mice that models human arthritis. To determine whether Prdml2 expression is modulated in such inflammatory condition, the inventors performed unilateral knee injections of CFA, 7 days after retrograde labeling of knee-innervating neurons by Fast Blue (FB). 24h after CFA injection, immunostaining was performed using anti-Prdm!2 and anti-Trpvl antibodies on whole DRG sections. Comparing the ipsilateral (CFA injected) and contralateral (Ctrl) sides of the injected mice, we observed a statistically significant decrease in the number of knee-innervating neurons that express Prdml2 while the number of Trpvl+ neurons was increased as expected (Fig. 8C).
To gain insight into the role of Prdml2 in mature nociceptors in adult mice, females carrying Prdml2 floxed alleles were crossed with tamoxifen (TAM)-inducible Cre driver mice: AvilCreERT2 that express Cre specifically in somatosensory neurons. Rosa26CreERT2 that express Cre ubiquitously following treatment with tamoxifen were also used given the very low transmission rate of the Cre with the AvilCreERT2. To ensure efficient excision of the floxed allele, mice with two Rosa26CreERT2 alleles were used (Sandlesh et al., PlosOne 2018). The resuitingconditional knockout offspring, AvilCreERT2; Prdml^A mice and Rosa26CreERT2,-Prdml2fl/fl mice are here designated Avil icKO and Rosa26 icKO. By immunostaining, we validated the tamoxifen induced loss of Prdml2 in the DRG of both Avil icKO and Rosa26 icKO (Fig. 1, and 14). Fig. 16 shows the validation of some of the identified DEGs as Prdml2 targets in Rosa26 icKO mice by RT-qPCR.
In contrast to Prdml2 ablation during development, we found no evidence for cell loss. In both icKO mice, using antibodies against Peripherin as a general marker of peripheral neurons, NF200 as a mechano-proprioceptive marker, Navi.8 marking all nociceptors, CGRP labeling peptidergic nociceptors and performing isolectin B4 (IB4) staining to visualize non-peptidergic nociceptors, no significant difference in the absolute number of cells positive with these markers was observed in DRG between mutant and control mice (Fig. 2 and 9A). No difference was also detected in the proportion of CGRP+ or IB4+ neurons among Peripherin+ cells (Fig. 2 and 9B) nor in the proportion of DRG neurons positive for Navl.8+ and NF200+ (Fig. 2 and 9C).
Given that CIP patients show a loss of intra-epidermal nerve endings, the inventors also performed immunohistochemical analysis of hind paw skin biopsies from both Prdml2 icKO and Prdin/ 2 iKO using Tubulin 1 (Tujl), a marker of intra-epidermal nerve fibers shows that in both icKO mice, normal epidermal Tuj 1+ innervation was observed (Fig.9D). Given that CIP patients show a loss of intraepidermal nerve endings, the inventors also performed immunohistochemical analysis of hind paw skin sections from both Avil icKO and Rosa26 icKO mice using blll-Tubulin, a marker of intraepidermal nerve fibers. In both the mutant mice, normal epidermal blll-Tubulinl in- nervation was observed (Fig. 9E). Thus, loss of Prdml2 appears dispensable for nociceptor survival in adult mice.
Example 2: Loss of Prdml2 affects gene expression in DRG nociceptors of adult mice
To determine whether Prdml2 plays a role in the control of gene expression in mature nociceptors, the inventors performed a transcriptomic analysis by bulk RNA-seq on dissected lumbar DRG of both Advil and Rosa26 icKO models and control mice one month after TAM injection. Using an FDR-adjusted p-value <0.05 and an absolute log2 Fold-Change cutoff of >0.449 (based on the Fold-Change of Ntrkl, recently reported as reduced in adult Prdml2 knockout mice (Landy et al., 2021) we obtained a list of 140 genes in Avil icKO and 134 in Rosa26 icKO that are dysregulated in the absence of Prdml2 (Fig. 3 and 10A and Table 2 and 3).
Comparing the datasets from both mouse lines, we identified 71 transcripts common to both models, among which 39 are downregulated and 33 upregulated by the loss of Prdml2 (Fig. 10B and Table 2 and 3). Most ( 87%) of these core DEGs are nociceptive neuronal genes (Fig. 10C). Gene expression distribution analysis of the DEGs in the major different DRG neuron subtypes revealed that they are not specific of a particular subtype (Fig. 10D). Gene ontology (GO) analysis based on molecular function of these common DEG revealed that a large number of downregulated DEGs encode channel proteins, transmembrane receptors and other membrane proteins, while some of the upregulated genes code for neurotransmitter receptors (Fig. 10E). Further GO analysis based on all categories indicates enrichment in genes involved in cathecolamine transport, detection of stimulus, detection/sensory perception of temperature stimulus, positive regulation of ion transport and blood vessel diameter (Fig. 15 A). KEGG pathway analysis revealed that the three most significantly deregulated pathways are neuroactive ligand-receptor interaction, calcium signaling and cholinergic synapse (Fig. 15B). Focusing on the neuroactive ligand-receptor interaction category, 6 genes appear upregulated while 3 appear downregulated (Fig. 15C). Besides Prdml2, among the core DEGs are genes encoding membrane proteins such as the NGF receptor Ntrkl/TrkA, the sensory specific G protein-coupled receptor Mrgprb5 (Liu et al., Cell 139, 13531365, 2019), the a 6 subunit of the nicotinic acetylcholine receptor Chrna6 that inhibits P2X2/3 receptors, the ionotropic glutamate receptor kainate 1 (Grikl/Glur5), the RET coreceptor GFRa3, the leukotriene receptor Cysltr2, the neuronal specific leucine-ricjh protein Lrm4 and the glycosylphosphatidylinositol-anchored protein OTOA (Zwaenepoel et al., PNAS 30, 6240- 6245, 2002), ions channel proteins such as the acid-sensing channel ASIC1, the cold-activated channel Trpm8, the potassium channels Kcnmbl and Kcngl (Smith et al., Frontiers in Neurosci. 14, 5664, 2020), extracellular proteins such as the secreted glycoprotein CREG2 (Kunita et al., Genmics 80, 456-460, 2002) and the secretory neuropeptide CALCB (Calcitonin Related Polypeptide Beta) (Wimalawansa et al., Endocr. Rev 1996, 17, 533-585, 1996) and enzymes such as A3galt2, Mettl7a3 and Stk32a. The differential expression of some of these genes has been confirmed by RT-qPCR and in situ hybridization on DRG section in Advil icKO and by RT-qPCR alone in Rosa26 icKO (Fig. 4-6 and 11 and 16). The consistency of the results obtained in the two mouse lines strongly support a role for Prdml2 in the transcriptional regulation of gene expression in mature nociceptors.
As TG have a more complex embryonic origin than DRG, being derived from both neural crest and placodal cells, the inventors also performed bulk RNA-seq analysis on TG samples from TAM-injected Rosa26 icKO and control mice. Using an FDR-adjusted P-value #0.05 and an absolute log2 fold-change cut-off of $0.31 (based on the fold-change of Mrgprb5 that appears as true DEG in DRG), we identified 125 DEGs, with 35 upregulated and 95 downregulated genes (Fig. 18A and Table 6 below). As in DRG, most (87%) of these DEGs are nociceptive neuronal genes, with many of them encoding proteins with channel activity (Fig. 18B). Comparing the 2 lists of DEG identified in DRG and TG of Rosa26 icKO mice, we found 41 common dysregulated transcripts (Fig. 18C). These transcripts common to both ganglia, designed here as Prdml2 core DEGs, are listed in Table 5 below. Together, these results strongly support a role for Prdml2 in mature nociceptors in the transcriptional regulation of a battery of genes essential for their function.
Example 3: Prdml2 icKO show altered sensitivity to capsaicin and formalin
To determine whether loss of Prdml2 in mature nociceptors of adult mice alters pain sensation, icKO mice and controls were injected with tamoxifen and used one month after TAM injection in different nociceptive assays. Given the difficulties encountered to generate Advil icKO mice due to non Mendelian tramsmission of the Cre and based on the similar transcriptional changes observed in DRG using the two mouse models, both Avil icKO and Rosa26 icKO mice were used. All behavior tests were performed using both male and female mice. To evaluate thermal nociception, the inventors performed thermal preference place in Avil icKO and tail flick test and cold plantar test in Rosa26 icKO. Mechanical nociception was evaluated in Rosa26 icKO in the mechanical conflict avoidance test. No difference was observed with regards to temperature place preference, tail flick, cold plantar test or mechanical conflict avoidance test between control and icKO mice (Fig. 12A-D and 19). No difference was also observed between icKO and control mice in the open field test used as a control for motor behavior (Fig. 17). We then tested the response of Rosa26 mice to capsaicin, the active component of chili that by binding to its receptor TRPV1 generates a painful sensation. Caspaicin was injected subcutaneously in the hind paw and the time the animals spent licking was recorded during 15 minutes following injection. We observed that the response of icKO mice was significantly reduced in comparison to control mice (Fig. 12E and 19) suggesting a downregulation of TRPV1 -related pain transmission in adult nociceptors lacking Prdml2.
Given the observation that Prdml2 expression is decreased in a model of joint inflammation, the inventors verified whether loss of Prdml2 might lead to changes in inflammation-related nociceptive behavior of adult mice, the inventors thus examined behavior responses of Advil icKO mice in the formalin test, a common model for tissue injury-induced pain, which produces paw swelling and an inflammatory response. This acute inflammation is caused by cell damage that provokes the production of endogenous mediators and subsequently the release of inflammatory mediators in the paw. In this test, two distinct periods of high licking activity of the injected paw can be identified. An early short phase of intense licking caused by the direct chemonociceptive effect of formalin on nociceptors followed by a short pause, and a second prolonged phase of continuous tonic licking induced at least in part by inflammatory responses caused by formalin induced cellular damages. Avil icKO mice showed similar responses to control animals during the first phase. However, they spent more time licking when compared to control mice during the second phase, suggesting a role for Prdml2 in mediating inflammatory pain (Fig. 7, 12F and 19). An increase of sensitivity in the second phase of the test was also observed in Rosa26 icKO mice (Fig. 12G).
Example 4: Loss of Prdml2 affects nociceptor functional properties of cultured DRG neurons
In light of the above results, the inventors next examined whether the loss of Prdml2 alters the excitability of DRG neurons. Current-clamp recordings were performed on cultured small diameter DRG neurons (i.e. putative nociceptors) from Advil icKO (marked by tdTomato+ fluorescence) or control mice. Measurements were done 24h after mechanical dissociation, which is known to induce hyperexcitability in DRG neurons by mechanisms that may present common points with in vivo inflammation-induced hyperexcitability. Representative action potential traces from control and Prdml2 icKO DRG neurons in response to depolarizing current steps showed that an action potential was elicited at a threshold of 1310 pA and 750 pA, respectively (Fig. 13 A). The mean current threshold for action potential firing in Prdml2 icKO neurons was significantly lower compared to control DRG neurons (Control, 1420 ± 161 pA, n=19; Prdml2 icKO, 850 ± 109 pA, n=30; Fig. 13B). Moreover, the absence of Prdml2 doubled the maximal number of action potentials evoked by a 1 sec-current step in small DRG neurons (Fig. 13C). There was no significant difference in resting membrane potential for DRG neurons isolated from control or Prdm 12 icKO mice (Fig. 13D). Thus, loss of Prdml2 leads to hypersensitivity of cultured DRGs. These results further demonstrate that Prdml2 is required for the functional properties of nociceptors in adult mice. They provide some first explanation to the hypersensitivity of icKO mice observed in the formalin test.
Table 2: List of DEG comparing AvilCreERT2 icKO with control, FDR-adjusted p-value > 0.05 and log2FoldChange > 0.449. In bold genes in common with the list of DEG from Rosa26CreERT2 icKO.
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000119_0003
Table 3: List of DEG comparing Rosa26CreERT2 icKO with control, FDR-adjusted p-value > 0.05 and log2FoldChange > 0.449. In bold genes in common with the list of DEG from AvilCreERT2 icKO.
Figure imgf000119_0002
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Table 4: List of primer pairs used for RT-qPCR analysis.
Figure imgf000122_0002
Figure imgf000123_0002
_
Table 5: List of transcripts common to both ganglia, designed here as Prdml2 core DEGs,
Figure imgf000123_0001
Table 6: List of DEG identified in TG of Rosa26 icKO. Genes in common with the list of DEGs from DRG of Rosa26 icKO are indicated in bold. Genes in common with the list of DEGs from DRG of Avil icKO are in italic.
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
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Claims

1. An in vitro method for identifying a molecule suitable as an analgesic, wherein said method comprises determining whether a candidate molecule modulates Prdml2 activity in nociceptors and/or dorsal root ganglia, preferably adult nociceptors and/or dorsal root ganglia.
2. The in vitro method according to claim 1, wherein said method comprises contacting a nociceptor and/or a dorsal root ganglion with a candidate Prdml2 modulator, preferably wherein said nociceptor or dorsal root ganglion expresses Prdml2.
3. The in vitro method according to claim 1 or 2, wherein Prdml2 activity is assessed by measuring G9a recruitment to histone H3 and/or the H3K9 methylation when compared to respectively G9a recruitment to histone H3 or H3K9 methylation in nociceptors and/or dorsal root ganglia of said subject before administration of the candidate modulator of Prdml2 activity.
4. The in vitro method according to claim 1 or 2, wherein Prdml2 activity is assessed by measuring expression of one or more genes selected from the group of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, MRGPRB5 Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
5. The in vitro method according to claim 4, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 activator when the expression of one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
6. The in vitro method according to claim 4, wherein the candidate modulator of Prdml2 activity is considered a Prdml2 inhibitor when the expression of one or more genes selected from the group consisting of: PRDM12, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 is increased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably at least about 100%; and/or one or more genes selected from the group consisting of: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5 is decreased by at least about 10%, at least about 25%, at least about 35%, preferably at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, most preferably about 100%, when compared to their respective expression levels before administration of the candidate modulator of Prdml2 activity.
7. The in vitro method according to any one of claims 1 to 6, wherein the nociceptors and/or dorsal root ganglia are human nociceptors and/or dorsal root ganglia.
8. A modulator of Prdml2 activity, preferably a modulator identified according to the method according to any one of claims 1 to 7, for use in treatment or prevention of a pain condition in a subject.
9. The modulator for use according to claim 8, wherein said modulator alters the expression level of Prdml2 in nociceptors of said subject.
10. The modulator for use according to claim 9, wherein said modulator increases Prdml2 expression in nociceptors of said subject, preferably wherein Prdml2 expression is increased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to Prdml2 expression in nociceptors of said subject before administration of said Prdml2 modulator. Preferably, said modulator is a Prdml2 protein or a polynucleotide encoding Prdml2, a functional fragment of Prdml2, or a polynucleotide encoding a functional fragment of Prdml2, a PRDM12 gene-expression or transcription targeting system, a transcription or expression inducer of the PRDM12 gene or any combination thereof.
11. The modulator for use according to claim 9, wherein said modulator decreases PRDM12 expression in nociceptors of said subject, preferably wherein PRDM12 expression is decreased by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to PRDM12 expression in nociceptors of said subject before administration of said PRDM12 modulator. Preferably, said modulator is a PRDM12 gene targeting system, inhibitory PRDM12 RNA or DNA systems, siRNA targeting PRDM12, antisense oligonucleotide targeting PRDM12, a zinc finger protein targeting PRDM12, optionally linked to a KRAB repressor, an enzymatically deficient Cas9 (dCas9) targeting PRDM12 fused to an activator (CRISPa) or a repressor (CRISPi)domain or any combination thereof.
12. The modulator for use according to claim 8, wherein the modulator alters the Prdml2- mediated G9a recruitment to histone H3, preferably wherein the modulator alters the methylation status of histone H3, preferably the methylation status of H3K9.
13. The modulator for use according to claim 12, wherein the modulator increases methylation of histone H3 in nociceptors of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, when compared to histone H3 methylation in nociceptors of said subject before administration of said Prdml2 modulator.
14. The modulator for use according to claims 12 or 13, wherein said modulator increases the transcription rate in nociceptors of at least one gene selected from the group of genes comprising: PRDM12, CREG2, Insrr, NTRK1, Kcnmbl, Gml3425, Smr2, GRIK1, CHRNA7, Gng8, Slc34a2, A3galt2, Fyb2, Mettl7a3, Chstl, Gml6364, Kcnck9, Otoa, MAL2, Nt5e, Gml8349, Tuftl, and MRGPRB5, by at least 25%, preferably at least 50%, more preferably at least 75% when compared to their transcription rate in nociceptors before administration of said Prdml2 modulator.
15. The modulator for use according to claim 12, wherein the modulator decreases methylation of histone H3 in nociceptors of said subject by at least 25%, at least 50%, at least 75%, more preferably at least 100%, most preferably inhibits methylation of histone H3, when compared to histone H3 methylation in nociceptors of said subject before administration of said Prdml2 modulator.
16. The modulator for use according to claims 12 or 15, wherein said modulator decreases the transcription rate in nociceptors of at least one gene selected from the group of genes comprising: PRDM12, Ntrk3, Casr, Arid5a, Agtrla, Chmb3, Cars, Neill, Steap3, Thsd7b, Cyp26bl, Cysltr2, Skor2, Drdl, Rgs9bp, Stk32a, Caleb, Atp6apall, Aldhla3, and Chma6 by at least 25%, preferably at least 50%, more preferably at least 75% when compared to their transcription rate in nociceptors before administration of said Prdml2 modulator.
17. The modulator for use according to any one of claims 8 to 16, wherein the subject is suffering from a pain condition caused or aggravated by altered Prdml2 expression versus Prdml2 expression in a subject not having said pain condition.
18. The modulator for use according to claim 17, wherein the subject is suffering from: chronic pain, operative pain, treatment-related pain, injury-related pain, trauma-related pain, or is a palliative subject, more preferably wherein the pain condition is a nociceptive pain, inflammatory pain, neuropathic pain, itch-related pain, inflammatory itch-related pain caused by e.g. atopic dermatitis, non-inflammatory itch-related pain caused by e.g. pruritus, neuropathic itch, inflammatory itch such as atopic dermatitis, non-inflammatory itch such as pruritus-related pain, or any combination thereof, more preferably wherein said pain condition is induced by nerve injury or inflammation or itch.
19. A pharmaceutical composition comprising Prdml2 and/or a Prdml2 modulator, preferably a modulator identified according to the method according to any one of claims 1 to 7, for use in treating, alleviating or preventing a pain condition in a subject, preferably wherein said pain condition is caused or aggravated by altered Prdml2 expression versus Prdml2 expression in a subject not having said pain condition.
20. The pharmaceutical composition of claim 19, wherein said Prdml2 modulator is a Prdml2 activator and wherein the pain condition is caused or aggravated by reduced or lost Prdml2 expression.
21. The pharmaceutical composition of claim 20, wherein the Prdml2 activator is retinoic acid (RA) or zinc finger protein of the cerebellum 1 (Zicl).
22. A viral vector encoding Prdml2, preferably wherein said modulator is aPrdml2 transgene, preferably wherein Prdml2 is human Prdml2 transgene, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or wherein the human PRDM12 transgene is defined by SEQ ID NO: 2 for use in treating, alleviating or preventing a pain condition in a subject, wherein the pain condition is caused or aggravated by reduced or lost Prdml2 expression.
23. The viral vector according to claim 22, wherein said viral vector backbone is an AAV vector backbone, such as an AAV5, AAV1, AAV6, AAV8, AAVPHPS, AAV2 and AAV9 vector backbone, more particularly a self-complementary AAV9 vector (scAAV9) viral vector backbone.
24. A non-viral vector system encoding Prdml2, preferably wherein said modulator is a Prdml2 transgene, preferably wherein Prdml2 is human Prdml2 transgene, more preferably wherein the human Prdml2 protein is characterised by SEQ ID NO: 1 and/or wherein the human PRDM12 transgene is defined by SEQ ID NO: 2, for use in treating, alleviating or preventing a pain condition in a subject, wherein the pain condition is caused or aggravated by reduced or lost Prdml2 expression or function.
25. The non-viral vector according to claim 24 which is selected from the group comprising: cationic lipids, liposomes, nanoparticles, PEG, PEI; plasmid vectors (e.g. pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequencesO, a mini circle, an episomal vector, or a transposon-based vector, such as a PiggyBac- based vector or a Sleeping Beauty-based vector.
26. The pharmaceutical composition of claim 20 or 21, or the viral or non-viral vector encoding Prdml2 according to any one of claims 22 to 25, wherein said pain condition is inflammatory pain or pain caused by itch or nerve injury, such as pain caused by nerve injury or by skin or j oint inflammation or itch, or a TRPV 1 -related pain condition, such as pain caused by cancer, neuropathic pain, osteoarthritic pain, postoperative pain, dysfunctional pain disorders and musculoskeletal pain.
27. The pharmaceutical composition according to claim 19, wherein said Prdml2 modulator is a Prdml2 inhibitor and wherein the pain condition is caused or aggravated by increased Prdml2 expression.
28. The pharmaceutical composition according to claim 27, wherein pain condition is heat- induced pain or chemically-induced pain or allodynia and/or hyperalgesia, or a pain that can be treated by capsaicin such as back pain, joint pain or headaches.
29. The pharmaceutical composition of claim 27 or 28, wherein the Prdml2 inhibitor is an inhibitor of alcohol and/or aldehyde dehydrogenases, for example citral.
30. A method for diagnosing a hypersensitivity to pain in a subject, the method comprising a step of determining the expression of Prdml2 in a biopsy, such as in a TG or DRG biopsy of said subject, wherein reduced expression of Prdml2 is indicative of a hypersensitivity to pain.
31. An analgesic comprising the vector according to any one of claims 22 to 25 or a modulator of Prdml2 activity as defined in any one of the previous claims.
PCT/EP2021/086946 2020-12-21 2021-12-21 Modulation of prdm12 for use in treatment of pain conditions WO2022136344A1 (en)

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