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WO2025168582A1 - Canaux de chlorure et leurs utilisations - Google Patents

Canaux de chlorure et leurs utilisations

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Publication number
WO2025168582A1
WO2025168582A1 PCT/EP2025/052851 EP2025052851W WO2025168582A1 WO 2025168582 A1 WO2025168582 A1 WO 2025168582A1 EP 2025052851 W EP2025052851 W EP 2025052851W WO 2025168582 A1 WO2025168582 A1 WO 2025168582A1
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Prior art keywords
seq
acid sequence
nucleic acid
cell
fusion protein
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Inventor
Luiz ALMEIDA SILVA
Steven Oliver DEVENISH
Dimitri Michael Kullmann
Andreas Lieb
Laura Vede USSINGKÆR
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UCL Business Ltd
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UCL Business Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to engineered glutamate-gated chloride channels that are useful in the treatment of epilepsy, epilepsy-related neurological disorders, as well as non-epilepsy related neurological disorders characterised by pathological neuronal overactivity and neuropsychiatric disorders characterised by pathological neuronal overactivity
  • Brain disorders are the most prevalent health concern in human populations, encompassing conditions such as stroke, dementia, epilepsy, schizophrenia and anxiety disorders (Charlson et al., 2018 Schizophr Bull 44(6)p1195-1203; G. B. D. N. S. D. Collaborators, 2024 Lancet Neurol 23(4)p344-381).
  • Epilepsy affects over 60 million people worldwide (Ngugi et al., 2010 Epilepsia 51(5)p883-90). Even with optimal treatment, -30% of patients remain resistant to pharmacotherapy (Kwan et al., 2011 N. Engl. J. Med. 8;365(10)p919-26; Picot et al., 2008 Epilepsia 49(7)p1230-8).
  • the development of new antiepileptic drugs in the last 20 years has had little impact on refractory epilepsy: people with inadequately controlled seizures continue to experience major co-morbidities, social exclusion, and an annual rate of sudden unexpected death in epilepsy (SUDEP) of 0.5-1% (Devinsky, 2011 N. Engl. J. Med.
  • WO2010042799 describes a family of chimeric receptors composed of a binding site from a nicotinic receptor and transmembrane segments from various ion channels, to be used with an exogenous ligand as a chemogenetic actuator.
  • WO2018175443, WO2017049252, WO2019104307 and WO2019094778 describe technologies and application closely related to WO2010042799.
  • Other chemogenetic tools are given in WO2017058926, which describes the use of a modified human glycine receptor delivered by a viral vector, which can be activated by glycine, and WO2014093251 , which describes a pH-regulated chimeric chloride channel.
  • a more refined gene therapy strategy relies on closed-loop suppression of neuronal circuit excitability triggered by episodes of abnormal excessive neuronal activity.
  • Two approaches to achieve cell- autonomous closed-loop modulation of excitability have been proposed:
  • therapeutic transgenes for example encoding a modified potassium channel
  • an activity-dependent promoter such as cFos (Qiu etal., 2022 Science 378(6619)p523-532). This is described in WO2021191474.
  • this approach is primarily suited to reducing the onset of subsequent seizures and may be too sluggish to activate immediately upon occurrence of pathological overactivity of neurons.
  • therapeutic transgenes encoding an inhibitory glutamate receptor may be utilised to open a transmembrane chloride conductance in response to a build-up of the (typically excitatory) neurotransmitter glutamate in the extracellular space, thus allowing for the closed-loop suppression of pathological circuit excitability.
  • inhibitory glutamate receptors significantly reduces the latency between excessive neuronal activity and initiation of neuronal inhibition (to a period of milliseconds), such that inhibitory glutamate receptors may be useful to abort ongoing seizures, as well as reducing the onset of further seizures.
  • eGluCI is a heteropentameric glutamate-gated chloride channel derived from Caenorhabditis elegans. It is made up of alpha and beta subunits (fused to fluorescent reporter proteins) and was optimised by inserting the L9’F mutation in the alpha subunit in order to increase its sensitivity to glutamate, such that the receptor exhibits an EC50 in the micromolar range.
  • Preclinical proof-of-concept experiments used a lentiviral vector to deliver the alpha and beta subunits into cells.
  • the fluorescent reporter proteins are not intrinsic to the function of the channel and limit the potential for clinical translation because of immunogenicity concerns. (Importantly, the fluorescent reporter proteins cannot be simply removed due to their role in mediating the aggregation and assembly of the monomeric channel subunits to form the heteromultimeric channel).
  • the transgenes encoding eGluCI are too big to fit into an adeno-associated viral vector, as would be preferred for clinical translation.
  • Neuropsychiatric conditions are common in people with epilepsy, as well as in people without epilepsy (Doherty et al., 2022 British Journal of Neuroscience Nursing 18(2); G. B. D. M. D. Collaborators 2022, Lancet Psychiatry 9(2)p137-150).
  • schizophrenia affects approximately 24 million people worldwide (Charlson et al., 2018 Schizophr Bull 44(6)p1195-1203).
  • Current treatment of schizophrenia is predominantly focused on managing symptoms of psychosis including hallucinations, delusions and psychomotor agitation.
  • antipsychotic medications are however associated with a high risk of adverse side effects such as parkinsonism, akathisia and tardive dyskinesia, and also reduce work functioning in the long-term (Ali et al., 2021 PLoS One 16(9)pe0257129; Harrow et al., 2017 Psychiatry Res 256p267-274).
  • Antipsychotic medications also fail to address, or can even worsen, negative symptoms such as social withdrawal and cognitive symptoms such as poor memory in patients with schizophrenia (Haddad et al., 2023 BMC Psychiatry 23(1)p61).
  • schizophrenia is marked by pathological neuronal overactivity for example in the hippocampus (McHugo et al., 2019 Am J Psychiatry 176(12)p1030-1038) . Interventions that reduce hippocampal activity alleviate cognitive symptoms in rodent models of schizophrenia (Donegan et al., 2019 Nat Commun 10(1)p2819).
  • GluCIs novel chimeric glutamate- gated chloride channels
  • Engineered GluCIs as disclosed herein achieve an EC50 in the micromolar range, high current density and minimal desensitization without constitutive activity.
  • the invention provides glutamate-gated chloride channel gene therapy useful for treating epilepsy and epilepsy-related neurological disorders, as well as non-epilepsy related neurological disorders and neuropsychiatric disorders characterised by pathological neuronal overactivity, such as schizophrenia.
  • the inventors have engineered a GluCI that incorporates: (i) a transmembrane domain derived from the transmembrane portion of a Homo sapiens GABA-Rho1 channel subunit, and (ii) a glutamate binding domain derived from the glutamate binding portion of a Haemonchus contortus GluCI.
  • GluRhol provides an engineered GluCI that is homopentameric and assembles without the need for fluorescent reporter proteins. GluRhol achieves adeno-associated virus (AAV) vector compatibility. The human origin of the transmembrane domain reduces the potential for immunogenicity.
  • AAV adeno-associated virus
  • Each subunit of GluRhol comprises the chimeric fusion protein ‘Short-HC-Rho1-E145G-P295G-395HA’, the amino acid sequence of which is set forth in SEQ ID NO:22.
  • Engineered GluCIs as disclosed herein are useful in the treatment of epilepsy and epilepsy-related neurological disorders as well as non-epilepsy related neurological disorders and neuropsychiatric disorders characterised by pathological neuronal overactivity, such as schizophrenia.
  • the original experimental data herein demonstrate that GluRhol has no detectable constitutive activity and exhibits anti-epileptic efficacy in vivo, without negatively affecting normal learning/memory behaviours. GluRhol rescues epilepsy comorbidities and protects against symptoms of schizophrenia.
  • a first aspect of the invention provides a chimeric fusion protein comprising: (i) a transmembrane domain and (ii) a glutamate binding domain, in which the transmembrane domain and the glutamate binding domain are heterologous to one-another.
  • a plurality of chimeric fusion proteins of the invention are capable of homologous or heterologous co-association in a membrane (e.g., in the membrane of a neuronal cell, in vivo or in vitro) to form a multimeric glutamate-gated chloride channel (GluCI).
  • the chimeric fusion protein comprises a transmembrane domain derived from the transmembrane portion of a Homo sapiens chloride channel subunit.
  • the chimeric fusion protein may comprise a transmembrane domain derived from the transmembrane portion of a Homo sapiens glycine receptor (GlyR) subunit.
  • the chimeric fusion protein may comprise a transmembrane domain derived from the transmembrane portion of a Homo sapiens gamma- aminobutyric acid (GABA) receptor subunit.
  • GABA gamma- aminobutyric acid
  • the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.
  • the transmembrane domain is derived from the transmembrane portion of a Homo sapiens GABA-Rho1 receptor subunit and comprises an amino acid substitution at position P295, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .
  • the transmembrane domain may comprise a Proline (P) Glycine (G) substitution (P295G) as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .
  • the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO:7.
  • the chimeric fusion protein comprises: (i) a transmembrane domain; (ii) a glutamate binding domain; and (iii) a peptide tag.
  • the chimeric fusion protein may comprise a hemagglutinin (HA) epitope peptide tag.
  • HA hemagglutinin
  • One suitable HA epitope peptide tag comprises the amino acid sequence set forth in SEQ ID NO:13.
  • the amino acid sequence of the peptide tag may be contiguous with the amino acid sequence of the transmembrane domain or may interrupt the amino acid sequence of the transmembrane domain (i.e. , amino acid sequence of the peptide tag may be embedded within the amino acid sequence of the transmembrane domain).
  • the amino acid sequence of the peptide tag interrupts the amino acid sequence of the transmembrane domain at position 395 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1).
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the glutamate binding domain is derived from the glutamate binding portion of a Haemonchus contortus glutamate receptor. In some embodiments, the glutamate binding domain is derived from the glutamate binding portion of a glutamate-gated chloride channel (GluCI) subunit. In some embodiments, the glutamate binding domain is derived from the glutamate binding portion of a Haemonchus contortus glutamate-gated chloride channel (GluCI) subunit.
  • the glutamate binding domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:19.
  • the glutamate binding domain may comprise the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:18.
  • the chimeric fusion protein comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .
  • the chimeric fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:21 , or SEQ ID NO:22.
  • the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.
  • a second aspect of the invention provides an engineered GluCI comprising two or more subunits, in which at least one subunit comprises a chimeric fusion protein of the first aspect of the invention.
  • the engineered GluCI exhibits a half-maximal effective concentration (EC50) for glutamate of between 1 and 100 ⁇ M, for example, an EC50 between 10 and 20 ⁇ M.
  • EC50 half-maximal effective concentration
  • a third aspect of the invention provides a nucleic acid encoding a chimeric fusion protein of the first aspect of the invention.
  • the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23.
  • the nucleic acid may comprise the nucleic acid sequence set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:44 or SEQ ID NO:45.
  • the nucleic acid comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:45.
  • a fourth aspect of the invention provides an expression vector, comprising a nucleic acid of the third aspect of the invention operably linked to a promoter.
  • the promoter is a regulatable promoter, a constitutive promoter, or a tissue-specific promoter.
  • the promoter comprises a human calcium-calmodulin (CaM)-dependent protein kinase II (hCaMKII) promoter.
  • CaM calcium-calmodulin
  • hCaMKII human calcium-calmodulin-dependent protein kinase II
  • One suitable hCaMKII promoter comprises the nucleic acid sequence set forth in SEQ ID NO:46.
  • the expression vector is a viral vector, preferably an adeno-associated virus (AAV) vector.
  • the expression vector may be an adeno-associated virus (AAV) vector selected from the group consisting of: rAAV2/1 , rAAV2, rAAV2/3, rAAV2/5, rAAV2/6, rAAV2/7, rAAV2/8, rAAV2/9 , AAVrh, AAVDJ, AAVDJ/8, AAVPhP.eB, AAVPhPS, and AAV2-retro.
  • the AAV vector is an rAAV2/9 vector.
  • the expression vector comprises an AAV2 inverted terminal repeat (ITR) sequence.
  • ITR inverted terminal repeat
  • an AAV2 ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:47 and/or SEQ ID NO:48.
  • the expression vector may comprise a Kozak sequence.
  • a Kozak sequence comprising the nucleic acid sequence set forth in SEQ ID NO:49.
  • the expression vector may comprise a woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence.
  • WPRE woodchuck hepatitis virus
  • WPRE sequence optimised to limit any potential oncogenic activity.
  • WPRE sequences comprises the nucleic acid sequence set forth in SEQ ID NQ:50.
  • the expression vector may comprise a human growth hormone polyadenylation signal (hGHpA) sequence.
  • hGHpA human growth hormone polyadenylation signal
  • a hGHpA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:51 .
  • the expression vector may comprise an F1 origin of replication.
  • an F1 origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:52.
  • the expression vector may comprise a neomycin or kanamycin resistance gene (NeoR/KanR) sequence.
  • a NeoR/KanR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:53.
  • the expression vector may comprise an origin of replication sequence.
  • an origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:54.
  • the expression vector may comprise one or more non-coding sequences.
  • the expression vector comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:55.
  • the expression vector may comprise the nucleic acid sequence set forth in SEQ ID NO:56 or SEQ ID NO:57.
  • the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:57.
  • the expression vector is encapsidated into a recombinant virus particle.
  • a fifth aspect of the invention provides a recombinant viral particle comprising an expression vector of the fourth aspect of the invention.
  • the recombinant viral particle is a recombinant adeno-associated virus (AAV) particle.
  • AAV adeno-associated virus
  • a virus particle selected from the group consisting of: rAAV2/1 , rAAV2, rAAV2/3, rAAV2/5, rAAV2/6, rAAV2/7, rAAV2/8, rAAV2/9, AAVrh, AAVDJ, AAVDJ/8, AAVPhP.eB, AAVPhPS, and AAV2-retro.
  • the recombinant viral particle is an rAAV2/9 virus particle.
  • a sixth aspect of the invention provides an in vitro method of preparing a recombinant virus particle, the method comprising: transducing a cell with an expression vector of the fourth aspect of the invention; expressing the viral packaging and envelope proteins necessary for the formation of a recombinant virus particle in the cell; and culturing the cell in a culture medium, such that the cell produces the recombinant virus particle.
  • the method comprises transducing the cell with one or more additional expression vectors that encode the viral packaging and envelope proteins necessary for formation of the recombinant virus particle. In some embodiments, the method comprises recovering recombinant virus particles from the cell culture medium and/or concentrating the recombinant virus particles.
  • a seventh aspect of the invention provides an engineered cell comprising one or more of: a chimeric fusion protein of the first aspect of the invention; an engineered GluCI of the second aspect of the invention; a nucleic acid of the third aspect of the invention; an expression vector of the fourth aspect of the invention, or a recombinant virus particle of the fifth aspect of the invention.
  • the engineered cell is a neuronal cell, for example a CA1 , CA2 or CA3 pyramidal cell, or an inhibitory interneuron cell.
  • the engineered cell may be a mammalian cell, preferably a human cell.
  • An eighth aspect of the invention provides a method of treating a disease in a subject in need thereof, the method comprising: administering a chimeric fusion protein of the first aspect of the invention; an engineered GluCI of the second aspect of the invention; a nucleic acid of the third aspect of the invention; an expression vector of the fourth aspect of the invention, a recombinant virus particle of the fifth aspect of the invention; or an engineered cell of the seventh aspect of the invention to the subject.
  • a chimeric fusion protein of the first aspect of the invention an engineered GluCI of the second aspect of the invention; a nucleic acid of the third aspect of the invention; an expression vector of the fourth aspect of the invention, a recombinant virus particle of the fifth aspect of the invention; or an engineered cell of the seventh aspect of the invention for use in a method of treating a disease in a subject in need thereof; the method comprising: administering a chimeric fusion protein of the first aspect of the invention; an engineered GluCI of the second aspect of the invention; a nucleic acid of the third aspect of the invention; an expression vector of the fourth aspect of the invention, a recombinant virus particle of the fifth aspect of the invention; an engineered cell of the seventh aspect of the invention to the subject.
  • a chimeric fusion protein of the first aspect of the invention an engineered GluCI of the second aspect of the invention; a nucleic acid of the third aspect of the invention; an expression vector of the fourth aspect of the invention, a recombinant virus particle of the fifth aspect of the invention; or an engineered cell of the seventh aspect of the invention in the preparation of a medicament for the treatment of a disease in a subject in need thereof is further disclosed.
  • the disease is an epilepsy, an epilepsy-related neurological disorder, or a neurological disorder characterised by pathological neuronal overactivity.
  • a ninth aspect of the invention provides an in vitro method of expressing a chimeric fusion protein in a cell, the method comprising: (i) transfecting the cell with a nucleic acid of the third aspect of the invention, an expression vector of the fourth aspect of the invention, or a recombinant virus particle of the fifth aspect of the invention, and culturing the cell in a culture medium, such that the cell expresses the chimeric fusion protein.
  • the invention includes any combination of the aspects and embodiments disclosed herein, except where such a combination is clearly impermissible or expressly avoided.
  • H. contortus GluCI (Hc-GluCI) variants were screened for increases in glutamate sensitivity and current density.
  • A Dose-response curves showing the relative current amplitudes (l/lmax) mediated by Hc-GluCI and the Hc-GluCI variant E145G in response to glutamate perfusion (3-3000 ⁇ M).
  • the dose-response curve for Hc-GluCI-E145G is shifted leftward.
  • D A schematic diagram summarising the characteristics of Hc-GluCI-E145G variants.
  • Hc-GluCI- E145G variant I335V
  • Three Hc-GluCI-E145G variants T93S; intracellular loop TM3-4 substitution from human GlyRal ; intracellular loop TM3-4 substitution from human GABA-Rho1
  • E Dose-response curves showing the relative current (l/lmax) mediated by Hc-GluCI-E145G variants in response to glutamate perfusion (3- 3000 ⁇ M).
  • FIG. 3 A first chimeric chloride channel comprising the E145G variant H. contortus GluCI glutamate binding domain and a transmembrane domain derived from the a1 subunit of the human glycine receptor (GlyRal) (termed Hc-GlyRa1) was generated and assessed for its suitability in chemogenetic applications.
  • a representative voltage clamp trace shows the response characteristics (pA/pF) of Hc-GlyRa1 following glutamate administration (10 ⁇ M).
  • a mean maximum current density of approximately 125 pA/pF was achieved (n 8).
  • C A representative voltage clamp trace shows the response characteristics of Hc-GlyRa1 following picrotoxin (PTX) administration (1000 ⁇ M) (in the absence of glutamate). As shown, Hc-GlyRa1 exhibits basal activity.
  • FIG. 4 A second chimeric chloride channel comprising the E145G variant H. contortus GluCI glutamate binding domain and a transmembrane domain derived the Rho1 subunit of the human GABAc channel (termed Hc-Rho1) was generated and assessed for its suitability in chemogenetic applications.
  • a representative voltage clamp trace shows the response characteristics (pA/pF) of Hc-Rho1 following glutamate administration (300 ⁇ M).
  • a mean maximum current density of approximately 70 pA/pF was achieved (n 6).
  • C A representative voltage clamp trace shows the response characteristics of Hc-Rho1 following picrotoxin (PTX) administration (1000 ⁇ M) (in the absence of glutamate). As shown, in contrast to Hc-GlyRa1 , Hc-Rho1 does not exhibit basal activity.
  • FIG. 5 The Hc-Rho1 variants L266F, P295F and T306A were screened for improvements in glutamate sensitivity and current density.
  • A Dose-response curves show the relative current amplitude (l/lmax) of the L266F, P295F and T306A variants following glutamate administration (1-1500 ⁇ M). The curves show a leftward displacement as compared to Hc-Rho1 .
  • B Maximum chloride ion current densities were measured following glutamate administration (1-1500 ⁇ M).
  • a representative voltage clamp trace shows the response characteristics of the P295F variant (30 ⁇ M glutamate).
  • a representative voltage clamp trace shows the response characteristics of the P295F variant following picrotoxin (PTX) administration (1000 ⁇ M) (in the absence of glutamate). As shown, the P295F variant exhibits basal activity.
  • FIG. 6 A further Hc-Rho1 variant (P295G) was screened for improvements in glutamate sensitivity and current density.
  • a dose-response curve shows the relative current amplitude (l/lmax) of the P295G variant following glutamate administration (1-3000 ⁇ M). The P295G curve shows a leftward displacement as compared to Hc-Rho1 .
  • B) An EC50 of approximately 20 ⁇ M was measured (n 9).
  • FIG. 7 The behaviour of Hc-Rho1-P295G was further assessed by way of comparison against alternative chloride channels.
  • A, B The basal activity of Hc-Rho1-P295G was analysed. As shown, Hc- Rho1-P295G and GABA-Rho1 exhibit no basal activity (PTX/cap approximately 0 pA/pF; PTX/lmax approximately 0%).
  • Hc- GlyRcd compared to GABA-Rho1 is ***, P ⁇ 0.05, Kruskal-Wallis test with Dunns post-hoc test and Hc- GlyRcd compared to Hc-Rho1-P295G is *, P ⁇ 0.05, Kruskal-Wallis test with Dunns post-hoc test; PTX/lmax: Hc-GlyRa1 compared to GABA-Rho1 is **, P ⁇ 0.05, Kruskal-Wallis test with Dunns post-hoc test).
  • Hc-Rho1-P295G was further engineered for attachment of an eGFP or hemagglutinin (HA) peptide tag.
  • A Dose-response curves show the relative current amplitude (l/lmax) of two Hc-Rho1- P295G-HA fusion proteins by glutamate application (1-10000 ⁇ M). The curves for Hc-Rho1-P295G, Hc- Rho1-P295G-395HA (HA fused to the TMD) and Hc-Rho1-P295G-HA (HA fused to the C-terminus of the protein) are closely aligned.
  • Hc-Rho1-P295G-395HA was further engineered by shortening the N-terminus of the protein. This created a new variant denoted ‘GluRhoT (Short-Hc-Rho1-E145G-P295G-395HA).
  • GluRhoT Short-Hc-Rho1-E145G-P295G-395HA
  • EC50 Glutamate sensitivity
  • P295G-395HA Glutamate sensitivity
  • P295G-395HA Glutamate sensitivity
  • P295G-395HA Glutamate sensitivity
  • FIG. 10 A I oss-of-fu notion Y186A GluRhol variant was engineered for use as a control sample.
  • a representative voltage clamp trace shows its response characteristics.
  • a first trace shows that GluRho1-Y186A ('Y186A') exhibits substantially no response following glutamate administration (1000 ⁇ M).
  • B A second trace shows that Y186A exhibits a strong response following emamectin administration (100 ⁇ M).
  • FIG. 11 Representative voltage clamp traces show the response characteristics of GluRhol and Y186A to glutamate (GLU, 1000 ⁇ M), pentylenetetrazol (PTZ, 1000 ⁇ M) or a mixture of both.
  • GLU 1000 ⁇ M
  • PTZ pentylenetetrazol
  • a first trace shows that GluRhol responds to glutamate, but not PTZ.
  • B A second trace shows that Y186A does not respond to glutamate or PTZ.
  • FIG. 13 GluRhol exhibits AAV compatibility.
  • a schematic diagram shows the encapsidation of GluRhol and Y186A transgenes (under control of hCaMKII promoters) into recombinant AAV2/9 viral particles.
  • B SDS-PAGE analysis shows bands corresponding to viral capsid proteins VP1 , VP2 and VP3.
  • FIG. 17 The efficacy of GluRhol to treat chronic epilepsy was assessed in an intra-amygdala kainate model of chronic drug-resistant temporal lobe epilepsy using a randomized blinded study design.
  • A Mice were injected with kainate in the amygdala, which causes the occurrence of generalized spontaneous recurrent seizures within two weeks.
  • a subcutaneous transmitter was implanted, and brain activity was recorded for 14 days (baseline electrocorticogram - ECoG) after which an AAV9 was injected in the ventral hippocampi of mice. After 14 days of virus expression, another 14 days of brain activity was recorded to capture the effect of the treatment on the seizure burden (post-AAV ECoG).
  • FIG 22 The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model as described in figure legend 20.
  • mice The first arm visited (“Left” or “Right”) was quantified for kainate and saline mice before and after AAV9 injection and/or re-testing (“Before” and “After”).
  • “( x )” denotes the original number of mice tested in the respective groups, whereas the digit preceding the parentheses denote the number of mice that were re-tested. The number of mice re-tested is lower than the original number of mice in two groups due to SUDEP. Only mice that were retested were included in statistical analyses.
  • FIG 24 The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model as described in figure legend 20.
  • Saline and kainate mice were subjected to spatial object recognition (SOR) testing as a measure of spatial memory. This test measures whether the mice are able to recognize that an object has been moved since they last saw it.
  • SOR spatial object recognition
  • a positive symptom of psychosis was evaluated on day 1
  • a negative symptom of schizophrenia was evaluated on day 9
  • a cognitive symptom of schizophrenia was evaluated on day 11 .
  • (B - left) Mice were subjected to large open field testing (large OFT) as a measure of psychomotor behaviour. Movements were tracked using ANY-maze software.
  • (B - middle) Representative plots show the movements in Y186A or GluRhol mice injected with saline or ketamine when psychomotor agitation is at its highest in the ketamine groups (15-20 minutes after I.P. injection, equivalent to minute ‘40’ of the test). An increase in rotations and movement is evident in ketamine plots.
  • Intraperitoneal ketamine injection caused a significant increase in rotations compared to saline injection in both Y186A and GluRhol groups (approximately 37 rotations twenty minutes after ketamine injection (minute ‘40’) compared to approximately 9 rotations twenty minutes after saline injection (minute ‘40’)), suggesting acute ketamine elicits psychomotor agitation, a symptom of psychosis, and that hippocampal GluRhol expression does not change this phenotype (p ⁇ 0.05 for ketamine groups compared to saline groups from minute ‘25’ to minute ‘50’ in a two-way repeated measures ANOVA with Tukey’s post-hoc tests).
  • SEQ ID NO:4 Short-Hc-Rho1-E145G-P295G protein sequence.
  • SEQ ID NO:5 Homo sapiens GABA-Rho1 subunit protein sequence.
  • SEQ ID NO:6 Homo sapiens GABA-Rho1 transmembrane portion protein sequence.
  • SEQ ID NO:7 GABA-Rho1-P295G transmembrane portion protein sequence.
  • SEQ ID NO:8 Homo sapiens GlyR a1 isoform 1 subunit protein sequence.
  • SEQ ID NO:9 Homo sapiens GlyR a1 isoform 1 transmembrane portion protein sequence.
  • SEQ ID NO:10 Homo sapiens GlyR a1 isoform 2 subunit protein sequence.
  • SEQ ID N0:11 Homo sapiens GlyR a1 isoform 2 transmembrane portion protein sequence.
  • eGFP Enhanced green fluorescent protein
  • SEQ ID NQ 20 Canonical Haemonchus contortus GluCI glutamate binding portion protein sequence.
  • SEQ ID NO:27 Homo sapiens GABA-Rho1 subunit nucleic acid sequence.
  • SEQ ID NO:28 Homo sapiens GABA-Rho1 transmembrane portion nucleic acid sequence.
  • SEQ ID NO:29 GABA-Rho1-P295G transmembrane portion nucleic acid sequence.
  • SEQ ID NQ:30 Homo sapiens GlyR a1 isoform 1 subunit nucleic acid sequence.
  • SEQ ID N0:31 Homo sapiens GlyR a1 isoform 1 transmembrane portion nucleic acid sequence.
  • SEQ ID NO:32 Homo sapiens GlyR a1 isoform 2 subunit nucleic acid sequence.
  • SEQ ID NO:33 Homo sapiens GlyR a1 isoform 2 transmembrane portion nucleic acid sequence.
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced green fluorescent protein
  • SEQ ID NO:38 Hemagglutinin (HA) nucleic acid sequence.
  • SEQ ID NO:39 GABA-Rho1-P295G-395HA transmembrane portion nucleic acid sequence.
  • SEQ ID NO:42 Short Haemonchus contortus GluCI E145G subunit nucleic acid sequence.
  • SEQ ID NO:45 Short-Hc-Rho1-E145G-P295G-395HA nucleic acid sequence.
  • SEQ ID NO:46 Homo sapiens CAMKII promoter nucleic acid sequence.
  • SEQ ID NO:47 AAV2 ITR #1 nucleic acid sequence.
  • SEQ ID NO:48 AAV2 ITR #2 nucleic acid sequence.
  • SEQ ID NO:49 Kozak nucleic acid sequence.
  • SEQ ID NO:54 Origin of replication nucleic acid sequence.
  • SEQ ID NO:57 Short-Hc-Rho1-E145G-P295G-395HA in AAV vector nucleic acid sequence.
  • SEQ ID NO:66 GSG linker protein sequence.
  • SEQ ID NO:67 GSG linker nucleic acid sequence.
  • SEQ ID NO:68 IRES nucleic acid sequence.
  • SEQ ID NO:69 Hexahistidine tag protein sequence.
  • the invention provides a chimeric fusion protein comprising: (i) a transmembrane domain and (ii) a glutamate binding domain, in which the transmembrane domain and the glutamate binding domain are heterologous to one-another.
  • chimeric fusion protein refers to a fusion protein comprising two or more domains derived from different species (for example, the chimeric fusion protein may comprise one domain from Homo sapiens and one domain from Haemonchus contortus). That is, chimeric fusion proteins disclosed herein comprise one or more domains that are heterologous to one another (i.e., are derived from different species).
  • transmembrane domain refers to the membrane-spanning region of a chimeric fusion protein disclosed herein.
  • transmembrane portion refers to the membrane-spanning region of an integral membrane protein (e.g., the transmembrane spanning region of a Homo sapiens chloride channel subunit, or a variant thereof).
  • the transmembrane region of a protein is readily identifiable by persons skilled in the art, for example via its density of hydrophobic (nonpolar) amino acid residues and/or via its membrane-spanning alpha helix or beta-barrel structures.
  • Chimeric fusion proteins of the invention may comprise a transmembrane domain derived from the transmembrane portion of an ion channel subunit.
  • the transmembrane domain may comprise the transmembrane portion of an ion channel subunit, or a variant thereof.
  • the transmembrane domain is derived from the transmembrane portion of a Homo sapiens ion channel subunit, or the transmembrane portion of an ion channel subunit of non-human origin.
  • the transmembrane domain may comprise the transmembrane portion of a Homo sapiens ion channel subunit or a variant thereof; or the transmembrane portion of an ion channel subunit of non- human origin, or a variant thereof.
  • the transmembrane domain is derived from the transmembrane portion of a ligand-gated anion channel subunit (e.g., a chloride channel subunit), such as the subunit of a glycine receptor (GlyR) or the subunit of a gamma-aminobutyric acid (GABA) receptor.
  • a ligand-gated anion channel subunit e.g., a chloride channel subunit
  • GlyR glycine receptor
  • GABA gamma-aminobutyric acid
  • the transmembrane domain may comprise the transmembrane portion of a ligand-gated anion channel subunit (e.g., a chloride channel subunit) or a variant thereof; such as the transmembrane portion of a glycine receptor (GlyR) subunit or a variant thereof; or the transmembrane portion of a gamma-aminobutyric acid (GABA) receptor subunit, or a variant thereof.
  • GABA receptors are GABA-responsive chloride channels which function as inhibitory receptors in the CNS.
  • the transmembrane domain may comprise the transmembrane portion a human ligand-gated anion channel subunit, or the subunit of a non-human ortholog thereof (e.g., Mus and Rattus rattus orthologs, or orthologs derived from rodent, canine, feline, equine, primate, simian, a monkey, or ape species), or a variant thereof.
  • a non-human ortholog thereof e.g., Mus and Rattus rattus orthologs, or orthologs derived from rodent, canine, feline, equine, primate, simian, a monkey, or ape species
  • the transmembrane domain is derived from the transmembrane portion of a GABAA or GABAc channel subunit (e.g., GABA-Rho1).
  • the transmembrane domain may comprise the transmembrane portion of a GABAA or GABAc channel subunit (e.g., GABA-Rho1), or a variant thereof.
  • GABAA and GABAc channels are involved in fast synaptic inhibition.
  • GABA-Rho1 js one of three isoforms of GABAc channel subunits, which are abundantly expressed in the retina and structurally homologous to GABAA receptors, but are able to assemble as homopentamers.
  • the transmembrane domain is derived from the transmembrane portion of a GABAA channel alpha (a1 , a2, a3, a4, a5, a6), beta (p1 , p2, p3), gamma (y1 , y2, y3), delta (5), epsilon (e), pi (IT) or theta (0) subunit, or from a GABAc channel rho (p1 , p2, p3) subunit.
  • the transmembrane domain may comprise the transmembrane portion of a GABAA channel alpha (a1 , a2, a3, a4, a5, a6), beta (p1 , p2, p3), gamma (y1 , y2, y3), delta (5), epsilon (e), pi (IT) or theta (0) subunit, or of a GABAc channel rho (p1 , p2, p3) subunit, or a variant thereof.
  • Transmembrane domains derived from the transmembrane portions of GABAc rho (p1 , p2, p3), GABAA beta (p1 , p2, p3) and glycine receptor alpha subunits (GlyRal , GlyRa2, GlyRa3) are preferable because these channels are known to form functional homopentamers.
  • the transmembrane domain is derived from the transmembrane portion of a human GABA-Rho1 subunit.
  • the amino acid sequence of the human GABA-Rho1 subunit is provided herein as SEQ ID NO:5.
  • the transmembrane domain may comprise the transmembrane portion of a Homo sapiens GABA-Rho1 subunit, or a sequence variant or derivative thereof. Suitable sequence variants or derivatives include those in which one or more amino acid residues in the native sequence are converted by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, and thus become different from the native sequence.
  • Transmembrane domains comprising truncated variants of the transmembrane portion of the human GABA-Rho1 subunit are explicitly envisaged.
  • the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.
  • the transmembrane domain may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.
  • the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:6.
  • the transmembrane domain may comprise a substitution at position P295 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1) in which proline (P, Pro) is substituted with an amino acid that is conservative with glycine (G, Gly).
  • the transmembrane domain may comprise a proline (P, Pro) cysteine (C, Cys) or proline (P, Pro) selenocysteine (U, Sec) substitution at position P295 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1).
  • the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.
  • the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence of the GlyR a1 isoform 2 subunit set forth in SEQ ID NO:11 .
  • the transmembrane domain may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11 .
  • the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:11
  • Sequence alignment and the calculation of percentage amino acid sequence identity is commonplace in the art, and forms part of the routine activity of persons skilled in the art.
  • percentage sequence identity is discussed in reference to amino acids it is recognised that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”.
  • Chimeric fusion protein peptide tags; linkers
  • the chimeric fusion protein further comprises a peptide tag.
  • the tag comprises the amino acid sequence set forth in SEQ ID NO:13.
  • Suitable alternative epitope tags may also include FLAG®-tag.
  • the peptide tag comprises a fluorescent or bioluminescent protein.
  • Suitable fluorescent proteins include green fluorescent protein (GFP) and derivatives thereof, such as mGreenLantern; mNeonGreen; DsRed, and derivatives thereof; such as mCherry and tdTomato; flavin mononucleotide-binding fluorescent protein (FbFP) and derivatives thereof; small ultra-red fluorescent protein (smURFP); and derivatives thereof; mRuby and derivatives thereof, TagRFP and derivatives thereof; and synthetic fluorescent proteins such as mScarlet and derivatives thereof.
  • Bioluminescent proteins are well-known in the art and include firefly luciferase, such as P.
  • the transmembrane domain comprises a peptide tag.
  • the peptide tag may be contiguous with (the N-terminus or the C-terminus of) the transmembrane domain.
  • the peptide tag may interrupt the transmembrane domain.
  • the amino acid sequence of the peptide tag may be embedded within the transmembrane domain amino acid sequence (e.g., at a position between the N-terminus and C-terminus of the transmembrane domain, such as at a position following a particular transmembrane-spanning sequence).
  • the peptide tag may interrupt the transmembrane domain amino acid sequence at position 395 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the chimeric fusion protein comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22.
  • the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.
  • the glutamate binding domain comprises a peptide tag.
  • the peptide tag may be contiguous with (the N-terminus or the C-terminus of) the glutamate binding domain.
  • the peptide tag may interrupt the glutamate binding domain.
  • the amino acid sequence of the peptide tag may be embedded within the amino acid sequence of the glutamate binding domain (e.g., at a position between the N-terminus and C-terminus of the glutamate binding domain).
  • the amino acid sequence of the peptide linker is contiguous with the N-terminus or the C- terminus of the chimeric fusion protein.
  • the chimeric fusion protein further comprises a peptide linker.
  • Peptide linkers may increase the solubility of the chimeric fusion protein by introducing charged and polar amino acids, thereby preventing the formation of protein aggregates, and increasing the bioavailability of the chimeric fusion protein in live cells.
  • peptide linkers may improve the flexibility of the chimeric fusion protein.
  • a suitable peptide linker may comprise or consist of 1 to 60 amino acids, such as 1 to 5 amino acids, 5 to 10 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, 20 to 25 amino acids, 25 to 30 amino acids, 30 to 35 amino acids, 35 to 40 amino acids, 45 to 50 amino acids, 50 to 55 amino acids, or 55 to 60 amino acids.
  • the peptide linker consists of between 1 to 30 amino acids, for example 5 to 25 amino acids or 5 and 15 amino acids.
  • Peptide linkers may be positioned between one or more (or all) of: (i) the transmembrane domain, (ii) the glutamate binding domain, and (iii) the peptide tag.
  • the peptide tag may be joined to the peptide linker.
  • the peptide tag may be linked to the N-terminus or the C-terminus of the peptide linker, or may be embedded within the peptide linker (e.g., at a position between the N-terminus and C-terminus of the peptide linker sequence).
  • the peptide linker comprises a ‘GSG’ linker, as set forth in SEQ ID NO:66.
  • the peptide linker comprises a self-cleaving viral “2A” linker, for example, a T2A linker (SEQ ID NO:58), a P2A linker (SEQ ID NO:60), an E2A linker (SEQ ID NO:62) or an F2A linker (SEQ ID NO:64).
  • the self-cleaving viral “2A” linker may be preceded by a ‘GSG’ linker as set forth in SEQ ID NO:66.
  • glutamate binding domain refers to the extracellular protein region of a chimeric fusion protein disclosed herein.
  • glutamate binding portion refers to the extracellular protein region of a glutamate receptor, or a variant thereof. Suitable glutamate binding portions may be found in ionotropic glutamate receptors (i.e. , glutamate-sensitive ion channels and transporters) or metabotropic glutamate receptors (i.e., glutamate-sensitive G-protein coupled receptors, GPCRs).
  • ionotropic glutamate receptors i.e. , glutamate-sensitive ion channels and transporters
  • metabotropic glutamate receptors i.e., glutamate-sensitive G-protein coupled receptors, GPCRs.
  • the glutamate binding region of a glutamate receptor is readily identifiable by persons skilled in the art, for example via its conserved glutamate binding pocket.
  • the glutamate binding domain is derived from the glutamate binding portion of a GRIA1-4, GRK1-5, GRIN1 , GRIN2A-D, or GRIN3A-B ionotropic glutamate receptor, or the glutamate binding portion of a GRM1-7 metabotropic glutamate receptor.
  • the glutamate binding domain may comprise the glutamate binding portion of a GRIA1-4, GRK1-5, GRIN1 , GRIN2A-D, or GRIN3A-B ionotropic glutamate receptor, or a variant thereof; or the glutamate binding portion of a GRM1-7 metabotropic glutamate receptor, or a variant thereof.
  • the glutamate binding domain is derived from the glutamate binding portion of a nematode or arthropod GluCI subunit, as reviewed in O'Halloran DM, 2022. G3(Bethesda)12(2):438 (incorporated herein by reference).
  • the glutamate binding domain may comprise the glutamate binding portion of a nematode or arthropod GluCI subunit, or a variant thereof.
  • the glutamate binding domain is derived from the glutamate binding portion of a mollusc, flatworm, tick, mite, insect or crustacean paralogue of a nematode or arthropod GluCI subunit.
  • the glutamate binding domain may comprise the glutamate binding portion of a mollusc, flatworm, tick, mite, insect or a crustacean paralogue of a nematode / arthropod GluCI subunit, or a variant thereof.
  • the glutamate binding domain is derived from a bacterial glutamate binding protein, as reviewed in Marvin et al. 2013 Nature Methods 10(2):162-70 (incorporated herein by reference).
  • the the glutamate binding domain may comprise the glutamate binding portion of a bacterial glutamate binding protein, or a variant thereof.
  • the glutamate binding domain is derived from the glutamate binding portion of a Haemonchus contortus glutamate receptor.
  • the glutamate binding domain may comprise the glutamate binding portion of a Haemonchus contortus glutamate receptor, or a variant thereof.
  • the glutamate binding domain is derived from the glutamate binding portion of a glutamate-gated chloride channel (GluCI) subunit.
  • the glutamate binding domain may comprise the glutamate binding portion of a GluCI subunit, or a variant thereof.
  • the glutamate binding domain is derived from the glutamate binding portion of a Haemonchus contortus glutamate-gated chloride channel (GluCI) subunit.
  • the glutamate binding domain may comprise the glutamate binding portion of a Haemonchus contortus glutamate-gated chloride channel (GluCI) subunit, or a variant thereof.
  • the canonical amino acid sequence of the H is derived from the glutamate binding portion of a glutamate-gated chloride channel (GluCI) subunit.
  • the glutamate binding domain may comprise the glutamate binding portion of a Haemonchus contortus glutamate-gated chloride channel (GluCI) subunit, or a variant thereof.
  • contortus GluCI subunit is provided herein as SEQ ID NO:19.
  • Variants or derivatives of the glutamate binding portions disclosed herein are suitable for use as glutamate binding domains in chimeric fusion proteins of in the invention. Suitable variants and derivatives include those in which one or more amino acid residues in the native sequence of a glutamate binding portion are converted by deletion, insertion, nonconservative or conservative substitution, or a combination thereof, and thus become different from the native sequence. Truncated variants of the glutamate binding portions disclosed herein are explicitly envisaged.
  • the glutamate binding domain may comprise a truncated variant of the glutamate binding portion of a H. contortus GluCI subunit.
  • the glutamate binding domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:20.
  • the glutamate binding domain may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NQ:20.
  • the glutamate binding domain comprises or consists of the glutamate binding domain set forth in SEQ ID NQ:20.
  • the glutamate binding domain is derived from the glutamate binding portion of a H. contortus GluCI subunit and comprises a single amino acid substitution at position E145, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .
  • the glutamate binding domain may comprise a glutamic acid (E, Glu) glycine (G, Gly) substitution at position E145 (E145G), as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .
  • the glutamate binding domain may comprise a substitution at position E145 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1) in which glutamic acid (E, Glu) is substituted with an amino acid that is conservative with glycine (G, Gly).
  • the glutamate binding domain may comprise a glutamic acid (E, Glu)-> cysteine (C, Cys) or glutamic acid (E, Glu)-> selenocysteine (U, Sec) substitution at position E145 (as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1).
  • the glutamate binding domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:16.
  • the glutamate binding domain comprises a truncated variant of any of the glutamate binding portions disclosed herein.
  • the glutamate binding domain may be a glutamate binding portion disclosed herein that is truncated by between 1 and 5; 1 and 10; 1 and 15; 1 and 20; 1 and 25 or 1 and 30 amino acids.
  • the glutamate binding domain may be a variant of any one of the glutamate binding portions disclosed herein that is truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the glutamate binding domain comprises a truncated variant of the H. contortus
  • the glutamate binding domain comprises a truncated variant of the H. contortus GluCI subunit glutamate binding portion having a single amino acid substitution at position E145, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 , as described herein.
  • the glutamate binding domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:18.
  • the chimeric fusion protein comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .
  • the chimeric fusion protein may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .
  • the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:21.
  • the chimeric fusion protein comprises a truncated variant of any of the chimeric fusion proteins disclosed herein.
  • a chimeric fusion protein may be truncated by between 1 and 5; 1 and 10; 1 and 15; 1 and 20; 1 and 25 or 1 and 30 amino acids.
  • the chimeric fusion protein may be a variant of any chimeric fusion protein disclosed herein that is truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the chimeric fusion protein comprises a truncated variant of the chimeric fusion protein set forth in SEQ ID NO:1 .
  • a variant of SEQ ID NO:1 that is truncated by between 1 and 5; 1 and 10; 1 and 15; 1 and 20; 1 and 25 or 1 and 30 amino acids.
  • the variant of SEQ ID NO:1 may be truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the chimeric fusion protein may comprise at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.
  • the chimeric fusion protein may comprise 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.
  • the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.
  • the chimeric fusion protein comprises or consist of the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:22.
  • the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.
  • the term “ion channel” refers to a multi-subunit protein having a transmembrane pore that facilitates the diffusion of one or more specific ions across a concentration gradient.
  • glutamate-gated chloride channel refers to a ligand-gated ion channel that is activated in the presence of glutamate, and that acts to facilitate the diffusion of chloride ions (Cl ) down their electrochemical gradient.
  • engineered GluCIs of the invention comprise two (2), three (3), four (4), five (5), six (6), seven (7) or more subunits.
  • one or both subunits may comprise a chimeric fusion protein as disclosed herein.
  • one, two or all subunits may comprise a chimeric fusion protein as disclosed herein.
  • one, two, three or all subunits may comprise a chimeric fusion protein as disclosed herein.
  • having five (5) subunits one, two, three, four or all subunits may comprise a chimeric fusion protein as disclosed herein.
  • one, two, three, four, five or all subunits may comprise a chimeric fusion protein as disclosed herein.
  • one, two, three, four, five, six, seven or all subunits may comprise a chimeric fusion protein as disclosed herein.
  • An engineered GluCI as disclosed herein may be heteromeric (comprising different subunits) or homomeric (comprising identical subunits).
  • the engineered GluCI is homopentameric (i.e., comprising 5 subunits), each subunit comprising the same chimeric fusion protein as disclosed herein.
  • the engineered GluCI exhibits a half-maximal effective concentration (EC50) for glutamate of between 1 and 100 ⁇ M.
  • the engineered GluCI may exhibit an EC50 for glutamate of between 1 and 90 ⁇ M, 1 and 80 ⁇ M, 1 and 70 ⁇ M, 1 and 60 ⁇ M, 1 and 50 ⁇ M, 1 and 40 ⁇ M, 1 and 30 ⁇ M, 1 and 20 ⁇ M or 1 and 10 ⁇ M.
  • the engineered GluCI may exhibit an EC50 for glutamate of between 5 and 100 ⁇ M, 5 and 90 ⁇ M, 5 and 80 ⁇ M, 5 and 70 ⁇ M, 5 and 60 ⁇ M, 5 and 50 ⁇ M, 5 and 40 ⁇ M, 5 and 30 ⁇ M, 5 and 20 ⁇ M or 5 and 10 ⁇ M.
  • the engineered GluCI may exhibit an EC50 for glutamate of between 10 and 100 ⁇ M, 10 and 90 ⁇ M, 10 and 80 ⁇ M, 10 and 70 ⁇ M, 10 and 60 ⁇ M, 10 and 50 ⁇ M, 10 and 40 ⁇ M or 10 and 30 ⁇ M.
  • the engineered GluCI may exhibit an EC50 for glutamate of between 5 and 50 ⁇ M.
  • the engineered GluCI may exhibit an EC50 for glutamate of between 10 and 20 ⁇ M. That is, the engineered GluCI may exhibit an EC50 for glutamate of approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 ⁇ M.
  • Table 1 (below) describes typical glutamate concentrations, as measured in rats and humans.
  • One aspect of the invention provides a nucleic acid encoding a chimeric fusion protein as disclosed herein.
  • the nucleic acid molecule may comprise DNA and/or RNA and may be partially or wholly synthetic. Reference to nucleic acids herein encompasses both DNA molecules with the specified sequence, and RNA molecules with the specified sequence in which U is substituted for T, unless context requires otherwise.
  • the nucleic acid may be codon optimised for expression in a mammalian cell, preferably a human cell. In some embodiments, the cell is a neuronal cell.
  • a hippocampal neuronal cell For example, a hippocampal neuronal cell, a hypothalamic neuronal cell, a thalamic neuronal cell, a basal ganglia neuronal cell, an amygdala neuronal cell or a cortical neuronal cell (e.g., a neuronal cell from the frontal cortex, temporal cortex, or olfactory cortex).
  • the cell may be a CA1 , CA2 or CA3 pyramidal cell.
  • the cell may be an inhibitory interneuron cell.
  • the cell may be a primary cell, isolated from a mammalian (e.g., a human) subject by in vivo harvesting (e.g., biopsy).
  • the cell is be an animal or a human cell line cell.
  • a HEK 293 human embryonic kidney
  • a CHO Choinese hamster ovary
  • the cell is a human cell.
  • the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23.
  • the nucleic acid may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:23.
  • the nucleic acid comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:23.
  • the nucleic acid may comprise or consist of the nucleic acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:44.
  • the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:25.
  • the nucleic acid may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:25.
  • the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO:25.
  • the nucleic acid may comprise or consist of the nucleic acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:45.
  • the nucleic acid comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:45.
  • the nucleic acid encoding the chimeric fusion protein may comprise specific sequence modifications to the 3’ terminus.
  • the nucleic acid may encode an additional Vai residue or an additional Met residue prior to the coding sequence. This may for example eliminate or substantially reduce translation from internal start sites that would lead to the production of truncated fusion proteins.
  • the nucleic acid comprises an IRES (internal ribosome entry site) sequence.
  • IRES internal ribosome entry site
  • a suitable IRES sequence is provided as SEQ ID NO:68.
  • Alternative suitable IRES sequences may differ from SEQ ID NO:68 by comprising a sequence of additional nucleotides at the 3’ end or the 5’ end of SEQ ID NO:68.
  • Alternative suitable IRES sequences may be variants or derivatives of SEQ ID NO:68 in which one or more nucleic acid residues are converted by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • suitable IRES sequences may comprise an adenine (a) guanine (g) substitution at position 474 as numbered with reference to SEQ ID NO:68, and/or a guanine (g) adenine (a) substitution at position 555 as numbered with reference to SEQ ID NO:68.
  • a particular nucleotide sequence variant may differ from any of the reference sequences shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or by 10 or more nucleotides. Due to the degeneracy of the genetic code, it is clear to persons skilled in the art that any nucleic acid sequence herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • percentage nucleic acid sequence identity refers to the percentage of nucleotides in a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins.
  • percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand.
  • the “percentage identity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent identity of the two sequences.
  • Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G-C, A-T, and A-U, through hydrogen binding.
  • the expression vector may be an adeno-associated virus (AAV) vector, such as an AAV vector selected from the group consisting of: rAAV2/1 , rAAV2, rAAV2/3, rAAV2/5, rAAV2/6, rAAV2/7, rAAV2/8, rAAV2/9 , AAVrh, AAVDJ, AAVDJ/8, AAVPhP.eB, AAVPhPS, and AAV2-retro.
  • AAV vector is an rAAV2/9 vector.
  • the expression vector may be a lentiviral vector, a retroviral vector, or an adenoviral vector.
  • the expression vector comprises a woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence optimised to limit any potential oncogenic activity.
  • WPRE woodchuck hepatitis virus
  • WPRE sequences comprises the nucleic acid sequence set forth in SEQ ID NQ:50.
  • the expression vector comprises a human growth hormone polyadenylation signal (hGHpA) sequence.
  • hGHpA human growth hormone polyadenylation signal
  • a hGHpA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:51.
  • the expression vector comprises an F1 origin of replication.
  • an F1 origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:52.
  • the expression vector comprises a neomycin or kanamycin resistance gene (NeoR/KanR) sequence.
  • a NeoR/KanR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:53.
  • the expression vector comprises an origin of replication sequence.
  • an origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:54.
  • the expression vector comprises one or more non-coding sequences.
  • the expression vector may comprise (in addition to a nucleic acid as disclosed herein, encoding a chimeric fusion protein as disclosed herein) one or more AAV2 ITR sequences, Kozak sequences, WPRE sequences, hGHpA sequences, F1 origin of replication sequences, NeoR/KanR sequences, origin of replication sequences or non-coding sequences, in any combination.
  • the expression vector comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:55.
  • the expression vector may comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:55.
  • the expression vector comprises or consists of the nucleic acid sequence set forth in SEQ ID NO:55.
  • the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:56 or SEQ ID NO:57.
  • the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:57.
  • the recombinant viral particle is a recombinant adeno-associated virus (AAV) particle.
  • AAV adeno-associated virus
  • a virus particle selected from the group consisting of: rAAV2/1 , rAAV2, rAAV2/3, rAAV2/5, rAAV2/6, rAAV2/7, rAAV2/8, rAAV2/9 , AAVrh, AAVDJ, AAVDJ/8, AAVPhP.eB, AAVPhPS, and AAV2-retro.
  • the recombinant viral particle is a rAAV2/9 virus particle.
  • the recombinant viral particle exhibits a trophism for neuronal cells.
  • the invention also provides an in vitro method of preparing a recombinant virus particle as disclosed herein, the method comprising: transducing a cell with an expression vector as disclosed herein; expressing the viral packaging and/or envelope proteins necessary for the formation of a recombinant virus particle in the cell; and culturing the cell in a culture medium, such that the cell produces the recombinant virus particle.
  • the method may further comprise transducing the cell with one or more additional expression vectors that encode the viral packaging and/or envelope proteins necessary for formation of the recombinant virus particle.
  • the method may further comprise recovering recombinant virus particles from the cell culture medium and/or concentrating the recombinant virus particles.
  • suitable viral packaging and/or envelope proteins and expression vectors encoding those proteins are commercially available, and are well known in the art.
  • the viral packaging and/or envelope proteins may include AAV Rep proteins Rep78, Rep68, Rep52 and Rep40; AAV capsid proteins VP1 , VP2 and VP3; and/or AAV accessory proteins MAAP & AAP.
  • the in vitro method comprises culturing the cell at 37°C in 5% CO2with saturating humidity, such that the cell produces the recombinant virus particle.
  • the cell is a neuronal cell.
  • a neuronal cell For example, a hippocampal neuronal cell, a hypothalamic neuronal cell, a thalamic neuronal cell, a basal ganglia neuronal cell, an amygdala neuronal cell or a cortical neuronal cell (e.g., a neuronal cell from the frontal cortex, temporal cortex, or olfactory cortex) .
  • the cell may be CA1 , CA2 or CA3 pyramidal cell, or an inhibitory interneuron cell.
  • the cell may be a primary cell, isolated from a mammalian (e.g., a human) subject by in vivo harvesting (e.g., biopsy).
  • the cell may be an animal or a human cell line cell.
  • a HEK 293 human embryonic kidney
  • a CHO Choinese hamster ovary
  • the cell is a human cell.
  • Methods of cell culture including methods of culturing neuronal cells, are commonplace in the art, and cell culture forms part of the routine activity of persons skilled in the art. Suitable cell culture media and supplements for use in such methods are commercially available and are known to persons skilled in the art.
  • the invention provides an in vitro method of expressing a chimeric fusion protein in a cell, the method comprising: (i) transfecting the cell with a nucleic acid as disclosed herein, the expression vector as disclosed herein; or the recombinant virus particle as disclosed herein, and culturing the cell in a culture medium, such that the cell expresses the chimeric fusion protein.
  • the in vitro method comprises culturing the cell at 37°C in 5% CO2with saturating humidity, such that the cell produces the chimeric fusion protein.
  • the engineered cell is a neuronal cell.
  • a hippocampal neuronal cell a hypothalamic neuronal cell, a thalamic neuronal cell, a basal ganglia neuronal cell, an amygdala neuronal cell or a cortical neuronal cell (e.g., a neuronal cell from the frontal cortex, temporal cortex, or olfactory cortex) .
  • the engineered cell may be CA1 , CA2 or CA3 pyramidal cell, or an inhibitory interneuron cell.
  • the engineered cell may be a primary cell, isolated from a mammalian (e.g., a human) subject by in vivo harvesting (e.g., biopsy).
  • the engineered cell may be an animal or a human cell line cell.
  • a HEK 293 human embryonic kidney
  • a CHO Choinese hamster ovary
  • the engineered cell is a human cell.
  • Methods of cell culture including methods of culturing neuronal cells, are commonplace in the art, and cell culture forms part of the routine activity of persons skilled in the art. Suitable cell culture media and supplements for use in such methods are commercially available and are known to persons skilled in the art.
  • the invention provides methods of treatment of a disease in a subject in need thereof.
  • a method of treating a disease in a subject in need thereof comprising: administering a chimeric fusion protein as disclosed herein to the subject;
  • the invention also provides:
  • a chimeric fusion protein as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the chimeric fusion protein to the subject;
  • an engineered GluCI as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the engineered GluCI to the subject;
  • nucleic acid as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the nucleic acid to the subject;
  • an expression vector as disclosed herein for use in a method of treating a disease in a subject comprising: administering the expression vector to the subject;
  • a recombinant virus particle as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the recombinant virus particle to the subject;
  • an engineered cell as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the engineered cell to the subject.
  • the invention further provides use of a chimeric fusion protein as disclosed herein; an engineered GluCI as disclosed herein; a nucleic acid as disclosed herein; an expression vector as disclosed herein, a recombinant virus particle as disclosed herein; or an engineered cell as disclosed herein in the preparation of a medicament for the treatment of a disease in a subject in need thereof.
  • the disease is a seizure disorder.
  • the disease is an epilepsy.
  • Epilepsies are characterised by the onset of recurrent, unprovoked seizures. Symptoms of seizures include confusion, visual disturbance, muscle contraction, uncontrollable movement of the arms and legs, loss of consciousness or awareness, and/or psychological symptoms (such as fear or anxiety).
  • the epilepsy may involve absence seizures, atonic seizures, atypical absence seizures, clonic seizures, epileptic or infantile spasms, secondary generalised seizures (focal bilateral tonic-clonic seizures), simple partial seizures (focal onset seizures with awareness), complex partial seizures (focal onset seizures with impaired awareness), gelastic or dacrystic seizures, myoclonic seizures, tonic-clonic seizures and/or tonic seizures.
  • the epilepsy involves seizures that are refractory to treatment using conventional anti-seizure agents and/or that cannot be treated by surgical intervention.
  • the epilepsy is a focal epilepsy, also termed “partial-onset” epilepsy.
  • the disease is an epilepsy-related neurological disorder.
  • an epilepsy-related neurological disorder characterised by abnormal excessive neuronal activity and/or abnormal neuronal circuit excitability.
  • the epilepsy-related neurological disorder is a neuropsychiatric comorbidity of an epilepsy, for example an attention-deficit/hyperactivity disorder, a cognitive impairment, a memory and learning deficit, an autism spectrum disorder, a schizophrenia, a depression or an anxiety disorder (such as agoraphobia, selective mutism, generalized anxiety disorder (GAD), social anxiety disorder, obsessive-compulsive disorder (OCD) and panic disorder).
  • GAD generalized anxiety disorder
  • OCD obsessive-compulsive disorder
  • the disease is a non-epilepsy-related neurological disorder characterised by pathological neuronal overactivity.
  • the disease may be Parkinson’s disease, primary cephalalgias such as cluster headache and migraine, and other pain conditions such as trigeminal neuralgia, post-herpetic neuralgia and radicular pain.
  • the disease is a non-epilepsy-related neuropsychiatric disorder characterised by pathological neuronal overactivity.
  • the disease may be an attention-deficit/hyperactivity disorder, a cognitive impairment, a memory and learning deficit, an autism spectrum disorder, a schizophrenia, a depression or an anxiety disorder (such as agoraphobia, selective mutism, generalized anxiety disorder (GAD), social anxiety disorder, obsessive-compulsive disorder (OCD) and panic disorder).
  • GAD generalized anxiety disorder
  • OCD obsessive-compulsive disorder
  • pathological neuronal overactivity refers to a disorder that is characterised by excessive, abnormal or dysregulated neuronal activity that goes beyond normal neuronal firing patterns and may result from dysfunctions in neuronal circuits, for example, caused by neurotransmitter imbalances or structural abnormalities.
  • Pathological neuronal overactivity is often associated with neurological and/or neuropsychiatric disorders as described herein including e.g. schizophrenia.
  • Suitable neuroimaging techniques include electroencephalography (EEG), magnetoencephalography (MEG), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), computerised tomography (CT) and functional near-infrared spectroscopy (fNIRS).
  • EEG electroencephalography
  • MEG magnetoencephalography
  • fMRI functional magnetic resonance imaging
  • PET positron emission tomography
  • CT computerised tomography
  • fNIRS functional near-infrared spectroscopy
  • Behavioural, neurological and/or psychiatric symptoms may also provide indirect evidence of pathological neuronal overactivity.
  • Suitable criteria for the diagnosis of epilepsies and their comorbidities are well known in the art (see for example Diagnostic and Statistical Manual of Mental Disorders, 5 th Edition, American Psychiatric Association 2013, Virginia USA).
  • the chimeric fusion proteins; engineered GluCIs; nucleic acids; expression vectors, recombinant virus particles or engineered cells as disclosed herein may be administered to a subject in a variety of ways, such as via direct injection to the brain, brainstem or spinal cord (stereotactic injection).
  • administration may involve direct injection to the cerebral cortex, in particular the neocortex of a subject, or direct injection to the hippocampus of a subject.
  • the administration may involve direct injection to a location in the brain believed to be functionally associated with an epilepsy or an epilepsy-related neurological disorder.
  • the treatment is for epilepsy
  • this may involve direct injection of the viral particles into the cortex or the hippocampus.
  • the administration may involve intrathecal or intracisternal injection.
  • the administration may also involve administration by convection-enhanced delivery.
  • the invention may be employed to treat multiple epileptic foci in a single subject simultaneously, by injection directly into the multiple identified loci.
  • the subject may be one who has been diagnosed with epilepsy, or one who exhibits drug-resistant or refractory epilepsy (i.e. , an epilepsy that continues despite the adequate administration of conventional anti-epileptic treatment).
  • the subject may be one who has been diagnosed as having focal epilepsy affecting a single area of the brain.
  • Focal epilepsies may arise, for example, from developmental abnormalities or following strokes, tumours, penetrating brain injuries or infections.
  • the recipient subject may exhibit a reduction in symptoms of the disease or disorder being treated. For example, for the subject may exhibit a reduction in the number of epileptic seizures, or a shortening of seizure duration.
  • the term "treatment,” describes the treatment or therapy of a mammalian subject, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition to be treated. This includes a reduction in the rate of progress, a halt in the rate of progress, a regression of the condition, an amelioration of the condition, and/or cure of the condition.
  • the mammalian subject may be a human patient, diagnosed with an epilepsy or an epilepsy- related neurological disorder, or suspected of having an epilepsy or an epilepsy-related neurological disorder. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included.
  • the chimeric fusion proteins; engineered GluCIs; nucleic acids; expression vectors, recombinant virus particles or engineered cells as disclosed herein are to be delivered in a therapeutically effective amount.
  • the term “therapeutically-effective amount” describes the amount of therapeutic agent to be administered that is effective for producing a desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • the term “prophylactically effective amount” describes the amount of therapeutic agent to be administered that is effective for producing a desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • the term “prophylaxis” in context of the present invention should not be understood to circumscribe complete success i.e. complete protection or complete prevention. Rather prophylaxis in the present context refers to a measure which is administered in advance of detection of a symptomatic condition with the aim of preserving health by helping to delay, mitigate or avoid the onset or pathophysiological progression of a particular condition.
  • the unit dose may be calculated in terms of the dose of virus particles being administered.
  • Viral doses may include a particular number of virus particles, virus genomes (vg) or plaque forming units (pfu).
  • exemplary unit doses include 10 3 , 10 4 , 10 5 , 10 s , 10 7 , 10 s , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 vg.
  • therapeutic agents of the present invention may be combined with other therapeutic agents, whether symptomatic or disease modifying (i.e., as combination therapies).
  • second/co-therapeutic agents are known to those skilled in the art and would be immediately recognisable on the basis of the disclosure herein.
  • the second/co-therapeutic agent may be any known agent in the art that is believed may give therapeutic effect in treating the diseases described herein, subject to the diagnosis of the individual being treated.
  • an epilepsy may be ameliorated by directly treating the underlying etiology, but also by way of administering conventional anti-seizure agents such as an anti-seizure agent selected from the group consisting of Acetazolamide, Brivaracetam, Cannabidiol, Carbamazepine, Cenobamate, Clobazam, Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Fenfluramine, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital Phenytoin, Piracetam, Pregabalin, Primidone, Rufinamide, Valproate, Stiripentol, Tiagabine, Topiramate, Valproic acid, Vigabatrin and Zonisamide.
  • an anti-seizure agent selected from the group consisting of Acetazolamide, Brivaracetam, Can
  • the invention includes any combination of the aspects and embodiments disclosed herein, except where such a combination is clearly impermissible or expressly avoided.
  • GluCIs glutamate-gated chloride channels
  • H. contortus GluCI (Hc-GluCI) variants were subsequently screened for increases in glutamate sensitivity and chloride ion current density (see Figure 2 legend).
  • a first chimeric chloride channel comprising the E145G variant H. contortus GluCI glutamate binding domain and a transmembrane domain derived from the a1 subunit of the human glycine receptor (GlyRal) (termed Hc-GlyRa1); and a second chimeric chloride channel comprising the E145G H. contortus GluCI glutamate binding domain and a transmembrane domain derived the Rho1 subunit of the human GABAc channel (termed Hc-Rho1) were subsequently generated. Both channels were assessed for their suitability in chemogenetic applications (see legends to Figures 3 and 4).
  • Hc-Rho1-E145G variants L266F, P295F, P295G and T306A were subsequently generated and screened for improvements in glutamate sensitivity and current density (see legends to Figures 5 and 6).
  • the P295G variant was further engineered for attachment of an eGFP or hemagglutinin (HA) peptide tag (see Figure 8 legend) and for shortening of the N-terminus of the protein (see Figure 9 legend).
  • the resulting fusion protein was termed “GluRhol” (Short-Hc-Rho1-E145G-P295G-385HA).
  • mice were administered GluRhol or Y186A GluRhol recombinant viral particles by intra-hippocampal stereotactic injection. Learning and memory behaviours were assessed at baseline (see Figure 14 legend).
  • mice were subsequently subject to an acute model of epilepsy.
  • IAK intra-amygdala kainate
  • TLE drug-resistant temporal lobe epilepsy
  • Epilepsy comorbidities including anxiety and memory were assessed in epileptic mice before and after injection with Y186A or GluRhol , and also compared to behaviours observed in naive mice (see legends of Figures 21 to 25).
  • engineered GluCIs are capable of operating with minimal desensitization and without constitutive activity.
  • the data describe the successful delivery of an AAV vector encoding an exemplary engineered GluCI (GluRhol) to the murine hippocampus, and demonstrate the anti-epileptic therapeutic efficacy of an engineered GluCI (GluRhol) in two in vivo models of epilepsy.
  • GluRhol did not affect the normal anxiety/learning/memory behaviour of non-epileptic test animals and successfully treated anxiety/learning/memory epilepsy comorbidities in epileptic test animals.
  • GluRhol was found to protect test animals from developing symptoms of schizophrenia.
  • Glutamate, GABA and PTZ were dissolved directly into the extracellular solution, whereas PTX and emamectin were first dissolved in DMSO.
  • Currents were amplified with Axopatch-1 D (Axon Instruments), recorded with WinEDR, analyzed in Excel and representative current traces were extracted in pCLAMP (Molecular Devices). Dose-response curves were drawn with a variable slope and ordinary fit in GraphPad Prism based on the % of maximum current amplitude achieved at a range of glutamate concentrations for a number of recorded cells.
  • the current density is, in all cases, the maximum current density achieved across a range of glutamate concentrations for a range of cells, and is determined by dividing the maximum current amplitude (pA) with the cell capacitance (pF). Basal activity was quantified by dividing the amplitude of the PTX- sensitive leak current (pA) with the cell capacitance (pF) or the maximum current recorded for any particular cell over a range of GABA or glutamate concentrations (pA). Error bars show the standard error of the mean (SEM).
  • HEK-T cells were transfected with transfer plasmid (pAAV-GluRho1 or pAAV-Y186A), miniDG9 capsidreplication plasmid and HGTI helper plasmid (Streck, et al. 2006 Cancer Gene Ther, 13(1), 99-106) using polyethyleneimine MAX (Generon, 24765). After 3-5 days of incubation (37°C, 5% CO2), the preparation was harvested and separated by centrifugation. The media fraction was incubated with ammonium sulfate (1-24 hours) and subsequently spun down.
  • transfer plasmid pAAV-GluRho1 or pAAV-Y186A
  • miniDG9 capsidreplication plasmid miniDG9 capsidreplication plasmid
  • HGTI helper plasmid HGTI helper plasmid
  • the 40% layer underwent buffer exchange to PBS containing 1 mM MgCh and 0.001% pluronic F68 (ThermoFisher, 24040032) using a vivaspin concentrator (Sartorius, VS2041).
  • the protein contents of the AAV vector preparations were assayed using SDS-PAGE and a SYPRO Ruby stain (ThermoFisher).
  • Vector titration was performed by qPCR on a QuantStudio system (Applied Biosystems) using SYBR green (Bio-Rad, 1725120) and AAV2 ITR primers (Aurnhammer et al. 2012 , Hum Gene Ther Methods, 23(1), 18-28).
  • mice 9-week-old male C57BL/6 mice (Charles River UK) received AAV9 injections in the ventral hippocampi.
  • mice were sedated with isoflurane, placed in a stereotaxic frame, had iodopovidone applied to the head and then received a subcutaneous injection of buprenorphine, metacam and saline.
  • An incision was made on the head and holes drilled at the site for injection (in mm: AP -3, ML -3 and 3).
  • 500 nL of 2 10 12 vg/mL AAV9 was injected at 100 nl/min per depth (in mm: DV -3.5, -3 and -2.5).
  • the skin was closed with simple interrupted sutures and lidocaine applied to the wound. Mice were earmarked and allowed to recover in a heat box at 37°C for 5 minutes before being moved back into their homecage.
  • mice were co-housed and handled multiple times per week. Before every behavioral test, mice were acclimatized to the experimental room for half an hour. In the open field test (OFT), mice were placed in a white arena (30x30x30 cm) for 10 minutes (one mouse per experiment) under dim light. The open field test was recorded with a USB camera and the arena was cleaned with 70% ethanol in-between trials. Any-maze software was used to detect the movement of mice in the periphery (9 cm) and center (12 cm) of the arena. In the novel object recognition (NOR) task, mice were habituated to the OFT arena three times (OFT, 5 minutes at 3-hour timepoint and 5 minutes at 24-hour timepoint).
  • OFT open field test
  • NOR novel object recognition
  • mice were placed in the arena containing two identical objects for 10 minutes (Lego towers or glass flasks filled with agarose). Each object was placed in adjacent corners 5 cm away from the walls.
  • mice were placed back into the arena now containing one familiar and one novel object (one Lego tower and one glass flask with agarose).
  • the arena was cleaned with 70% ethanol in-between trials.
  • mice were placed in an arena containing a brightly lid light chamber (32x25x32 cm) with a 7 cm wide door leading to a covered dark chamber (16x25x25 cm). To start the test, mice were placed into the light chamber facing the door and then allowed 10 minutes to explore the arena. The number of entries into the light chamber was counted (defined as moving all four paws from the dark chamber into the light chamber), as well as the time spent in the light chamber (determined with a stopwatch). The arena was cleaned with 70% ethanol in-between trials.
  • mice were placed at the distal end of a “T” shaped arena facing away from the track.
  • the long track was 50x1 1x25 cm, whilst the short arms were 20x11x25 cm each (LxWxH).
  • the mouse was allowed to move through the T-maze until it first entered one of the short arms.
  • a transparent plastic insert was lowered to trap the mouse in the chosen arm for 30 seconds, which marked the end of the trial. 10 consecutive trials were performed, and the spontaneous alternation rate was counted based on how many times the mouse chose to go into the other arm, as opposed to the arm it had visited in the previous trial.
  • the arena was cleaned with water in-between trials.
  • mice were given an IP injection of 50 mg/kg pentylenetetrazol (PTZ) and placed in an empty cage for observation.
  • PTZ pentylenetetrazol
  • the onset and progression of seizures were monitored by a researcher blinded to the treatment groups for 30 minutes using a modified Racine scale (Van Erum et al. 2019 Epilepsy Behav, 95, 51-55).
  • mice were sacrificed using transcardiac perfusion to harvest the brains for immunostaining analysis. All graphs and accompanying statistics were created/computed in GraphPad Prism. Error bars show the standard error of the mean (SEM).
  • Brain slices were permeabilized in 0.3% triton in PBS (PBST) for 30 minutes, blocked in 0.3% PBST with 8% normal goat serum (NGS) for 1 hour and then incubated in 0.2% PBST with 4% NGS and 1 :1000 dilutions of primary antibodies anti-HA.11 (BioLegend, 901501) and antiGABA (Sigma, A2052) at 4°C shaking overnight.
  • PBST triton in PBS
  • NGS normal goat serum
  • the chronic IAK model of drug-resistant TLE was initiated with a surgical injection of 200 nL 7.15 mM synthetic kainate (Bio-Techne, 7065/10) in the right basolateral amygdala (in mm: AP -1 , ML 2.85, and DV in mm from dura: -3.75) in 9-week-old male C57BL/6 mice (Charles River UK). Kainate was injected at a rate of 200 nL/min, and the needle withdrawn 2 minutes later. The head wound was closed using a combination of sutures and fast-acting cyanoacrylate adhesive to reduce the surgery time.
  • mice After 5 minutes in a heated chamber (37°C), mice were moved to a clean cage only containing bedding and acute seizures scored using the 5-stage Racine scale. 40 minutes after kainate injection, mice were given diazepam by I.P. (0.05 mL of a 5 mg/ml stock) to terminate seizure activity. Unlike for AAV9 injections, Metacam is given at the end of the IAK surgery protocol. One week later, mice were singlehoused and floor-fed. Another week later, epileptic mice were selected to undergo transmitter implantation based on observed signs of epilepsy: hyperactivity, avoidance behaviour, prolonged periods of freezing, ear trembling, forelimb clonus, tonic-clonic seizures, poor nesting and poor cage hygiene.
  • mice were prepared for surgery as previously described and burr holes drilled above the ventral hippocampi (in mm: AP -3, ML -3 and 3). An incision was made in the skin on the back and a subcutaneous ECoG transmitter (Open Source Instruments Inc., single channel 256 Hz A3048S2-AA- C45-D) inserted. A short recording electrode was inserted into the right burr hole and secured in place using Medbond glue. A reference electrode was inserted into the left burr hole.
  • ECoG transmitter Open Source Instruments Inc., single channel 256 Hz A3048S2-AA- C45-D
  • Betamox was given to prevent infection (0.1 mL of a 150 mg/mL stock).
  • ECoG was sampled at 256 Hz and later reviewed in PyECog (www.pyecog.com/) by an experimenter blind to treatment group.
  • Generalized seizures were identified using an algorithm trained to recognize the classic electrographic patterns accompanying tonic-clonic seizures, which consisted of high-amplitude (>2x baseline), high-frequency (>5 Hz) polyspike events with a duration of at least 10 seconds and was mostly followed by a post-ictal depression (EEG suppression within 15 seconds of seizure cessation).
  • mice were injected with AAV9 in the ventral hippocampi through pre-implanted guide cannulas (DV: -10.56, -10.06, -9.56 relative to the top of the guide cannula).
  • 500 nL AAV9 was injected at each depth at 100 nL/min.
  • Epilepsy comorbidities were assessed in a chronic IAK model of drug-resistant TLE. Some mice received an IAK injection, whereas other mice received an intra-amygdala saline injection as a control. Epileptic mice and saline mice were single-housed one week after amygdala injection. Three weeks after amygdala injection, mice underwent a battery of behavioural tests including the OFT, LDB, T-maze and spatial object recognition (SOR) test. When testing had completed, epileptic mice were injected with AAV9 in the ventral hippocampi. Saline mice did not receive an AAV9 injection. Three weeks later, the same battery of behavioural tests were repeated in epileptic and saline mice.
  • mice were habituated to a rectangular white box (30x50x50 cm) containing an orientation marker as described in the NOR test.
  • mice were placed into the arena containing three identical objects (towers or flasks; see NOR objects), aligned 5 cm away from one of the long walls.
  • Mice were allowed to explore the box with the objects for 8 minutes in a training session at hour 25 before being moved back into their home cage to rest.
  • mice were returned to the same arena, which now contained two objects in their original position and one object that had been displaced.
  • Mice were again allowed 8 minutes to explore the objects in the box, which was marked by sniffing. Time spent climbing and chewing the objects was not counted.
  • the object type and the location of the displaced object was counterbalanced across cohorts and across repeated measures.
  • the discrimination index (DI) was calculated as follows:
  • %TD (Timeospiaced I (Timeospiaced + TimeNon-dis P iaced#i + TimeNon-dis P iaced#2)) x100%
  • DI (%TDTrial — %TDTraining) — (%TANTrial — %TANTraining)
  • mice 7- to 8-week-old female C57BL/6 mice were injected with AAV9 in ventral hippocampus, and then received ketamine or saline injections 2.5 weeks later.
  • psychomotor agitation was investigated using the large OFT. Mice received saline or ketamine daily for another six days (one week in total).
  • sociality was assessed in the social interaction test (SIT).
  • SIT social interaction test
  • recognition memory was assessed in the NOR test.
  • mice were habituated to a large arena (90x30x30 cm) for 20 minutes, given an LP. injection of saline or 30 mg/kg ketamine (Ketamidor, Chanelle Pharma) and then placed back into the arena for another 40-minutes.
  • Any-maze software was used to automatically detect movement. Nose tracking was used to account for distance travelled while rotating.
  • mice were habituated to a white 3-chamber arena containing two empty 8x10 cm wired cups in adjacent corners of the furthermost chambers for 6 minutes. Each chamber was 30x30x30 cm and connected via 7 cm doorways. Mice were always introduced to the arena via the medial chamber. 1-2 minutes after the habituation session, mice explored the arena again now containing one wired cup with a plastic object ( ⁇ 7 cm tall) and one wired cup with a gender- and age-matched stranger mouse for 6 minutes in a social interaction test. Interaction with the mouse or object, marked by sniffing, during the first 5 minutes of each trial was quantified. Time spent climbing and chewing the wired cups was not counted. The location of the wired cups and the stimulus was randomized between cages to reduce the risk of place preference biasing read-outs.
  • Table 2 Glutamate sensitivities of GluCI channels derived from C. eleqans, H. contortus & C. roqercresseyi.
  • Table 4 Change in the basal activity of H. contortus GluCI TM34 loop variants.
  • Table 5 Change in basal activity of Hc-GlyaR1 variants.
  • Table 7 Relative activities (%) of Ce-GluCI loss-of-function variants.
  • SEQ ID NO:6 Homo sapiens GABA-Rho1 transmembrane portion protein sequence (200aa)
  • eGFP Enhanced green fluorescent protein
  • SEQ ID NO:28 Homo sapiens GABA-Rho1 transmembrane portion nucleic acid sequence (603nt)
  • SEQ ID NO:30 Homo sapiens GIvR o1 isoform 1 subunit nucleic acid sequence (1374nt)
  • SEQ ID NO:31 Homo sapiens GIvR o1 isoform 1 transmembrane portion nucleic acid sequence
  • GlyR a1 isoform 2 transmembrane portion nucleic acid sequence (612nt) CAGATGGGTTACTACCTGATTCAGATGTATATTCCCAGCCTGCTCATTGTCATCCTCTCATGGATCTCCT
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced green fluorescent protein
  • eGFP Enhanced qreen fluorescent protein
  • GAGAGGAGC T C CC C AC AGAGG AAAAGT C AGAGAAGC AGC TAT GT GAGCAT GAGAAT CG AC AC CC AC GC C A
  • SEQ ID NO:46 Homo sapiens CAMKII promoter nucleic acid sequence (1300nt)
  • SEQ ID NO:54 Origin of replication nucleic acid sequence (589nt) TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

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Abstract

L'invention concerne des canaux de chlorure dépendants du glutamate modifiés (GluCl) utiles dans le traitement d'une maladie. L'invention concerne des protéines de fusion chimériques, des acides nucléiques, des vecteurs d'expression, des particules virales recombinantes et des cellules modifiées conçues pour coder ou exprimer les protéines de fusion chimériques et/ou les GluCl modifiés. L'invention concerne également des méthodes de traitement et des utilisations correspondantes des GluCl modifiés, des protéines de fusion chimériques, des acides nucléiques, des vecteurs d'expression, des particules virales recombinantes et des cellules modifiées.
PCT/EP2025/052851 2024-02-05 2025-02-04 Canaux de chlorure et leurs utilisations Pending WO2025168582A1 (fr)

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