WO2025168582A1 - Chloride channels and uses thereof - Google Patents

Abstract

Engineered glutamate-gated chloride channels (GluCl) useful in the treatment of disease are disclosed herein. Chimeric fusion proteins are described, alongside nucleic acids, expression vectors, recombinant virus particles and engineered cells configured to encode or express the chimeric fusion proteins and/or engineered GluCls. Corresponding methods of treatment and uses of the engineered GluCls, chimeric fusion proteins, nucleic acids, expression vectors, recombinant virus particles and engineered cells are also provided.

Description

Chloride channels and uses thereof

Cross-reference to Related applications

This application claims the right of priority of GB patent application no. 2401492.0 entitled “Chloride channels and uses thereof’ as filed 05 February 2024, the entire contents of which are incorporated herein by reference.

Cross-reference to Sequence Listing

The contents of the electronically submitted sequence listing (“[FINAL]_008739351_ST26_SEQL.xml“; Size: 113,841 bytes; Date of creation 03 February 2025) are herein incorporated by reference in their entirety.

Field of the Invention

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

Background

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.

365(19)p1801 -11; Hoppe and Eiger, 2011 Nat. Rev. Neurol. 7(8)p462-72).

For example, surgery to remove the seizure-generating tissue is irreversible and is limited by damage to normal brain functions. Less invasive surgeries such as laser interstitial thermal therapy are less effective and remain destructive and irreversible. Gene therapy is a promising alternative for patients with treatment-resistant epilepsy. Conventional gene therapies aim to decrease neuronal circuit excitability constitutively, controllably or on demand. An example of constitutive decrease of excitability to treat epilepsy is given in WO2018229254, which describes the overexpression of a potassium channel to treat epilepsy (see also Snowball et al,. 2019, J. Neurosci. 39(16)p3159-3169).

An example of controllable or on-demand decrease of excitability is given in WO2015136247, which describes the use of a modified muscarinic receptor together with an exogenous ligand to treat epilepsy. This is known as chemogenetics (see also Katzel et al., 2014 Nat. Commun. 5:3847). The therapeutic effect can be controlled by adjusting the dose of the ligand, or the drug can be used on-demand. Other examples of chemogenetic tools are given in Magnus et al., 2011 Science 2;333(6047)p1292-6, and WO2022238513, which describes a refinement of WO2015136247 in which the ligand of the modified receptor is an over-the-counter drug. 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.

Conventional chemogenetic approaches using modified receptors require the administration of an exogenous ligand, which may be associated with adverse events.

Moreover, neither the constitutive overexpression of potassium channels nor conventional chemogenetics permit rapid closed-loop suppression of excitability. In addition, both approaches affect all neurons that express the therapeutic transgene, whether they participate in seizures or not.

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:

First, therapeutic transgenes (for example encoding a modified potassium channel) can be put under the control of an activity-dependent promoter such as cFos (Qiu etal., 2022 Science 378(6619)p523-532). This is described in WO2021191474. However, due the activation latency (in the region of minutes) between the occurrence of a seizure and the upregulation of promoter activity, 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.

Second, 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. The use of 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. The expression of one exemplary inhibitory glutamate receptor (“enhanced GluCI” or eGluCI) has recently been shown to be effective in a rodent model of epilepsy (Lieb et al., 2018 Nat. Med. 24(9)p1324-1329). 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.

However, there are two main disadvantages of using eGluCI as a therapeutic tool in the treatment of epilepsy. First, 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). Second, 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). For example, 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. The use of antipsychotic medications is 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). Like epilepsy, 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).

Consequently, there remains a need in the art for improved gene therapy tools for treating epilepsy and 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, such as schizophrenia.

Summary of the Invention

The inventors believe that an improved way to treat such disorders is through closed-loop suppression of neuronal circuit excitability using a quick-to-act gene therapy tool to normalise network activity without disrupting normal brain activity. Accordingly, the inventors have engineered novel chimeric glutamate- gated chloride channels (“GluCIs") that incorporate: (i) a transmembrane domain derived from the transmembrane portion of a Homo sapiens chloride channel subunit; and (ii) a glutamate binding domain derived from the glutamate binding portion of a glutamate receptor. 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.

In one embodiment, 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.

A specific embodiment of the invention, termed “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. 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).

In some embodiments, the chimeric fusion protein comprises a transmembrane domain derived from the transmembrane portion of a Homo sapiens chloride channel subunit. For example, the chimeric fusion protein may comprise a transmembrane domain derived from the transmembrane portion of a Homo sapiens glycine receptor (GlyR) subunit. Alternatively, the chimeric fusion protein may comprise a transmembrane domain derived from the transmembrane portion of a Homo sapiens gamma- aminobutyric acid (GABA) receptor subunit.

In some embodiments, the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

In some embodiments, 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 . For example, 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 . For example, the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO:7.

In some embodiments, the chimeric fusion protein comprises: (i) a transmembrane domain; (ii) a glutamate binding domain; and (iii) a peptide tag. For example, the chimeric fusion protein may comprise a hemagglutinin (HA) epitope peptide tag. 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). In some embodiments, 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). In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:14.

In some embodiments, 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.

In some embodiments, the glutamate binding domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:19. For example, the glutamate binding domain may comprise the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:18.

In some embodiments, the chimeric fusion protein comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 . For example, 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. Preferably, 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.

In some embodiments, the engineered GluCI exhibits a half-maximal effective concentration (EC50) for glutamate of between 1 and 100 μM, for example, an EC50 between 10 and 20 μM.

A third aspect of the invention provides a nucleic acid encoding a chimeric fusion protein of the first aspect of the invention.

In some embodiments, the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23. For example, 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. Preferably, 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.

In some embodiments, the promoter is a regulatable promoter, a constitutive promoter, or a tissue-specific promoter. In some embodiments, the promoter comprises a human calcium-calmodulin (CaM)-dependent protein kinase II (hCaMKII) promoter. One suitable hCaMKII promoter comprises the nucleic acid sequence set forth in SEQ ID NO:46.

In some embodiments, the expression vector is a viral vector, preferably an adeno-associated virus (AAV) vector. For example, 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. In preferred embodiments, the AAV vector is an rAAV2/9 vector.

In some embodiments, the expression vector comprises an AAV2 inverted terminal repeat (ITR) sequence. For example, an AAV2 ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:47 and/or SEQ ID NO:48.

In some embodiments, the expression vector may comprise a Kozak sequence. For example, a Kozak sequence comprising the nucleic acid sequence set forth in SEQ ID NO:49.

In some embodiments, the expression vector may comprise a woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence. For example, a WPRE sequence optimised to limit any potential oncogenic activity. One suitable WPRE sequences comprises the nucleic acid sequence set forth in SEQ ID NQ:50.

In some embodiments, the expression vector may comprise a human growth hormone polyadenylation signal (hGHpA) sequence. For example, a hGHpA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:51 .

In some embodiments, the expression vector may comprise an F1 origin of replication. For example, an F1 origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:52.

In some embodiments, the expression vector may comprise a neomycin or kanamycin resistance gene (NeoR/KanR) sequence. For example, a NeoR/KanR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:53.

In some embodiments, the expression vector may comprise an origin of replication sequence. For example, an origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:54.

In addition, the expression vector may comprise one or more non-coding sequences.

In some embodiments, the expression vector comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:55. For example, the expression vector may comprise the nucleic acid sequence set forth in SEQ ID NO:56 or SEQ ID NO:57. Preferably, the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:57.

In some embodiments, the expression vector is encapsidated into a recombinant virus particle.

Accordingly, a fifth aspect of the invention provides a recombinant viral particle comprising an expression vector of the fourth aspect of the invention.

In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (AAV) particle. For example, 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. In preferred embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

Accordingly disclosed herein are 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.

Use 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, 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.

In some embodiments of the eighth aspect of the invention, 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.

In some embodiments, the cell is a neuronal cell, for example CA1 , CA2 or CA3 pyramidal cell, or an inhibitory interneuron cell. The cell may be a mammalian cell, preferably a human cell.

Importantly, the invention includes any combination of the aspects and embodiments disclosed herein, except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention are discussed with reference to the accompanying figures.

Figure 1. Glutamate-gated chloride channels (GluCIs) from different species were characterised in HEK293 cells. (A) A dose-response curve shows the relative current amplitude (l/lmax) of GluCI from C. elegans evoked by glutamate perfusion (30-4000 μM). The EC50 was estimated at approximately 740 μM (n=4). (B) Representative voltage clamp trace showing the current mediated by GluCI from C. elegans evoked by 3000 μM glutamate. The maximum current density was estimate at approximately 85 pA/pF (n=4). (C) Dose-response curve for the relative current amplitude (l/lmax) mediated by GluCI from H. contortus evoked by glutamate (3-1000 μM). The EC50 was estimated at approximately 133 μM (n=4). (D) Representative voltage clamp trace for GluCI from H. contortus in response to 1000 μM glutamate perfusion. The mean maximum current density was estimated at approximately 98 pA/pF (n=4). Figure 2. 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 μM). The dose-response curve for Hc-GluCI-E145G is shifted leftward. (B) EC50 values obtained with 3-3000 μM glutamate perfusion. The mean EC50 for Hc-GluCI-E145G was estimated at approximately 60 μM (n=10). (C) Maximum current densities were estimated in response to glutamate perfusion (3-3000 μM). The maximum current density was estimated at approximately 160 pA/pF for Hc-GluCI-E145G (n=10). Hc-GluCI-E145G was used for all subsequent experiments. (D) A schematic diagram summarising the characteristics of Hc-GluCI-E145G variants. Numerous point mutations led to loss-of-function (disturbance of function or loss of conductance) or had no effect on glutamate sensitivity. One Hc-GluCI- E145G variant (I335V) exhibited a reduced glutamate sensitivity. Three Hc-GluCI-E145G variants (T93S; intracellular loop TM3-4 substitution from human GlyRal ; intracellular loop TM3-4 substitution from human GABA-Rho1) exhibited enhanced glutamate sensitivity. (E) Dose-response curves showing the relative current (l/lmax) mediated by Hc-GluCI-E145G variants in response to glutamate perfusion (3- 3000 μM). Curves for the T93S, TM3-4 GlyRal and TM3-4 Rho1 variants showed a leftward shift. (F) Maximum current densities in response to glutamate perfusion (3-3000 μM). The following mean maximum current densities were estimated: T93S, approximately 40 pA/pF (n=3); TM3-4 GlyRal , approximately 35 pA/pF (n=9); TM3-4 Rho1 approximately 70 pA/pF (n=3). (G) Representative voltage clamp traces showing the response characteristics of Hc-GluCI-E145G and its variants T93S, TM3-4 GlyRal and TM3-4 Rho1 . The T93S variant exhibited a trace that was similar in shape to that of Hc- GluCI-E145G. The TM3-4 GlyRal and TM3-4 Rho1 variants showed non-desensitizing currents. (H) Representative voltage clamp traces showing the response characteristics of the TM3-4 GlyRal and TM3-4 Rho1 Hc-GluCI-E145G variants following picrotoxin (PTX) administration (1000 μM) (in the absence of glutamate). The TM3-4 GlyRal and TM3-4 Rho1 Hc-GluCI-E145G variants exhibited basal activity.

Figure 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) A dose-response curve shows the relative current amplitude (l/lmax) of Hc-GlyRa1 following glutamate administration (0.1-100 μM). An EC50 of approximately 4 μM was measured (n=8). (B) A representative voltage clamp trace shows the response characteristics (pA/pF) of Hc-GlyRa1 following glutamate administration (10 μM). 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 μM) (in the absence of glutamate). As shown, Hc-GlyRa1 exhibits basal activity.

Figure 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) A dose-response curve shows the relative current amplitude (l/lmax) of Hc-Rho1 following glutamate administration (3-1000 μM). An EC50 of approximately 30 μM was measured (n=6). (B) A representative voltage clamp trace shows the response characteristics (pA/pF) of Hc-Rho1 following glutamate administration (300 μM). 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 μM) (in the absence of glutamate). As shown, in contrast to Hc-GlyRa1 , Hc-Rho1 does not exhibit basal activity.

Figure 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 μM). The curves show a leftward displacement as compared to Hc-Rho1 . (B) Maximum chloride ion current densities were measured following glutamate administration (1-1500 μM). The following mean maximum current densities were achieved: L266F, approximately 50 pA/pF, n=3; P295F, approximately 180 pA/pF, n=3; T306A, approximately 45 pA/pF, n=3. (C) A representative voltage clamp trace shows the response characteristics of the P295F variant (30 μM glutamate). (D) A representative voltage clamp trace shows the response characteristics of the P295F variant following picrotoxin (PTX) administration (1000 μM) (in the absence of glutamate). As shown, the P295F variant exhibits basal activity.

Figure 6. A further Hc-Rho1 variant (P295G) was screened for improvements in glutamate sensitivity and current density. (A) A dose-response curve shows the relative current amplitude (l/lmax) of the P295G variant following glutamate administration (1-3000 μM). The P295G curve shows a leftward displacement as compared to Hc-Rho1 . (B) An EC50 of approximately 20 μM was measured (n=9). (C) Maximum chloride ion current densities were measured following glutamate administration (1-3000 μM). A mean current density of approximately 210 pA/pF was achieved (n=9). (D) Representative voltage clamp trace showing the response characteristics of the P295G variant following glutamate administration (3, 30, 300, 1000 and 3000 μM). (E) A representative voltage clamp trace shows the response characteristics of Hc-Rho1-P295G following picrotoxin (PTX) administration (1000 μM). As shown, the P295G variant does not exhibit basal activity.

Figure 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%). However, eGluCI (approximately 3 pA/pF; approximately 3% PTX/lmax) and Hc- GlyRcd (approximately 5 pA/pF; approximately 4% PTX/lmax) exhibit PTX-sensitive leaks (PTX/cap: 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). (C) Maximum chloride ion current densities were measured following glutamate administration (1- 3000 μM) or GABA administration for GABA-Rho1 (3-10 μM). The following mean maximum current densities were achieved: GABA-Rho1 , approximately 215 pA/pF (n=6); Hc-GluCI-E145G, approximately 160 pA/pF (n=10); Hc-Rho1 , approximately 70 pA/pF (n=6); Hc-Rho1-P295G, approximately 200 pA/pF (n=9); eGluCI, approximately 195 pA/pF (n=5). (D) Representative voltage clamp traces show the response characteristics of GABA-Rho1 , Hc-GluCI-E145G, Hc-Rho1 , Hc-Rho1-P295G and eGluCI in the presence of glutamate (300-3000 μM) or GABA (10 μM).

Figure 8. 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 μM). 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. (B) The curve for Hc-Rho1-P295G-395eGFP (eGFP fused to the TMD) shows a rightward displacement as compared to Hc-Rho1-P295G. The curves for Hc-Rho1-P295G and Hc-Rho1-P295G-eGFP (eGFP fused to the C-terminus of the protein) are closely aligned. (C) Maximum chloride current densities evoked by glutamate application (1-10000 μM). The following mean maximum current densities were achieved: Hc-Rho1-P295G, approximately 200 pA/pF, n=9; Hc-Rho1-P295G- 395HA, approximately 220 pA/pF, n=7; Hc-Rho1-P295G-HA, approximately 230 pA/pF, n=7; Hc-Rho1- P295G-395eGFP, approximately 130 pA/pF, n=9; Hc-Rho1-P295G-eGFP, approximately 90 pA/pF n=8.

Figure 9. 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). (A) Glutamate sensitivity (EC50) of ‘full-length’ Hc-Rho1 -P295G-395HA (‘P295G-395HA’) and GluRhol were normalised to the average EC50 of the parent channel (‘P295G’). P295G, P295G-395HA and GluRhol were compared in interleaved experiments, and were equally sensitive to glutamate. (B) Current densities of P295G-395HA and GluRhol were normalised to the current density of P295G. There was no significant difference between the channels (one-way ANOVA: p=0.17). (C,D) None of the channels exhibited basal activity, whether the PTX-sensitive current was related to cell capacitance or maximal current (PTX/cap, all approximately 0-1 pA/pF; PTX/lmax, all approximately 0-0.6%). (E) A representative voltage clamp trace shows the response characteristics of GluRhol following glutamate application (3, 30, 300, 1000 and 3000 μM). (F) A representative voltage clamp trace shows the response characteristics of GluRhol to PTX application (1000 μM). GluRhol exhibits no detectable basal activity.

Figure 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) A first trace shows that GluRho1-Y186A ('Y186A') exhibits substantially no response following glutamate administration (1000 μM). (B) A second trace shows that Y186A exhibits a strong response following emamectin administration (100 μM). A mean maximum current density of approximately 235 pA/pF was measured (n=3, insert).

Figure 11 . Representative voltage clamp traces show the response characteristics of GluRhol and Y186A to glutamate (GLU, 1000 μM), pentylenetetrazol (PTZ, 1000 μM) or a mixture of both. (A) 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.

Figure 12. Representative voltage clamp traces show the response characteristics of GABA-Rho1 , GluRhol and Y186A to glutamate (GLU, 1000 μM) and GABA (10 μM). (A) A first trace shows that GABA-Rho1 responds to GABA, but not glutamate. (B) A second trace shows that GluRhol responds to glutamate, but not GABA. (C) A third trace shows that Y186A does not respond to glutamate or GABA.

Figure 13. GluRhol exhibits AAV compatibility. (A) 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.

Figure 14. Mice were administered GluRhol or Y186A recombinant viral particles by intra-hippocampal stereotactic injection. Learning and memory behaviours were assessed at baseline. Mice were first subject to open field testing: (A) mean distance travelled was unchanged between groups (approximately 25m, n=10 per group); (B) mean thigmotaxis was also unchanged (approximately 0.94, n=10 per group). Mice were subsequently subject to light/dark box testing: (C) mean number of light chamber entries was unchanged between groups (approximately 20 entries, n=5 per group); (D) mean duration of light chamber occupancy was also unchanged (approximately 100 seconds, 5 minutes; approximately 250 seconds, 10 minutes; n=5 per group). Mice were subsequently subject to novel object testing: (E) novel object exploration time was unchanged between groups (approximately 5-20 seconds for familiar objects, and approximately 10-35 seconds for novel objects, n=10 per group); (F) discrimination index was also unchanged between groups (approximately 0.63, n=10 per group). Mice were subsequently subject to T- maze testing: (G) mean spontaneous alternation rate was unchanged between groups (approximately 0.7, n=10). Data in figure 14, A-D and F-G was assessed using two-tailed unpaired t-tests, and no significant differences were observed in any test. Data in figure 14E was assessed using a two-way ANOVA with repeated measured in one condition and the effect of object novelty was significant, p < 0.0001.

Figure 15. Mice expressing GluRhol or Y186A were then subject to an acute model of epilepsy. Animals were administered 50 mg/kg PTZ via intraperitoneal injection (n=9 Y186A, n=10 GluRhol) and the anti-epileptic activity of GluRhol was assessed over a period of 30 minutes. (A) Clonus latency (s) was unchanged between GluRhol or Y186A expressing mice (P=0.271 , Two-tailed Mann-Whitney U test). (B) Latency (s) to tonic-clonic seizure was significantly increased in GluRhol -expressing mice (P=0.006, Two-tailed Mann-Whitney U test) as compared to mice expressing Y186A. (C) Seizure severity (Racine score) was significantly reduced in GluRhol -expressing mice (P=0.033, Fisher’s exact test for reaching Racine score of >5 or not) as compared to mice expressing Y186A. (D) A bar chart shows the percentage (%) of mice exhibiting symptoms consistent with a Racine score of 3 (myoclonic seizure), 4 (clonic seizure) or 5 to 7 (tonic-clonic seizure). As shown, fewer GluRhol -expressing mice exhibit a Racine score of 5, 6 or 7 than mice expressing Y186A.

Figure 16. Immunohistochemistry shows the presence of GluRhol ⁺GABA⁺ cells in the hippocampus. As indicated (white arrows), approximately 0.6% of GluRhol ⁺ cells in the dentate gyrus, and 3.6% of GluRhol ⁺ cells in the hippocampal subfield CA3 exhibit colocalised staining with GABA.

Figure 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). (B-D) Epileptic mice had tonic-clonic seizures that were approximately 46 seconds long (n=13 mice), and were followed by a post-ictal depression in -95% of cases (n=13 mice). (E) Seizures occurred more frequently during the light phase when mice were asleep (p=0.02 in a two-tailed paired t-test, n=13 mice). (F- left) A violin plot shows the spread of generalized seizures per week including the first quartile (bottom line), median (middle line) and third quartile (top line). The numbers of generalized seizures per week in baseline ECoG recordings did not follow a normal distribution when tested with normality tests (NT; p<0.05 in D’Agostino & Pearson test, Anderson-Darling test, Shapiro-Wilk test and Kolmogorov-Smirnov test, n=13 mice). (F - right) Data was transformed using a Log(x+1) function, where “x” is seizures/week. A violin plot shows the spread of generalized seizures per week including the first quartile (bottom line), median (middle line) and third quartile (top line). The transformed dataset was normal when tested with normality tests (NT; (NT; p>0.05 in D’Agostino & Pearson test, Anderson-Darling test, Shapiro-Wilk test and Kolmogorov- Smirnov test, n=13 mice).

Figure 18. (A) Shown are the number of seizures per day in the baseline and post-AAV ECoG recordings of AAV9-hCaMKII-Y186A (Y186A) mice (grey, n=7) and in AAV9-hCaMKII-GluRho1 (GluRhol) mice (purple, n=6). Mice that died from ‘Sudden Unexpected Death in Epilepsy’ (SUDEP) are shown as double-crossed boxes. (B) Whilst Y186A does not affect the seizure frequency, GluRhol gene therapy causes a significant reduction in the seizure burden (p=0.6 and p=0.0005 respectively in a two- way repeated-measures ANOVA with Sidak's post-hoc test). (C) The change in the number of seizures in the post-AAV recordings compared to baseline recordings is significantly lower in GluRhol mice (approximately -76%, n=6) compared to Y186A mice (approximately +55%, n=7) (p=0.02 in a two-tailed unpaired t-test with Welch’s correction). The change in the number of seizures in the post-GluRho1 recordings compared to baseline recordings is significantly different from 0% (p=0.0005 in a one sample t-test). (D) The seizure burden in GluRhol mice is reduced by approximately 84% compared to Y186A mice.

Figure 19. (A) Most mice treated with the Y186A gene therapy experienced no change or a decrease in seizure-free days (71%), whilst a minority experienced more seizure-free days (29%). In contrast, most mice treated with the GluRhol gene therapy experienced an increase in seizure-free days (50%), some became seizure free (36%), and a minority had the same or fewer seizure-free days (14%). (B) Treatment with GluRhol leads to a significant increase in the percentage of seizure-free days (approximately +78%) compared to Y186A (approximately -10%) (p=0.03 in a two-tailed unpaired t-test with Welch’s correction). The percentage change in the number of seizure-free days in the post-GluRho1 recordings compared to baseline recordings is significantly different from 0% (p=0.04 in a one sample t- test, n=6 mice). Figure 20. (A) The cumulative time spent in seizures (normalized to the total seizure time in two weeks of baseline recording) increases by 2- and 2.5-fold in Y186A-treated mice 1 and 2 weeks into post-AAV recordings, respectively. In contrast, there is a negligible increase in seizure time in GluRhol mice in the same time period. Compared to Y186A-treated mice, GluRhol mice spend a significantly less amount of time in seizures both 1 and 2 weeks into post-AAV recordings (p=0.02 and p<0.0001 at 1 and 2 weeks, respectively, in a two-way repeated-measures ANOVA with Sidak's post-hoc test). (B) Treatment with Y186A or GluRhol gene therapy does not change the seizure duration (p=0.7 and p=0.6, respectively, in a two-way repeated-measures ANOVA with Sidak's post-hoc test). The two GluRhol mice that did not have seizures in post-AAV9 recordings are shown as empty circles but were not included in the statistical analysis.

Figure 21. The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model of chronic drug-resistant temporal lobe epilepsy using a randomized blinded study design. 3 weeks after intra-amygdala kainate or saline injection, baseline behaviours were measured to identify psychiatric and cognitive deficits in epileptic mice. Following baseline assessments, epileptic mice (kainate) were injected with AAV9-hCaMKII-Y186A (Y186A) or AAV9-hCaMKII-GluRho1 (GluRhol) and then tested in the same tests after 3 weeks of virus expression to learn whether the comorbidities were still present. (A - left) Saline and kainate mice were subject to an open field test (OFT) as a measure of anxiety. Movements were tracked with ANY-maze software, and representative movement plots are shown. (A - middle) The mean distance was significantly higher for kainate mice (approximately 36 m, n=29) compared to saline mice (approximately 29 m, n=18), suggesting the epileptic mice are hyperactive (p=0.03 in a two-tailed unpaired t-test). (A - right) The mean thigmotaxis was significantly higher for kainate mice (approximately 0.94, n=29) compared to saline mice (approximately 0.91 , n=18), suggesting the epileptic mice are anxious (p=0.03 in a two-tailed unpaired t-test). (B - left) The mean distance was significantly lower for Y186A (n=7), GluRhol (n=10) and Saline (n=12) groups when re-tested (p=0.006, p=0.01 and p=0.001 , respectively, in two-way repeated measures ANOVA with Tukey’s post-hoc test), suggesting all groups of mice experienced a non-specific habituation effect to the test that made them calmer when re-tested. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis. (B - right) The mean thigmotaxis was significantly lower for Y186A (n=7), GluRhol (n=10) and Saline (n=12) groups when re-tested (p=0.0007, p=0.002 and p=0.0008, respectively, in two- way repeated measures ANOVA with Tukey’s post-hoc test), suggesting all groups of mice experienced a non-specific habituation effect to the test that made them less anxious when re-tested. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis.

Figure 22. The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model as described in figure legend 20. (A - left) Saline and kainate mice were subjected to light/dark box (LDB) testing as a measure of anxiety. (A - middle left) Kainate mice spent less time in the light chamber compared to saline mice. (A - middle right) The mean duration of light chamber occupancy was significantly decreased in kainate mice (approximately 40 seconds, n=29) compared to saline mice (approximately 89 seconds, n=18) during the first 5 minutes of the test, suggesting the epileptic mice are anxious (p<0.0001 in a two-tailed unpaired t-test). (A - right) The mean duration of light chamber occupancy was significantly decreased in kainate mice (approximately 103 seconds, n=29) compared to saline mice (approximately 213 seconds, n=18) during the 10 minutes of the test, suggesting the epileptic mice are anxious (p<0.0001 in a two-tailed unpaired t-test). (B - left) The mean light chamber occupancy duration was unchanged after Y186A treatment (n=7), significantly increased after GluRhol treatment (n=10) and unchanged in saline mice (n=12) in the first 5 minutes of the LDB test (p=0.4, p=0.0003 and p=0.3, respectively, in two-way repeated measures ANOVA with Tukey’s post-hoc test), suggesting treatment with Y186A and re-testing has no effect on anxiety, whereas treatment with GluRhol reduces anxiety. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis. (B - right) The mean light chamber occupancy duration was unchanged after Y186A treatment (n=7), significantly increased after GluRhol treatment (n=10) and unchanged in saline mice (n=12) during 10 minutes of the LDB test (p=0.3, p=0.002 and p=0.9, respectively, in two-way repeated measures ANOVA with Tukey’s post-hoc test), suggesting treatment with Y186A and re-testing has no effect on anxiety, whereas treatment with GluRhol reduces anxiety. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis.

Figure 23. The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model as described in figure legend 20. (A - left) Saline and kainate mice were subjected to T- maze testing as a measure of spatial working memory. (A - middle) The mean spontaneous alternation rate was significantly lower in kainate mice (approximately 0.5, n=22) compared to saline mice (approximately 0.7, n=12), suggesting the epileptic mice have impaired spatial working memory (p=0.002 in a two-tailed unpaired t-test). (A - right) The mean spontaneous alternation rate was unchanged after Y186A treatment (n=7), significantly increased after GluRhol treatment (n=10) and unchanged in saline mice (n=12) (p=0.9, p=0.01 and p=0.6, respectively, in two-way repeated measures ANOVA with Tukey’s post-hoc test), suggesting treatment with Y186A and re-testing has no effect on spatial working memory, whereas treatment with GluRhol rescues impaired spatial working memory. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis. (B) 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. Before Y186A or GluRhol treatments, epileptic mice visited the right arm of the T-maze more frequently than the left arm (Y186A left = 3, Y186A right = 8; GluRhol left = 2, GluRhol right = 9). In contrast, saline mice visited each arm an equal number of times (Saline left = 6, saline right = 6). The side preference observed after AAV treatment or re-testing was compared to the side preference observed in the baseline test in GluRhol and saline groups. It was not possible to evaluate whether the side preference had changed before and after Y 186A treatment due to the high rate of SUDEP in this group (4/11 mice = 36%), which annulled the side preference observed before treatment. Whilst saline mice did not have a side preference in the baseline test (“Before”) or in the second test (“After”), GluRhol treatment removed the side preference observed in the baseline test (p=0.4 and p=0.002 in a two-tailed binomial test). These data suggests unilateral intra-amygdala kainate injection may cause a side preference in the T-maze test, whereas unilateral intra-amygdala saline injection does not. Treatment with GluRhol rescues this bias.

Figure 24. The efficacy of GluRhol to treat epilepsy comorbidities was assessed in an intra-amygdala kainate model as described in figure legend 20. (left) 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. (middle) The mean discrimination index percentage score was significantly lower in kainate mice (approximately -11%, n=22) compared to saline mice (approximately +29%, n=12), suggesting the epileptic mice have impaired spatial memory (p<0.0001 in a two-tailed unpaired t-test). (right) The mean discrimination index percentage score was unchanged after Y186A treatment (n=7), significantly increased after GluRhol treatment (n=10) and unchanged in saline mice (n=12) (p=0.4, p=0.0003 and p=0.7, respectively, in two- way repeated measures ANOVA with Tukey’s post-hoc test), suggesting treatment with Y186A and retesting has no effect on spatial memory, whereas treatment with GluRhol rescues impaired spatial memory. Crosses denote mice that died from SUDEP, which were not included in the statistical analysis.

Figure 25. The effect of Y186A and GluRhol treatment on survival rate was monitored in the intra- amygdala kainate model used to investigate epilepsy comorbidities. Following intra-amygdala kainate (IAK) injection at week 0 and AAV9 injection at week 4, survival was monitored until week 8 where the behavioural study ended. The absolute percentage survival was higher in the GluRhol group compared to the Y186A group (91% and 64%, respectively).

Figure 26. The efficacy of GluRhol at treating symptoms of schizophrenia was assessed in a ketamine model of schizophrenia using a randomized blinded study design. (A) Mice were injected with AAV9 in the ventral hippocampi. After 2.5 weeks of viral expression, mice received an intraperitoneal (I.P.) injection with either saline or 30 mg/kg ketamine daily for 7 days (D1-7) followed by no treatment for 4 days (D8-11) to elicit acute symptoms of psychosis followed by sub-chronic symptoms of schizophrenia. A positive symptom of psychosis (psychomotor agitation) was evaluated on day 1 , a negative symptom of schizophrenia (asociality) was evaluated on day 9 and a cognitive symptom of schizophrenia (memory deficits) 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. (B - right) Mice rotated an equal number of times during a 20-minute habituation session (approximately 20 rotations at minute ‘5’, 16 rotations at minute ’10’, 14 rotations at minute ‘15’ and 13 rotations at minute ‘20’; n=7 for saline groups and n=11 for ketamine groups). 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). (C) Ketamine injection caused a significant increase in the total distance travelled compared to saline injection in both Y186A and GluRhol groups (approximately 221 seconds and 133 seconds, comparatively), supporting the conclusion that acute ketamine elicits psychomotor agitation (p<0.001 for ketamine groups compared to saline groups in a one-way ANOVA with Tukey’s post-hoc tests). (D - left) Mice were subjected to a social interaction test (SIT) as a measure of sociality. (D - right) Saline-injected mice spent significantly more time interacting with a stranger mouse compared to a Lego object (approximately 40 seconds and 20 seconds, comparatively), suggesting they prefer to be social (p=0.009 for Y186A saline and p=0.03 for GluRhol saline in a two-way ANOVA with Sidak's post-hoc test, n=7 mice per group). Whilst a social preference was retained in GluRhol mice injected with ketamine (p<0.0001 in a two-way ANOVA with Sidak's post-hoc test, n=7 mice), it was absent in Y186A mice injected with ketamine (approximately 30 seconds for stranger mouse and 25 seconds for Lego object, p=0.4 in a two-way repeated measures ANOVA with Sidak's post-hoc test, n=7 mice), suggesting GluRhol protects mice from developing asociality, a negative symptom of schizophrenia. (E - left) Mice were subjected to novel object recognition (NOR) testing as a measure of recognition memory, a subtype of declarative memory. (E - middle) Saline-injected mice spent significantly more time interacting with a novel object compared to a familiar object (approximately 25 seconds and 10 seconds, comparatively), suggesting they have intact recognition memory (p<0.0001 for Y186A saline and p<0.0001 for GluRhol saline in a two-way ANOVA with Sidak's post-hoc test, n=7 mice per group). Whilst recognition memory was likewise intact in GluRhol mice injected with ketamine (p<0.0001 in a two-way ANOVA with Sidak's post-hoc test, n=7 mice), it was impaired in Y186A mice injected with ketamine (approximately 14 seconds for familiar object and 16 seconds for novel object, p=0.6 in a two-way repeated measures ANOVA with Sidak's post-hoc test, n=7 mice), suggesting GluRhol protects mice from developing memory deficits, a cognitive symptom of schizophrenia.

Summary of the Sequences:

SEQ ID N0:1 Hc-Rho1-E145G protein sequence.

SEQ ID N0:2 Hc-Rho1-E145G-P295G protein sequence.

SEQ ID N0:3 Short-Hc-Rho1-E145G protein sequence.

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.

SEQ ID N0:12 Enhanced green fluorescent protein (eGFP) protein sequence.

SEQ ID N0:13 Hemagglutinin (HA) protein sequence.

SEQ ID N0:14 GABA-Rho1-P295G-395HA transmembrane portion protein sequence.

SEQ ID N0:15 Haemonchus contortus GluCI E145G subunit protein sequence.

SEQ ID N0:16 Haemonchus contortus GluCI E145G glutamate binding portion protein sequence.

SEQ ID N0:17 Short Haemonchus contortus GluCI E145G subunit protein sequence.

SEQ ID NO:18 Short Haemonchus contortus GluCI E145G glutamate binding portion protein sequence.

SEQ ID N0:19 Canonical Haemonchus contortus GluCI subunit protein sequence.

SEQ ID NQ:20 Canonical Haemonchus contortus GluCI glutamate binding portion protein sequence.

SEQ ID N0:21 Hc-Rho1-E145G-P295G-395HA protein sequence.

SEQ ID NO:22 Short-Hc-Rho1-E145G-P295G-395HA protein sequence.

SEQ ID NO:23 Hc-Rho1-E145G nucleic acid sequence.

SEQ ID NO:24 Hc-Rho1-E145G-P295G nucleic acid sequence.

SEQ ID NO:25 Short-Hc-Rho1-E145G nucleic acid sequence.

SEQ ID NO:26 Short-Hc-Rho1-E145G-P295G nucleic acid 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.

SEQ ID NO:34 Enhanced green fluorescent protein (eGFP) nucleic acid sequence.

SEQ ID NO:35 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (no start codon).

SEQ ID NO:36 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (no stop codon).

SEQ ID NO:37 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (no start or stop codon).

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:40 Haemonchus contortus E145G GluCI subunit nucleic acid sequence.

SEQ ID NO:41 Haemonchus contortus GluCI E145G glutamate binding portion nucleic acid sequence.

SEQ ID NO:42 Short Haemonchus contortus GluCI E145G subunit nucleic acid sequence.

SEQ ID NO:43 Short Haemonchus contortus GluCI E145G glutamate binding portion nucleic acid sequence.

SEQ ID NO:44 Hc-Rho1- E145G-P295G-395HA 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:50 WPRE nucleic acid sequence.

SEQ ID NO:51 hGHpA nucleic acid sequence.

SEQ ID NO:52 F1 origin of replication nucleic acid sequence.

SEQ ID NO:53 NeoR/KanR nucleic acid sequence.

SEQ ID NO:54 Origin of replication nucleic acid sequence.

SEQ ID NO:55 Short-Hc-Rho1-E145G in AAV vector nucleic acid sequence.

SEQ ID NO:56 Short-Hc-Rho1-E145G-P295G in AAV vector nucleic acid sequence.

SEQ ID NO:57 Short-Hc-Rho1-E145G-P295G-395HA in AAV vector nucleic acid sequence.

SEQ ID NO:58 T2A linker protein sequence.

SEQ ID NO:59 T2A linker nucleic acid sequence.

SEQ ID NO:60 P2A linker protein sequence.

SEQ ID NO:61 P2A linker nucleic acid sequence.

SEQ ID NO:62 E2A linker protein sequence.

SEQ ID NO:63 E2A linker nucleic acid sequence.

SEQ ID NO:64 F2A linker protein sequence.

SEQ ID NO:65 F2A linker 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.

Detailed Description of the Invention

Chimeric fusion protein: transmembrane domains

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.

In context of the present invention, the term “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).

The term “transmembrane domain" refers to the membrane-spanning region of a chimeric fusion protein disclosed herein. In contrast, the term “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.

In some embodiments, 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.

In some embodiments, 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. 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 be derived from the transmembrane portion of 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). 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.

In preferred embodiments, 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.

In some embodiments, 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.

In some embodiments, the transmembrane domain is derived from the transmembrane portion of a glycine receptor (GlyR) alpha (a1 , a2, a3, a4) and beta (p) subunit. The transmembrane domain may comprise the transmembrane portion of a glycine receptor (GlyR) alpha (a1 , a2, a3, a4) and beta (p) 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.

In some embodiments, 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.

In some embodiments, the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. For example, 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. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the transmembrane domain is derived from the transmembrane portion of a human GABA-Rho1 subunit and comprises a single amino acid substitution at position P295, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 . For example, the transmembrane domain may comprise a proline (P, Pro) glycine (G, Gly) substitution at position P295 (P295G), as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 . Alternatively, 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). For example, 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). In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7.

In some embodiments, the transmembrane domain is derived from the transmembrane portion of a human GABA-Rho1 subunit and comprises a mutation in the amino acid sequence at positions 429-440, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1 .

In some embodiments, the transmembrane domain is derived from the transmembrane portion of a human GlyR a1 subunit. The amino acid sequence of the human GlyR a1 isoform 1 subunit is provided herein as SEQ ID NO:8. The amino acid sequence of the human GlyR a1 isoform 2 subunit is provided herein as SEQ ID NQ:10. The transmembrane domain may comprise the transmembrane portion of a Homo sapiens GlyR a1 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 GlyR a1 subunit are explicitly envisaged.

In some embodiments, the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence of the GlyR a1 isoform 1 subunit set forth in SEQ ID NO:9. For example, 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:9. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:9.

In some embodiments, 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 . For example, 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 . In some embodiments, 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. When 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. When 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”.

For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST), etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or amino acid sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm (see e.g., Chenna et al. ,2003 Nucleic Acids Res. 31p3497-3500; Thompson et al., 1994 Nucleic Acids Res. 22p4673-4680; Larkin et al., 2007 Bioinformatics 23p2947-48; and Altschul et al. 1990 J. Mol. Biol. 215p403-41O).

Chimeric fusion protein: peptide tags; linkers

In one embodiment, the chimeric fusion protein further comprises a peptide tag.

In context of the present invention, the term “peptide tag” refers to a peptide that facilitates detection and/or identification of the chimeric fusion protein. For example, the peptide tag may generate a detectable signal (e.g., a fluorescent signal) and/or may comprise an epitope tag (e.g., a hemagglutinin tag).

In embodiments comprising a hemagglutinin (HA) epitope 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.

In some embodiments, 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. pyralis luciferase; Renilla luciferase, such as R. reniformis luciferase and photoproteins, such as aequorin. Other suitable bioluminescent proteins include NanoLuc and derivatives thereof (Hall, M. 2012 ACS Chem. Biol., 7, 11p1848-1857; Suzuki, K. 2016 Nat Commun. 7p13718). In preferred embodiments, the peptide tag comprises an enhanced green fluorescent protein (eGFP) tag. One suitable eGFP tag comprises the amino acid set forth in SEQ ID NO:12. In some embodiments, the peptide tag comprises a polyhistidine (e.g. a hexahistidine (SEQ ID NO:69)) ‘His-tag’ or a peptide tag derived from haloalkane dehalogenase (‘HaloTag’).

In some embodiments, the transmembrane domain comprises a peptide tag. For example, the peptide tag may be contiguous with (the N-terminus or the C-terminus of) the transmembrane domain. Alternatively, 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). For example, 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 . In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:14.

In some embodiments, the chimeric fusion protein comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22. Preferably, the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.

In some embodiments, the glutamate binding domain comprises a peptide tag. For example, the peptide tag may be contiguous with (the N-terminus or the C-terminus of) the glutamate binding domain. Alternatively, 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). In some embodiments, the amino acid sequence of the peptide linker is contiguous with the N-terminus or the C- terminus of the chimeric fusion protein.

In one embodiment, 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. In addition, 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. In some embodiments, 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.

In some embodiments, the peptide tag may be joined to the peptide linker. For example, 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).

In some embodiments, the peptide linker comprises a ‘GSG’ linker, as set forth in SEQ ID NO:66. In some embodiments, 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). In such embodiments, the self-cleaving viral “2A” linker may be preceded by a ‘GSG’ linker as set forth in SEQ ID NO:66.

Chimeric fusion proteins: glutamate binding domains

In context of the present invention, the term “glutamate binding domain" refers to the extracellular protein region of a chimeric fusion protein disclosed herein. In contrast, the term “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). 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. 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 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. 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. For example, the glutamate binding domain may comprise a truncated variant of the glutamate binding portion of a H. contortus GluCI subunit.

In some embodiments, 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. In some embodiments, the glutamate binding domain comprises or consists of the glutamate binding domain set forth in SEQ ID NQ:20.

In some embodiments, 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 . For example, 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 . Alternatively, 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). For example, 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). For example, the glutamate binding domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:16.

In some embodiments, the glutamate binding domain comprises a truncated variant of any of the glutamate binding portions disclosed herein. For example, 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.

In some embodiments, the glutamate binding domain comprises a truncated variant of the H. contortus

GluCI subunit glutamate binding portion set forth in SEQ ID NO:19. In some embodiments, 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. For example, the glutamate binding domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO:18.

Chimeric fusion proteins

In some embodiments, the chimeric fusion protein comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 . For example, 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 . In some embodiments, the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:21.

In some embodiments, the chimeric fusion protein comprises a truncated variant of any of the chimeric fusion proteins disclosed herein. For example, 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.

In one embodiment, the chimeric fusion protein comprises a truncated variant of the chimeric fusion protein set forth in SEQ ID NO:1 . For example, 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.

For example, 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. In some embodiments, the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:3.

In some embodiments, the chimeric fusion protein comprises or consist of the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:22. Preferably, the chimeric fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.

Glutamate-gated chloride channels

The invention further provides an engineered glutamate-gated chloride channel (GluCI) comprising two or more subunits, in which at least one subunit comprises a chimeric fusion protein as disclosed herein. Chimeric fusion proteins as disclosed herein are capable of co-association in a membrane to form a glutamate-gated chloride channels (GluCIs).

In context of the present invention, 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. The term “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.

In some embodiments, engineered GluCIs of the invention comprise two (2), three (3), four (4), five (5), six (6), seven (7) or more subunits. In embodiments having two (2) subunits, one or both subunits may comprise a chimeric fusion protein as disclosed herein. In embodiments having three (3) subunits, one, two or all subunits may comprise a chimeric fusion protein as disclosed herein. In embodiments having four (4) subunits, one, two, three or all subunits may comprise a chimeric fusion protein as disclosed herein. In embodiments having five (5) subunits, one, two, three, four or all subunits may comprise a chimeric fusion protein as disclosed herein. In embodiments having six (6) subunits, one, two, three, four, five or all subunits may comprise a chimeric fusion protein as disclosed herein. In embodiments having seven (7) or more subunits, 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). In preferred embodiments, the engineered GluCI is homopentameric (i.e., comprising 5 subunits), each subunit comprising the same chimeric fusion protein as disclosed herein.

In some embodiments, the engineered GluCI exhibits a half-maximal effective concentration (EC50) for glutamate of between 1 and 100 μM. For example, the engineered GluCI may exhibit an EC50 for glutamate of between 1 and 90 μM, 1 and 80 μM, 1 and 70 μM, 1 and 60 μM, 1 and 50 μM, 1 and 40 μM, 1 and 30 μM, 1 and 20 μM or 1 and 10 μM. The engineered GluCI may exhibit an EC50 for glutamate of between 5 and 100 μM, 5 and 90 μM, 5 and 80 μM, 5 and 70 μM, 5 and 60 μM, 5 and 50 μM, 5 and 40 μM, 5 and 30 μM, 5 and 20 μM or 5 and 10 μM. The engineered GluCI may exhibit an EC50 for glutamate of between 10 and 100 μM, 10 and 90 μM, 10 and 80 μM, 10 and 70 μM, 10 and 60 μM, 10 and 50 μM, 10 and 40 μM or 10 and 30 μM. The engineered GluCI may exhibit an EC50 for glutamate of between 5 and 50 μM. The engineered GluCI may exhibit an EC50 for glutamate of between 10 and 20 μM. 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 μM. Table 1 (below) describes typical glutamate concentrations, as measured in rats and humans.

Table 1 : Typical glutamate concentrations.

Rats: Medina-Ceja et al. 2015

Humans: Cavus et al. 2005

Methods suitable for assessing the EC50 of a ligand-gated ion channel are well known in the art (see e.g. Rang & Dale’s Pharmacology 9th Edition, Elsevier Churchill Livingstone 2019, England, UK). Exemplary methodologies are provided in the experimental examples.

Nucleic acids

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. 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. In some embodiments, 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).

In some embodiments, the cell is be an animal or a human cell line cell. For example, a HEK 293 (human embryonic kidney) or a CHO (Chinese hamster ovary) cell. Preferably, the cell is a human cell.

In some embodiments, the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23. For example, 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. In some embodiments, the nucleic acid comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:23. For example, the nucleic acid may comprise or consist of the nucleic acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:44.

In some embodiments, the nucleic acid comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:25. For example, 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. In some embodiments, the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO:25. For example, the nucleic acid may comprise or consist of the nucleic acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:45. Preferably, 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. For example, 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.

In some embodiments, the nucleic acid comprises an IRES (internal ribosome entry site) sequence. 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. For example, 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. Other 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.

Sequence alignment and the calculation of percentage nucleic acid sequence identity is commonplace in the art, and forms part of the routine activity of persons skilled in the art. When percentage sequence identity is discussed in reference to nucleic acids it is recognised that the percentage 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. Such a 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. If the “percentage identity" is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), the “percentage identity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

Suitable methods for the introduction of heterologous nucleic acids into cells are also well-known in the art, and are described in more detail below. In some embodiments, the nucleic acid encoding the chimeric fusion protein described herein may be introduced directly into cells using gene editing techniques.

Expression vectors

The invention further provides an expression vector, comprising a nucleic acid encoding a chimeric fusion protein disclosed herein, operably linked to a promoter.

In context of the present invention, the term “expression vector³’ refers to a nucleic acid molecule that is used to transfer and express foreign genetic material in a cell. Such vectors include a promoter sequence operably linked to the gene encoding the protein to be expressed. The expression vector may further comprise include a termination codon and/or one or more expression enhancers.

In context of the present invention, the term "operably linked' refers to the covalent linkage of the selected gene and the promoter in such a way as to place expression of the gene under the influence or control of the promoter. Thus, a promoter is operably linked to a chimeric fusion protein nucleic acid sequence if the promoter is capable of effecting transcription of the chimeric fusion protein nucleic acid sequence into RNA in a cell. Where appropriate, the resulting RNA transcript may then be translated into a chimeric fusion protein as disclosed herein. Promoters disclosed herein are suitable to effect expression of the operably linked chimeric fusion protein nucleic acid sequence in a mammalian cell. In some embodiments, the cell is 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).

In some embodiments, the cell may be an animal or a human cell line cell. For example, a HEK 293 (human embryonic kidney) or a CHO (Chinese hamster ovary) cell. Preferably, the cell is a human cell.

In some embodiments, the promoter comprises a human calcium-calmodulin (CaM)-dependent protein kinase II (hCaMKII) promoter. One suitable hCaMKII promoter comprises the nucleic acid sequence set forth in SEQ ID NO:46.

Suitable vectors include plasmids, binary vectors, phage, phagemids, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes or bacterial artificial chromosomes). The design and construction of nucleic acid vectors is commonplace in the art, and forms part of the routine activity of persons skilled in the art.

As is described in detail below, preferred expression vectors include viral expression vectors. For example, 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. In preferred embodiments, the AAV vector is an rAAV2/9 vector. Alternatively, the expression vector may be a lentiviral vector, a retroviral vector, or an adenoviral vector.

In some embodiments, the expression vector comprises an AAV2 inverted terminal repeat (ITR) sequence. For example, an AAV2 ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:47 and/or SEQ ID NO:48.

In some embodiemnts, the expression vector comprises a Kozak sequence. For example, a Kozak sequence comprising the nucleic acid sequence set forth in SEQ ID NO:49.

In some embodiments, the expression vector comprises a woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence optimised to limit any potential oncogenic activity. One suitable WPRE sequences comprises the nucleic acid sequence set forth in SEQ ID NQ:50.

In some embodiments, the expression vector comprises a human growth hormone polyadenylation signal (hGHpA) sequence. For example, a hGHpA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:51.

In some embodiments, the expression vector comprises an F1 origin of replication. For example, an F1 origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:52.

In some embodiments, the expression vector comprises a neomycin or kanamycin resistance gene (NeoR/KanR) sequence. For example, a NeoR/KanR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:53.

In some embodiments, the expression vector comprises an origin of replication sequence. For example, an origin of replication sequence comprising the nucleic acid sequence set forth in SEQ ID NO:54.

In some embodiments, 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.

In some embodiments, the expression vector comprises at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:55. For example, 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. In some embodiments, the expression vector comprises or consists of the nucleic acid sequence set forth in SEQ ID NO:55. In some embodiments, the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:56 or SEQ ID NO:57. Preferably, the expression vector comprises or consist of the nucleic acid sequence set forth in SEQ ID NO:57.

In some embodiments, the expression vector is encapsidated into a recombinant virus particle.

Recombinant viral particles; Methods of preparation thereof

Accordingly, the invention further provides a recombinant viral particle comprising an expression vector as disclosed herein.

The recombinant virus particle may be a recombinant retrovirus, lentivirus, adenovirus or adeno- associated virus particle. Recombinant virus particles of the invention are recombinant insofar as they comprise a viral expression vector comprising a nucleic acid as disclosed herein (encoding a chimeric fusion protein as described herein). The design and construction of recombinant viruses is commonplace in the art, and forms part of the routine activity of persons skilled in the art.

In some embodiments, the recombinant viral particle is a recombinant adeno-associated virus (AAV) particle. For example, 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. In preferred embodiments, the recombinant viral particle is a rAAV2/9 virus particle. In some embodiments, 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. Examples of 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.

In some embodiments, 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.

In some embodiments, the cell is 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).

In some embodiments, the cell may be an animal or a human cell line cell. For example, a HEK 293 (human embryonic kidney) or a CHO (Chinese hamster ovary) cell. Preferably, 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.

Engineered cells

The invention further provides an engineered cell comprising one or more of: a chimeric fusion protein as disclosed herein; an engineered GluCI as disclosed herein; a nucleic acid (encoding a chimeric fusion protein) as disclosed herein, an expression vector as disclosed herein, or a recombinant virus particle as disclosed herein.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the engineered cell is 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 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).

In some embodiments, the engineered cell may be an animal or a human cell line cell. For example, a HEK 293 (human embryonic kidney) or a CHO (Chinese hamster ovary) cell. Preferably, 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.

Epilepsies; Neurological disorders & Methods of treatment thereof

The invention provides methods of treatment of a disease in a subject in need thereof.

For example, disclosed herein are: (i) a method of treating a disease in a subject in need thereof, the method comprising: administering a chimeric fusion protein as disclosed herein to the subject;

(ii) a method of treating a disease in a subject in need thereof, the method comprising: administering an engineered GluCI as disclosed herein to the subject;

(iii) a method of treating a disease in a subject in need thereof, the method comprising: administering a nucleic acid as disclosed herein to the subject;

(iv) a method of treating a disease in a subject in need thereof, the method comprising: administering an expression vector as disclosed herein to the subject;

(v) a method of treating a disease in a subject in need thereof, the method comprising: administering a recombinant virus particle as disclosed herein to the subject; and

(vi) a method of treating a disease in a subject in need thereof, the method comprising: administering an engineered cell as disclosed herein to the subject.

Accordingly, the invention also provides:

(i) 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;

(ii) (ii) 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;

(iii) a 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;

(iv) an expression vector as disclosed herein for use in a method of treating a disease in a subject, the method comprising: administering the expression vector to the subject;

(v) 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; and

(vi) 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.

In some embodiments, the disease is a seizure disorder. In some embodiments, 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. In some embodiments, the epilepsy involves seizures that are refractory to treatment using conventional anti-seizure agents and/or that cannot be treated by surgical intervention. Preferably, the epilepsy is a focal epilepsy, also termed “partial-onset" epilepsy.

In some embodiments, the disease is an epilepsy-related neurological disorder. For example, an epilepsy-related neurological disorder characterised by abnormal excessive neuronal activity and/or abnormal neuronal circuit excitability. In some embodiments, 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).

In some embodiments, the disease is a non-epilepsy-related neurological disorder characterised by pathological neuronal overactivity. For example, 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.

In some embodiments, the disease is a non-epilepsy-related neuropsychiatric disorder characterised by pathological neuronal overactivity. For example, 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).

The term “characterised by 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.

Various clinical and experimental techniques are available in the art to assess the electrical and metabolic behaviour of neurons, for example neuroimaging techniques. 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). 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).

Methods of administration; Subjects to be treated; Pharmaceutical compositions and formulations

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). For example, 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. For example, where 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 selection of and appropriate method and route of administration for a given therapeutic agent is commonplace in the art, and forms part of the routine activity of persons skilled in the art. That is, selecting the particular route of administration is left to the discretion of the practicing physician, who employs their common general knowledge to select administration methods and routes as appropriate for the individual subject to be treated.

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.

Following administration of the chimeric fusion proteins; engineered GluCIs; nucleic acids; expression vectors, recombinant virus particles or engineered cells as disclosed herein, 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.

In context of the present invention, 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.

In methods of the invention, 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.

In context of the present invention, 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. Similarly, 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.

Whilst it is possible for a therapeutic agent of the invention to be employed (e.g., administered) alone, it is often preferable to administer agents of the invention as a composition or formulation e.g. with a pharmaceutically acceptable carrier or diluent.

As described in W02008096268, in embodiments employing the delivery of a recombinant virus particle, 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). For embodiments involving the administration of recombinant AAV particles, exemplary unit doses include 10³, 10⁴, 10⁵, 10^s, 10⁷, 10^s, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ vg.

In some embodiments, therapeutic agents of the present invention may be combined with other therapeutic agents, whether symptomatic or disease modifying (i.e., as combination therapies).

Therapeutic agents of the invention may be administered with a second therapeutic agent, simultaneously, sequentially or separately. For example, it may be beneficial to combine therapeutic agents of the invention with one or more other (e.g., 1 , 2, 3, 4) therapeutic agents.

In some embodiments, therapeutic agents of the invention may be activated by co-administration a second therapeutic agent (an activating agent). In some embodiments, the activating agent is emamectin. Such embodiments may be advantageous in that the second therapeutic agent (activating agent) may be used to modulate the activity of the gene therapy.

Appropriate examples of second/co-therapeutic agents are known to those skilled in the art and would be immediately recognisable on the basis of the disclosure herein. Typically, 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. For example 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.

The selection of an appropriate dose and dosage schedule for a given therapeutic agent is commonplace in the art, and forms part of the routine activity of persons skilled in the art.

Importantly, the invention includes any combination of the aspects and embodiments disclosed herein, except where such a combination is clearly impermissible or expressly avoided.

Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, can, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth herein are illustrative and not limiting. Various changes to the described embodiments can be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors are not bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Experimental

Developing an autoregulatory gene therapy for epilepsy using a chimeric glutamate-gated GABAA- Rho1 channel (GluRhol)

The activity of glutamate-gated chloride channels (GluCIs) from different species were first characterised in HEK293 cells (see Figure 1 legend).

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 behaviour of Hc-Rho1- E145G-P295G was assessed by way of comparison against alternative chloride channels (see Figure 7 legend).

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).

Loss-of-function Y186A GluRhol variants were subsequently generated for use as control samples (see legends to Figures 10, 11 and 12), and the AAV compatibility of GluRhol and Y186A GluRhol was also investigated (see legends to Figure13).

The function of GluRhol was assessed in vivo. In brief, 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. In brief, animals were administered 50 mg/kg PTZ via intraperitoneal injection (n=9 Y186A GluRhol , n=10 GluRhol) and the anti-epileptic activity of GluRhol was assessed over a period of 30 minutes (see Figure 15 legend).

Ex vivo immunostaining was performed to quantify the presence of GluRhol ⁺GABA⁺ cells in the hippocampus of test animals (see Figure 16 legend).

The efficacy of GluRhol for treating chronic epilepsy was assessed in an intra-amygdala kainate (IAK) model of drug-resistant temporal lobe epilepsy (TLE). Mice that developed generalized spontaneous recurrent seizures after IAK injection were implanted with subcutaneous transmitters, and ECoG was then sampled before and after injection with Y186A or GluRhol to understand their effect on the seizure burden (see legends of Figures 17 to 20).

The effect of GluRhol was also tested on epilepsy comorbidities in the IAK model of drug-resistant TLE. 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).

The efficacy of GluRhol to protect against schizophrenia was subsequently tested in a mouse model where symptoms of acute psychosis (psychomotor agitation) and symptoms of schizophrenia (asociality and memory deficits) were elicited using acute and sub-chronic ketamine dosing (see legend to Figure 26).

Summary

In combination, these data describe the production and optimisation of engineered GluCIs of the invention. As shown, 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. In addition, GluRhol was found to protect test animals from developing symptoms of schizophrenia.

Materials and methods

In brief: 9.6 cm2 dishes of HEK cells were co-transfected with 0.4 pg copGFP and 1 .2 pg transgene using TurboFect transfection reagent (ThermoFisher, R0532). For eGFP fusions, equimolar amounts of transgene was used compared to GluRhol . One day after transfection, cells were replated onto flame- sterilized coverslips. 1-2 days after replating, individual HEK cells were patched using thin-walled glass pipettes (2.5-4.5 MQ) in whole-cell mode and chloride currents measured at a holding potential of -60 mV using an intracellular (135 mM CsCI, 4 mM MgCI2, 1 mM EGTA, 10 mM HEPES, 4 mM Na2-ATP, pH 7.35) and an extracellular solution (150 mM NaCI, 2.8 mM KCI, 2 mM CaCI2, 2 mM MgCI2, 10 mM HEPES, pH 7.35) except for basal activity experiments where a holding potential of 0 mV was combined with a CsMeS intracellular solution (148 mM CsMes, 0.1 mM CaCI2, 2 mM MgCI2, 1 mM EGTA, 2 mM Mg-ATP, 10 mM HEPES, pH 7.35). 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. Cell and ammonium-sulfate pellets were then resuspended in lysis buffer (140 mM NaCI, 25 mM tris base, 5 mM KCI, 3.5 mM MgCh, 0.7 mM K2HPO4, pH 7.5), separately, and placed in a -80°C freezer. The cell fraction then underwent three freeze-thaw- vortex cycles. Finally, both fractions were treated with Benzonase and MgCh at 37°C before AAV particles were isolated using an iodixanol gradient (60%, 40%, 25% and 15%) and ultracentrifugation. 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).

9-week-old male C57BL/6 mice (Charles River UK) received AAV9 injections in the ventral hippocampi. In preparation of this surgery, 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 10¹² vg/mL AAV9 (GluRhol or Y186A) was injected at 100 nl/min per depth (in mm: DV -3.5, -3 and -2.5). After the last AAV injection, 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). At the 25-hour timepoint, 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. At the 26- hour timepoint, mice were placed back into the arena now containing one familiar and one novel object (one Lego tower and one glass flask with agarose). Time spent exploring each object in 8 minutes, marked by sniffing, was scored by a researcher blind to the treatments using the discrimination index (DI = TimeNovei/(TimeNovei+Timefamiiiar)). Time spent climbing and chewing the objects was not counted. The arena was cleaned with 70% ethanol in-between trials.

In the light-dark box (LDB) test, 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.

In the T-maze test, 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). During a trial, the mouse was allowed to move through the T-maze until it first entered one of the short arms. At this point, 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.

In the acute PTZ model of epilepsy, mice were given an IP injection of 50 mg/kg pentylenetetrazol (PTZ) and placed in an empty cage for observation. 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). At the end of the experiment, 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).

For immunostaining, brains were fixed in 4% paraformaldehyde and subsequently sliced to a 30 pm thickness with a vibratome (Leica). 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. Slices were then washed with PBS four times, incubated in PBS with 2% NGS and 1 :1000 dilutions of secondary mouse and rabbit antibodies (ThermoFisher, A-11001 and Abeam, ab150079) for 2 hours. After another four PBS washes, slices were mounted with Fluoroshield (Abeam, ab104139) and then imaged with a LSM 710 confocal microscope (Zeiss, ZEN 2009 software) using a 488 nm argon laser and a 633 nm HeNe2 laser. Confocal images were merged in Imaged (Rasband, W. S. (1997-2018).

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. 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. Guide cannulas (Bilaney, C200GS-5/SPCn) were centred on top of the burr holes and cemented in place using RelyX cement (Dental Sky, 123-0009) cured under blue light, and closed with a dummy cannula. A head plate was created using Simplex bone cement, which covered all of the exposed skull as well as the base of the cannulas. A thin layer of Medbond glue was applied to the edges of the headplate to help secure it in place. The back wound was sutured, and lidocaine applied. A subcutaneous injection of Betamox was given to prevent infection (0.1 mL of a 150 mg/mL stock).

Using LWDAQ Software (Open Source Instruments Inc.), 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). After two weeks of ECoG acquisition, 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. The project was designed to achieve a power greater than 0.8 with an a= 0.05.

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.

For the SOR test, mice were habituated to a rectangular white box (30x50x50 cm) containing an orientation marker as described in the NOR test. One hour after the last habituation session, 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. After a one-hour interval, 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:

Percentage Time displaced (%TD) = (Timeospiaced I (Timeospiaced + TimeNon-dis_Piaced#i + TimeNon-dis_Piaced#2)) x100%

Percentage Time Average Non-displaced (%TAN) = (100 - %TD) 12

DI = (%TDTrial — %TDTraining) — (%TANTrial — %TANTraining)

To assess symptoms of psychosis and schizophrenia, 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. On day 1 , psychomotor agitation was investigated using the large OFT. Mice received saline or ketamine daily for another six days (one week in total). On day 9 (2 days after the last treatment), sociality was assessed in the social interaction test (SIT). On day 11 (4 days after the last treatment), recognition memory was assessed in the NOR test.

In the large OFT, 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.

In the SIT, 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.

Tables

The following tables describe further characteristics of particular GluCI channels and variants thereof.

Table 2: Glutamate sensitivities of GluCI channels derived from C. eleqans, H. contortus & C. roqercresseyi.

Table 3: Changes in the glutamate (Glu) sensitivity (EC50) and basal activity of C. eleqans GluCI variants.

Table 4: Change in the basal activity of H. contortus GluCI TM34 loop variants.

Bertozzi et al., 2016

Table 5: Change in basal activity of Hc-GlyaR1 variants.

Table 6: Changes in the glutamate (Glu) sensitivity (EC50) and current density (CD) of Hc-Rho1 GluCI variants.

Table 7: Relative activities (%) of Ce-GluCI loss-of-function variants.

Li, Slimko & Lester, 2002

Sequences

The following amino acid and nucleic acid sequence further describe the GluCI channels disclosed herein.

SEQ ID NO:1 Hc-Rho1-E145G protein sequence (459aa)

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF QNEKGARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH IFFFLLQTYF PATLMVML S W VSFWIDRRAVPARVPLGITTVLTMSTI ITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTT

VQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASERSSPQRKS QRSSYVSMRIDTHAIDKYSRI IFPAAYILFNLIYWSI FS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295

SEQ ID NO:2 Hc-Rho1-E145G-P295G protein sequence (459aa)

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF QNEKGARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH IFFFLLQTYF PATLMVML S W VSFWIDRRAVPARVGLGITTVLTMSTI ITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTT VQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASER

SSPQRKSQRSSYVSMRIDTHAIDKYSRII FPAAYILFNLIYWSI FS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G

SEQ ID NO:3 Short-Hc-Rho1-E145G protein sequence (446aa)

MLALICTVSTIMSAVEAKRKLKEQEIIQRILNNYDWRVRPRGLNASWPDTGGPVLVTVNIYLRSISKIDD

VNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFFQNEKGARRHLIDK

PNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEWKEQNPVQQKDGLR

QSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH I FFFLLQTY FPATLMVMLSWVS FWI DRRAVPAR

VPLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKL

PCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTH

AIDKYSRII FPAAYILFNLIYWSIFS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295

SEQ ID NO:4 Short-Hc-Rho1-E145G-P295G protein sequence (446aa)

MLALICTVSTIMSAVEAKRKLKEQEIIQRILNNYDWRVRPRGLNASWPDTGGPVLVTVNIYLRSISKIDD

VNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFFQNEKGARRHLIDK

PNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEWKEQNPVQQKDGLR

QSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH I FFFLLQTY FPATLMVMLSWVS FWI DRRAVPAR

VGLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKL

PCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKPDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTH

AIDKYSRII FPAAYILFNLIYWSIFS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G

SEQ ID NO:5 Homo sapiens GABA-Rho1 subunit protein sequence (479aa)

MLAVPNMRFGI FLLWWGWVLATESRMHWPGREVHEMSKKGRPQRQRREVHEDAHKQVSPILRRSPDITKS

PLTKSEQLLRIDDHDFSMRPGFGGPAIPVGVDVQVESLDSISEVDMDFTMTLYLRHYWKDERLSFPSTNN

LSMTFDGRLVKKIWVPDMFFVHSKRSFIHDTTTDNVMLRVQPDGKVLYSLRVTVTAMCNMDFSRFPLDTQ

TCSLEIESYAYTEDDLMLYWKKGNDSLKTDERISLSQFLIQEFHTTTKLAFYSSTGWYNRLYINFTLRRH

I FFFLLQTY FPATLMVMLSWVS FWIDRRAVPARVPLGITTVLTMST I ITGVNASMPRVSYIKAVDIYLWV

SFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEKP

DRMMVQLTLASERSSPQRKSQRSSYVSMRIDTHAIDKYSRII FPAAYILFNLIYWSIFS

SEQ ID NO:6 Homo sapiens GABA-Rho1 transmembrane portion protein sequence (200aa)

HI FFFLLQTY FPATLMVMLSWVS FWIDRRAVPARVPLGITTVLTMST I ITGVNASMPRVSYIKAVDIYLW

VSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEK

PDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTHAIDKYSRI IFPAAYILFNLIYWSI FS

Key: P295

SEQ ID NO:7 GABA-Rho1-P295G transmembrane portion protein sequence (200aa) HI FFFLLQTYFPATLMVMLSWVSFWIDRRAVPARVGLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLW

VSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPENGEK

PDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTHAIDKYSRI IFPAAYILFNLIYWSI FS

Key: P295G

SEQ ID NO:8 Homo sapiens GlyR a1 isoform 1 subunit protein sequence (457aa)

MYSFNTLRLYLWETIVFFSLAASKEAEAARSAPKPMSPSDFLDKLMGRTSGYDARIRPNFKGPPVNVSCN

IFINSFGSIAETTMDYRVNIFLRQQWNDPRLAYNEYPDDSLDLDPSMLDSIWKPDLFFANEKGAHFHEIT

TDNKLLRISRNGNVLYSIRITLTLACPMDLKNFPMDVQTCIMQLESFGYTMNDLIFEWQEQGAVQVADGL

TLPQFILKEEKDLRYCTKHYNTGKFTCIEARFHLERQMGYYLIQMYIPSLLIVILSWISFWINMDAAPAR

VGLGITTVLTMTTQSSGSRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLRFRRKRR

HHKSPMLNLFQEDEAGEGRFNFSAYGMGPACLQAKDGISVKGANNSNTTNPPPAPSKSPEEMRKLFIQRA

KKIDKISRIGFPMAFLI FNMFYWIIYKIVRREDVHNQ

SEQ ID NO:9 Homo sapiens GlyR a1 isoform 1 transmembrane portion protein sequence (211aa)

QMGYYLIQMYIPSLLIVILSWISFWINMDAAPARVGLGITTVLTMTTQSSGSRASLPKVSYVKAIDIWMA

VCLLFVFSALLEYAAVNFVSRQHKELLRFRRKRRHHKSPMLNLFQEDEAGEGRFNFSAYGMGPACLQAKD

GISVKGANNSNTTNPPPAPSKSPEEMRKLFIQRAKKIDKISRIGFPMAFLIFNMFYWI IYKIVRREDVHN

Q

SEQ ID NO:1Q Homo sapiens GlyR a1 isoform 2 subunit protein sequence (449aa)

MYSFNTLRLYLWETIVFFSLAASKEAEAARSAPKPMSPSDFLDKLMGRTSGYDARIRPNFKGPPVNVSCN

IFINSFGSIAETTMDYRVNIFLRQQWNDPRLAYNEYPDDSLDLDPSMLDSIWKPDLFFANEKGAHFHEIT

TDNKLLRISRNGNVLYSIRITLTLACPMDLKNFPMDVQTCIMQLESFGYTMNDLIFEWQEQGAVQVADGL

TLPQFILKEEKDLRYCTKHYNTGKFTCIEARFHLERQMGYYLIQMYIPSLLIVILSWISFWINMDAAPAR

VGLGITTVLTMTTQSSGSRASLPKVSYVKAIDIWMAVCLLFVFSALLEYAAVNFVSRQHKELLRFRRKRR

HHKEDEAGEGRFNFSAYGMGPACLQAKDGISVKGANNSNTTNPPPAPSKSPEEMRKLFIQRAKKIDKISR

IGFPMAFLI FNMFYWIIYKIVRREDVHNQ

SEQ ID NO:11 Homo sapiens GlyR a1 isoform 2 transmembrane portion protein sequence (203aa)

QMGYYLIQMYIPSLLIVILSWISFWINMDAAPARVGLGITTVLTMTTQSSGSRASLPKVSYVKAIDIWMA

VCLLFVFSALLEYAAVNFVSRQHKELLRFRRKRRHHKEDEAGEGRFNFSAYGMGPACLQAKDGISVKGAN

NSNTTNPPPAPSKSPEEMRKLFIQRAKKIDKISRIGFPMAFLIFNMFYWI IYKIVRREDVHNQ

SEQ ID NO:12 Enhanced green fluorescent protein (eGFP) protein sequence (239aa)

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ

CFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH

KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK

DPNEKRDHMVLLEFVTAAGITLGMDELYK

SEQ ID NO:13 Hemagglutinin (HA) protein sequence (9aa) YPYDVPDYA

SEQ ID NO:14 GABA-Rho1 -P295G-395HA transmembrane

HI FFFLLQTYFPATLMVMLSWVSFWIDRRAVPARVGLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLW

VSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPYPYDV

PDYAENGEKPDRMMVQLTLASERSSPQRKSQRSSYVSMRIDTHAIDKYSRII FPAAYILFNLIYWSIFS

Key: P295G: HA tag

SEQ ID NO:15 Haemonchus contortus GluCI E145G subunit

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT

VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF

QNEKGARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW

KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRREYSYYLIQLYIPCIMLVVVSW

VSFWLDKDAVPARVSLGVTTLLTMTTQASGINSKLPPVSYIKAVDVWIGVCLAFIFGALLEYAVVNYYGR

KEFLRKEKKKKTRLDDCVCPSERPALRLDLSNYRRRGWTPLNRLLDMLGRNADLSRRVDLMSRITFPSLF

TAFLVFYYSVYVKQSNLD

SEQ ID NO:16 Haemonchus contortus GluCI E145G

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT

VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF

QNEKGARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW

KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRR

SEQ ID NO:17 Short Haemonchus contortus GluCI E145G subunit

MLALICTVSTIMSAVEAKRKLKEQEIIQRILNNYDWRVRPRGLNASWPDTGGPVLVTVNIYLRSISKIDD

VNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFFQNEKGARRHLIDK

PNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEWKEQNPVQQKDGLR

QSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRREYSYYLIQLYIPCIMLVVVSWVSFWLDKDAVPAR

VSLGVTTLLTMTTQASGINSKLPPVSYIKAVDVWIGVCLAFI FGALLEYAVVNYYGRKEFLRKEKKKKTR

LDDCVCPSERPALRLDLSNYRRRGWTPLNRLLDMLGRNADLSRRVDLMSRITFPSLFTAFLVFYYSVYVK

QSNLD

SEQ ID NO:18 Short Haemonchus contortus GluCI E145G

MLALICTVSTIMSAVEAKRKLKEQEIIQRILNNYDWRVRPRGLNASWPDTGGPVLVTVNIYLRSISKIDD

VNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFFQNEKGARRHLIDK

PNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEWKEQNPVQQKDGLR

QSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRR

SEQ ID NO:19 Canonical Haemonchus contortus GluCI subunit uence MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT

VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF

QNEKEARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW

KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRREYSYYLIQLYIPCIMLVVVSW

VSFWLDKDAVPARVSLGVTTLLTMTTQASGINSKLPPVSYIKAVDVWIGVCLAFIFGALLEYAVVNYYGR

KEFLRKEKKKKTRLDDCVCPSERPALRLDLSNYRRRGWTPLNRLLDMLGRNADLSRRVDLMSRITFPSLF

TAFLVFYYSVYVKQSNLD

SEQ ID NQ:20 Canonical Haemonchus contortus GluCI glutamate binding portion protein seguence (259aa)

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT

VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF

QNEKEARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW

KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRR

SEQ ID NO:21 Hc-Rho1 -E145G-P295G-395HA protein seguence (468aa)

MRNSVPLATRIGPMLALICTVSTIMSAVEAKRKLKEQEI IQRILNNYDWRVRPRGLNASWPDTGGPVLVT

VNIYLRSISKIDDVNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFF

QNEKGARRHLIDKPNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEW

KEQNPVQQKDGLRQSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH I F F FLLQT Y F PATLMVML S W

VSFWIDRRAVPARVGLGITTVLTMSTI ITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTT

VQERKEQKLREKLPCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPYPYDVPDYAENGEKPDRMMVQLTLAS

ERSSPQRKSQRSSYVSMRIDTHAIDKYSRII FPAAYILFNLIYWSI FS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G; HA tag

SEQ ID NO:22 Short-Hc-Rho1 -E145G-P295G-395HA protein seguence (455aa)

MLALICTVSTIMSAVEAKRKLKEQEIIQRILNNYDWRVRPRGLNASWPDTGGPVLVTVNIYLRSISKIDD

VNMEYSAQFTFREEWVDARLAYGRFEDESTEVPPFVVLATSENADQSQQIWMPDTFFQNEKGARRHLIDK

PNVLIRIHKDGSILYSVRLSLVLSCPMSLEFYPLDRQNCLIDLASYAYTTQDIKYEWKEQNPVQQKDGLR

QSLPSFELQDVVTKYCTSKTNTGEYSCARVKLLLRRH I FFFLLQTY FPATLMVMLSWVS FWI DRRAVPAR

VGLGITTVLTMSTIITGVNASMPRVSYIKAVDIYLWVSFVFVFLSVLEYAAVNYLTTVQERKEQKLREKL

PCTSGLPPPRTAMLDGNYSDGEVNDLDNYMPYPYDVPDYAENGEKPDRMMVQLTLASERSSPQRKSQRSS

YVSMRIDTHAIDKYSRI IFPAAYILFNLIYWSIFS

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G; HA tag

SEQ ID NO:23 Hc-Rho1-E145G nucleic acid seguence (1380nt)

ATGCGCAATTCCGTCCCTCTGGCGACTCGAATAGGGCCTATGCTGGCCCTCATCTGTACTGTCAGCACAA

TTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTA

CGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACG GTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTT

TTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCC

GTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTC

CAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACG

GCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTT

GGATCGACAGAACTGCCTTATCGATCTCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGG

AAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATG

TCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTT

GCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGG

GTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCCCCTTAGGTATCACAACGGTGCTGACCA

TGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTA

CCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACT

GTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCA

CTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGG

AGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGT

CAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTC

CAGCAGCATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295;

SEQ ID NO:24 Hc-Rho1 -E145G-P295G nucleic acid sequence (1380nt)

ATGCGCAATTCCGTCCCTCTGGCGACTCGAATAGGGCCTATGCTGGCCCTCATCTGTACTGTCAGCACAA

TTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTA

CGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACG

GTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTT

TTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCC

GTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTC

CAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACG

GCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTT

GGATCGACAGAACTGCCTTATCGATCTCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGG

AAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATG

TCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTT

GCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGG

GTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCA

TGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTA

CCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACT

GTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCA

CTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGG

AGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGT CAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTC

CAGCAGCATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G;

SEQ ID NO:25 Short-Hc-Rho1-E145G nucleic acid sequence (1341 nt)

ATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAAC

AGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTG

GCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGAC

GTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCC

GTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTC

ACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAG

CCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGT

CCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATCTCGCATCATATGC

GTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGT

CAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAG

AATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTT

CCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGA

GTCCCCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGC

GCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCT

GGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTT

CCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGA

ATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGC

CTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCAC

GCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGCATACATTTTATTCAATTTAATATACTGGTCTA

TTTTCTCCTAG

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295;

SEQ ID NO:26 Short-Hc-Rho1 -E145G-P295G nucleic acid sequence (1341 nt)

ATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAAC

AGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTG

GCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGAC

GTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCC

GTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTC

ACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAG

CCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGT

CCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATCTCGCATCATATGC

GTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGT

CAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAG

AATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTT CCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGA

GTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGC

GCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCT

GGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTT

CCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGA

ATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGC

CTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCAC

GCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGCATACATTTTATTCAATTTAATATACTGGTCTA

TTTTCTCCTAG

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G;

SEQ ID NO:27 Homo sapiens GABA-Rho1 subunit nucleic acid sequence (1440nt)

ATGTTGGCTGTCCCAAATATGAGATTTGGCATCTTTCTTTTGTGGTGGGGATGGGTTTTGGCCACTGAAA

GCAGAATGCACTGGCCCGGAAGAGAAGTCCACGAGATGTCTAAGAAAGGCAGGCCCCAAAGACAAAGACG

AGAAGTACATGAAGATGCCCACAAGCAAGTCAGCCCAATTCTGAGACGAAGTCCTGACATCACCAAATCG

CCTCTGACAAAGTCAGAACAGCTTCTGAGGATAGATGACCATGATTTCAGCATGAGGCCTGGCTTTGGAG

GCCCTGCCATTCCTGTTGGTGTGGATGTGCAGGTGGAGAGTTTGGATAGCATCTCAGAGGTTGACATGGA

CTTTACGATGACCCTCTACCTGAGGCACTACTGGAAGGACGAGAGGCTGTCTTTTCCAAGCACCAACAAC

CTCAGCATGACGTTTGATGGCCGGCTGGTCAAGAAGATCTGGGTCCCTGACATGTTTTTCGTGCACTCCA

AACGCTCCTTCATCCACGACACCACCACAGACAACGTCATGTTGCGGGTCCAGCCTGATGGGAAAGTGCT

CTATAGTCTCAGGGTTACAGTAACTGCAATGTGCAACATGGACTTCAGCCGATTTCCCTTGGACACACAA

ACGTGCTCTCTTGAAATTGAAAGCTATGCCTATACAGAAGATGACCTCATGCTGTACTGGAAAAAGGGCA

ATGACTCCTTAAAGACAGATGAACGGATCTCACTCTCCCAGTTCCTCATTCAGGAATTCCACACCACCAC

CAAACTGGCTTTCTACAGCAGCACAGGCTGGTACAACCGTCTCTACATTAATTTCACGTTGCGTCGCCAC

ATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCT

GGATCGACCGCAGAGCCGTGCCTGCCAGAGTCCCCTTAGGTATCACAACGGTGCTGACCATGTCCACCAT

CATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTC

AGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGA

GGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCT

GGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAGCCC

GACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCA

GC T AT GT GAGCAT GAGAAT CG AC AC CC AC GC CAT T GAT AAAT AC T C C AGG AT CAT C T T T C C AGC AGC AT A

CATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

SEQ ID NO:28 Homo sapiens GABA-Rho1 transmembrane portion nucleic acid sequence (603nt)

CACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCT

TCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCCCCTTAGGTATCACAACGGTGCTGACCATGTCCAC CATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGG

GTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGG

AGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAAT

GCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAG

CCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAA

GCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGC

ATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

Key: P295

SEQ ID NO:29 GABA-Rho1-P295G transmembrane portion nucleic acid sequence (603nt)

CACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCT

TCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCAC

CATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGG

GTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGG

AGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAAT

GCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAG

CCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAA

GCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGC

ATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

Key: P295G

SEQ ID NO:30 Homo sapiens GIvR o1 isoform 1 subunit nucleic acid sequence (1374nt)

ATGTACAGCTTCAATACTCTTCGACTCTACCTTTGGGAGACCATTGTATTCTTCAGCCTTGCTGCTTCTA

AGGAGGCTGAAGCTGCTCGCTCCGCACCCAAGCCTATGTCACCCTCGGATTTCCTGGATAAGCTAATGGG

GAGAACCTCCGGATATGATGCCAGGATCAGGCCCAATTTTAAAGGTCCCCCAGTGAACGTGAGCTGCAAC

ATTTTCATCAACAGCTTTGGTTCCATTGCTGAGACAACCATGGACTATAGGGTCAACATCTTCCTGCGGC

AGCAATGGAACGACCCCCGCCTGGCCTATAATGAATACCCTGACGACTCTCTGGACCTGGACCCATCCAT

GCTGGACTCCATCTGGAAACCTGACCTGTTCTTTGCCAACGAGAAGGGGGCCCACTTCCATGAGATCACC

ACAGACAACAAATTGCTAAGGATCTCCCGGAATGGGAATGTCCTCTACAGCATCAGAATCACCCTGACAC

TGGCCTGCCCCATGGACTTGAAGAATTTCCCCATGGATGTCCAGACATGTATCATGCAACTGGAAAGCTT

TGGATATACGATGAATGACCTCATCTTTGAGTGGCAGGAACAGGGAGCCGTGCAGGTAGCAGATGGACTA

ACTCTGCCCCAGTTTATCTTGAAGGAAGAGAAGGACTTGAGATACTGCACCAAGCACTACAACACAGGTA

AATTCACCTGCATTGAGGCCCGGTTCCACCTGGAGCGGCAGATGGGTTACTACCTGATTCAGATGTATAT

TCCCAGCCTGCTCATTGTCATCCTCTCATGGATCTCCTTCTGGATCAACATGGATGCTGCACCTGCTCGT

GTGGGCCTAGGCATCACCACTGTGCTCACCATGACCACCCAGAGCTCCGGCTCTCGAGCATCTCTGCCCA

AGGTGTCCTATGTGAAAGCCATTGACATTTGGATGGCAGTTTGCCTGCTCTTTGTGTTCTCAGCCCTATT

AGAATATGCTGCCGTTAACTTTGTGTCTCGGCAACATAAGGAGCTGCTCCGATTCAGGAGGAAGCGGAGA

CATCACAAGAGCCCCATGTTGAATCTATTCCAGGAGGATGAAGCTGGAGAAGGCCGCTTTAACTTCTCTG

CCTATGGGATGGGCCCAGCCTGTCTACAGGCCAAGGATGGCATCTCAGTCAAGGGCGCCAACAACAGTAA CACCACCAACCCCCCTCCTGCACCATCTAAGTCCCCAGAGGAGATGCGAAAACTCTTCATCCAGAGGGCC

AAGAAGATCGACAAAATATCCCGCATTGGCTTCCCCATGGCCTTCCTCATTTTCAACATGTTCTACTGGA

TCATCTACAAGATTGTCCGTAGAGAGGACGTCCACAACCAGTGA

SEQ ID NO:31 Homo sapiens GIvR o1 isoform 1 transmembrane portion nucleic acid sequence

CAGATGGGTTACTACCTGATTCAGATGTATATTCCCAGCCTGCTCATTGTCATCCTCTCATGGATCTCCT

TCTGGATCAACATGGATGCTGCACCTGCTCGTGTGGGCCTAGGCATCACCACTGTGCTCACCATGACCAC

CCAGAGCTCCGGCTCTCGAGCATCTCTGCCCAAGGTGTCCTATGTGAAAGCCATTGACATTTGGATGGCA

GTTTGCCTGCTCTTTGTGTTCTCAGCCCTATTAGAATATGCTGCCGTTAACTTTGTGTCTCGGCAACATA

AGGAGCTGCTCCGATTCAGGAGGAAGCGGAGACATCACAAGAGCCCCATGTTGAATCTATTCCAGGAGGA

TGAAGCTGGAGAAGGCCGCTTTAACTTCTCTGCCTATGGGATGGGCCCAGCCTGTCTACAGGCCAAGGAT

GGCATCTCAGTCAAGGGCGCCAACAACAGTAACACCACCAACCCCCCTCCTGCACCATCTAAGTCCCCAG

AGGAGATGCGAAAACTCTTCATCCAGAGGGCCAAGAAGATCGACAAAATATCCCGCATTGGCTTCCCCAT

GGCCTTCCTCATTTTCAACATGTTCTACTGGATCATCTACAAGATTGTCCGTAGAGAGGACGTCCACAAC CAGTGA

SEQ ID NO:32 Homo sapiens GIvR o1 isoform 2 subunit nucleic acid sequence (1350nt)

ATGTACAGCTTCAATACTCTTCGACTCTACCTTTGGGAGACCATTGTATTCTTCAGCCTTGCTGCTTCTA

AGGAGGCTGAAGCTGCTCGCTCCGCACCCAAGCCTATGTCACCCTCGGATTTCCTGGATAAGCTAATGGG

GAGAACCTCCGGATATGATGCCAGGATCAGGCCCAATTTTAAAGGTCCCCCAGTGAACGTGAGCTGCAAC

ATTTTCATCAACAGCTTTGGTTCCATTGCTGAGACAACCATGGACTATAGGGTCAACATCTTCCTGCGGC

AGCAATGGAACGACCCCCGCCTGGCCTATAATGAATACCCTGACGACTCTCTGGACCTGGACCCATCCAT

GCTGGACTCCATCTGGAAACCTGACCTGTTCTTTGCCAACGAGAAGGGGGCCCACTTCCATGAGATCACC

ACAGACAACAAATTGCTAAGGATCTCCCGGAATGGGAATGTCCTCTACAGCATCAGAATCACCCTGACAC

TGGCCTGCCCCATGGACTTGAAGAATTTCCCCATGGATGTCCAGACATGTATCATGCAACTGGAAAGCTT

TGGATATACGATGAATGACCTCATCTTTGAGTGGCAGGAACAGGGAGCCGTGCAGGTAGCAGATGGACTA

ACTCTGCCCCAGTTTATCTTGAAGGAAGAGAAGGACTTGAGATACTGCACCAAGCACTACAACACAGGTA

AATTCACCTGCATTGAGGCCCGGTTCCACCTGGAGCGGCAGATGGGTTACTACCTGATTCAGATGTATAT

TCCCAGCCTGCTCATTGTCATCCTCTCATGGATCTCCTTCTGGATCAACATGGATGCTGCACCTGCTCGT

GTGGGCCTAGGCATCACCACTGTGCTCACCATGACCACCCAGAGCTCCGGCTCTCGAGCATCTCTGCCCA

AGGTGTCCTATGTGAAAGCCATTGACATTTGGATGGCAGTTTGCCTGCTCTTTGTGTTCTCAGCCCTATT

AGAATATGCTGCCGTTAACTTTGTGTCTCGGCAACATAAGGAGCTGCTCCGATTCAGGAGGAAGCGGAGA

CATCACAAGGAGGATGAAGCTGGAGAAGGCCGCTTTAACTTCTCTGCCTATGGGATGGGCCCAGCCTGTC

TACAGGCCAAGGATGGCATCTCAGTCAAGGGCGCCAACAACAGTAACACCACCAACCCCCCTCCTGCACC

ATCTAAGTCCCCAGAGGAGATGCGAAAACTCTTCATCCAGAGGGCCAAGAAGATCGACAAAATATCCCGC

ATTGGCTTCCCCATGGCCTTCCTCATTTTCAACATGTTCTACTGGATCATCTACAAGATTGTCCGTAGAG

AGGACGTCCACAACCAGTGA

SEQ ID NO:33 Homo sapiens GlyR a1 isoform 2 transmembrane portion nucleic acid sequence (612nt) CAGATGGGTTACTACCTGATTCAGATGTATATTCCCAGCCTGCTCATTGTCATCCTCTCATGGATCTCCT

TCTGGATCAACATGGATGCTGCACCTGCTCGTGTGGGCCTAGGCATCACCACTGTGCTCACCATGACCAC

CCAGAGCTCCGGCTCTCGAGCATCTCTGCCCAAGGTGTCCTATGTGAAAGCCATTGACATTTGGATGGCA

GTTTGCCTGCTCTTTGTGTTCTCAGCCCTATTAGAATATGCTGCCGTTAACTTTGTGTCTCGGCAACATA

AGGAGCTGCTCCGATTCAGGAGGAAGCGGAGACATCACAAGGAGGATGAAGCTGGAGAAGGCCGCTTTAA

CTTCTCTGCCTATGGGATGGGCCCAGCCTGTCTACAGGCCAAGGATGGCATCTCAGTCAAGGGCGCCAAC

AACAGTAACACCACCAACCCCCCTCCTGCACCATCTAAGTCCCCAGAGGAGATGCGAAAACTCTTCATCC

AGAGGGCCAAGAAGATCGACAAAATATCCCGCATTGGCTTCCCCATGGCCTTCCTCATTTTCAACATGTT

CTACTGGATCATCTACAAGATTGTCCGTAGAGAGGACGTCCACAACCAGTGA

SEQ ID NO:34 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (720nt)

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA

ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT

CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG

TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG

TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC

AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG

TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC

CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA

GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

TGGACGAGCTGTACAAGTAA

SEQ ID NO:35 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (no start codon)

(717nt)

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACG

GCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAT

CTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC

TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA

CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG

CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA

ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCC

CATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC

CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGG

ACGAGCTGTACAAGTAA

SEQ ID NO:36 Enhanced green fluorescent protein (eGFP) nucleic acid sequence (no stop codon)

(717nt) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA

ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTT

CATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG

TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG

TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG

CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC

AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG

TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC

CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA

GACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

TGGACGAGCTGTACAAG

SEQ ID NO:37 Enhanced qreen fluorescent protein (eGFP) nucleic acid sequence (no start or stor

GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACG

GCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCAT

CTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC

TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGA

CACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG

CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGA

ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCC

CATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC

CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGG ACGAGCTGTACAAG

SEQ ID NO:38 Hemaqqlutinin (HA) nucleic acid sequence (27nt)

TACCCATACGATGTTCCAGATTACGCT

SEQ ID NO:39 GABA-Rho1-P295G-395HA transmembrane portion nucleic acid sequence (630nt)

CACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCT

TCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCAC

CATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGG

GTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGG

AGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAAT

GCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCATACCCATACGATGTT

CCAGATTACGCTGAGAATGGAGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGA

GCTCCCCACAGAGGAAAAGTCAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAA

ATACTCCAGGATCATCTTTCCAGCAGCATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG

Key: P295G: HA tag SEQ ID NO:40 Haemonchus contortus E145G GluCI subunit nucleic acid uence

ATGCGCAATTCCGTCCCTCTGGCGACTCGAATAGGGCCTATGCTGGCCCTCATCTGTACTGTCAGCACAA

TTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTA

CGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACG

GTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTT

TTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCC

GTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTC

CAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACG

GCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTT

GGATCGACAGAACTGCCTTATCGATCTCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGG

AAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATG

TCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTT

GCGAAGAGAGTACAGTTACTACCTCATCCAGCTCTACATTCCATGTATTATGCTCGTTGTGGTTTCATGG

GTTTCTTTCTGGCTCGATAAGGATGCCGTACCAGCTCGAGTGTCTCTGGGTGTCACGACACTGCTCACAA

TGACAACTCAGGCAAGCGGTATCAACTCCAAACTCCCACCTGTCTCTTACATCAAGGCTGTGGACGTGTG

GATCGGCGTATGTTTGGCGTTCATTTTCGGAGCTCTACTTGAATATGCCGTTGTGAATTATTATGGCCGC

AAGGAATTTCTTCGGAAGGAGAAAAAGAAGAAAACGCGTCTGGACGACTGCGTTTGCCCGTCTGAACGTC

CTGCTCTACGGCTTGACTTGAGCAATTATCGTCGACGGGGTTGGACTCCGCTGAATAGGCTATTGGATAT

GTTGGGTCGAAATGCGGATCTCTCACGTAGGGTGGACTTAATGTCACGGATCACTTTTCCCTCACTGTTT

ACAGCATTTCTAGTGTTCTACTATTCAGTGTACGTGAAACAAAGCAACCTCGACTGA

SEQ ID NO:41 Haemonchus contortus GluCI E145G qlutamate bindinq portion nucleic acid

ATGCGCAATTCCGTCCCTCTGGCGACTCGAATAGGGCCTATGCTGGCCCTCATCTGTACTGTCAGCACAA

TTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTA

CGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACG

GTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTT

TTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCC

GTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTC

CAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACG

GCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTT

GGATCGACAGAACTGCCTTATCGATCTCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGG

AAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATG

TCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTT GCGAAGA

SEQ ID NO:42 Short Haemonchus contortus GluCI E145G subunit nucleic acid

ATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAAC

AGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTG GCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGAC

GTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCC

GTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTC

ACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAG

CCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGT

CCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATCTCGCATCATATGC

GTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGT

CAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAG

AATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGAGAGTACAGTTACTACCTCATCCAGCTCTACAT

TCCATGTATTATGCTCGTTGTGGTTTCATGGGTTTCTTTCTGGCTCGATAAGGATGCCGTACCAGCTCGA

GTGTCTCTGGGTGTCACGACACTGCTCACAATGACAACTCAGGCAAGCGGTATCAACTCCAAACTCCCAC

CTGTCTCTTACATCAAGGCTGTGGACGTGTGGATCGGCGTATGTTTGGCGTTCATTTTCGGAGCTCTACT

TGAATATGCCGTTGTGAATTATTATGGCCGCAAGGAATTTCTTCGGAAGGAGAAAAAGAAGAAAACGCGT

CTGGACGACTGCGTTTGCCCGTCTGAACGTCCTGCTCTACGGCTTGACTTGAGCAATTATCGTCGACGGG

GTTGGACTCCGCTGAATAGGCTATTGGATATGTTGGGTCGAAATGCGGATCTCTCACGTAGGGTGGACTT

AATGTCACGGATCACTTTTCCCTCACTGTTTACAGCATTTCTAGTGTTCTACTATTCAGTGTACGTGAAA

CAAAGCAACCTCGACTGA

SEQ ID NO:43 Short Haemonchus contortus GluCI E145G glutamate binding portion nucleic acid seguence (738nt)

ATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAAC

AGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTG

GCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGAC

GTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCC

GTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTC

ACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAG

CCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGT

CCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATCTCGCATCATATGC

GTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGT

CAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAG

AATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGA

SEQ ID NO:44 Hc-Rho1- E145G-P295G-395HA nucleic acid seguence (1407nt)

ATGCGCAATTCCGTCCCTCTGGCGACTCGAATAGGGCCTATGCTGGCCCTCATCTGTACTGTCAGCACAA

TTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTA

CGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACG

GTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTT

TTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCC

GTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTC CAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACG

GCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTT

GGATCGACAGAACTGCCTTATCGATCTCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGG

AAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATG

TCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTT

GCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGG

GTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCA

TGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTA

CCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACT

GTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCA

CTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGAATGACCTGGACAACTACATGCCATACCCATA

CGATGTTCCAGATTACGCTGAGAATGGAGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCA

GAGAGGAGC T C CC C AC AGAGG AAAAGT C AGAGAAGC AGC TAT GT GAGCAT GAGAAT CG AC AC CC AC GC C A

T T GAT AAAT AC T C C AGG AT CAT C T T T C C AGC AGC AT AC AT T T T AT T C AAT T T AAT AT ACT GGT C T AT T T T

CTCCTAG

Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G; HA tag

SEQ ID NO:45 Short-Hc-Rho1 -E145G-P295G-395HA nucleic acid sequence (1368nt)

ATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGGAAACTCAAAGAAC

AGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGATTAAATGCTTCCTG

GCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTCAAAAATTGACGAC

GTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGGCTTGCCTACGGCC

GTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGAATGCCGACCAGTC

ACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACATCTCATAGACAAG

CCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTATCTCTGGTGCTGT

CCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATCTCGCATCATATGC

GTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAAGGACGGCTTACGT

CAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAAACCAATACCGGAG

AATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGCTCCAAACTTATTT

CCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGCCGTGCCTGCCAGA

GTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAATGCCTCCATGCCGC

GCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGTTCCTCTCGGTGCT

GGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCTGCGGGAGAAGCTT

CCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGTGATGGGGAGGTGA

ATGACCTGGACAACTACATGCCATACCCATACGATGTTCCAGATTACGCTGAGAATGGAGAGAAGCCCGA

CAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCAGC

TAT GT GAGCAT GAGAAT CG AC AC CC AC GC CAT T G AT AAAT AC T C C AGG AT CAT C T T T C C AGC AGC AT AC A

TTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAG Key: H.c. GluCI glutamate binding portion; GABA-Rho1 transmembrane portion; P295G; HA tag

SEQ ID NO:46 Homo sapiens CAMKII promoter nucleic acid sequence (1300nt)

TAAATAAATAAATAAATAATATAAATAATAAATGTCCAGGAATCAGAGCTCAAACTCAGATCCTTAGTCT

TAAACTCCAGTCCCTTTTCTTCCTAACTCCAAGACCTTGGAGTAAGATCTTGTGGCTGTAGGTATGGCTG

ATGCCCTGAAGAGTTGAAGTTGGCAGGGAAGGTGCCCAGAAAATTTTGGATTGAAGATTTCATGGCAAGT

CTCTGGCCAGTGGCCTAGCCCGGGTAAGCCATGCTATGCTCACCTCCCCACAGCCCCCTCTCGCCTTTTT

TTTTTTTTTTTTTTTTACCTTGACTGGAAGCACAAGCAGAAACTGGGACATGAGCACCAGGAGACCAGAT

TTCCATGGTCCCGTTGGGGGCATGGGGTTGGGGAGAGGTTGCAGAGGAGGGCTCTGGAGGGGAGCAACTG

TCACAGCTGTGAGAGGTGGGGGTGAGCAGGCAGTCAGGGCTGTTCCCTCCAGAATCCTGGGGTGTCCTCT

GCACTTCTGCGCCAAGCTGGAGTGCTAGTGTGATGGACAAGGTGGTAAGAGAGCTGAAAGAGCACGAGCA

ACAGAGACTACAGTGATCCACAGAGGGAGAGCCATCCCTGTGAATTAGCCATCATTTCCCTGTAAACCTT

AGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTCTCTAGAGATGAAGTTGCCAGGGTAACTG

CATCCTGTCATTCGTTCCTGGGGACCATCCGGAATGCGGCACCCACTGGCTGTTACCATGGCAACTGCCT

TTTTGCCCCACTTAATCCCATCCCGTCTGCTACAAGGGCCCCACAGTTGGAGGTGGGGGAGGTGGGAAGA

GAAAAGATCACTTGTGGACAAAGTTTGCTCTATTCCACCTCCTCCAGGCCCTCCTTGGGTCCATCACCCC

AGGGGTGCTGGGTCCATCCCACCCCCAGGCCCACACAGGCTTGCAGTATTGTGTGCGGTATGGTCAGGGC

GTCCGAGAGCAGGTTTCGCAGTGGAAGGCAGGCAGGTGTTGGGGAGGCAGTTACCGGGGCAACGGGAACA

GGGCGTTTTGGAGGTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGCCAC

AGTGCCCTGCTCAGAAGCCCCGGGCTCGTCAGTCAAACCGGTTCTCTGTTTGCACTCGGCAGCACGGGCA

GGCAAGTGGTCCCTAGGTTCGGGAGCAGAGCAGCAGCGCC

SEQ ID NO:47 AAV2 ITR #1 nucleic acid sequence (141 nt)

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTT

TGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCC

T

SEQ ID NO:48 AAV2 ITR #2 nucleic acid sequence (141 nt)

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC

AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAG

G

SEQ ID NO:49 Kozak nucleic acid sequence (12nt)

GCCGCCACCATG

SEQ ID NQ:50 WPRE nucleic acid sequence (589nt)

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGC

TATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTC

CTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTG TGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGAA

CTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGG

GGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTC

GCCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGG

ACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAG

TCGGATCTCCCTTTGGGCCGCCTCCCCGC

SEQ ID NO:51 hGHpA nucleic acid sequence (477nt)

GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCA

GCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTG

GAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAA

CCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCC

TGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTG

GTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCT

TGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT

SEQ ID NO:52 F1 origin of replication nucleic acid sequence (456nt)

ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC

CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT

CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAAC

TTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGA

GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCT

TTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA

AC GC GAAT T T T AAC AAAAT AT T AAC GC T T AC AAT T T

SEQ ID NO:53 NeoR/KanR nucleic acid sequence (795nt)

ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACT

GGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCT

TTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTG

GCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTAT

TGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGC

TGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGC

ATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGG

GGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGAC

CCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGC

CGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCG

GCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTA

TCGCCTTCTTGACGAGTTCTTCTGA

SEQ ID NO:54 Origin of replication nucleic acid sequence (589nt) TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT

TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA

ATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT

CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA

AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGGTCGTGCACACAGCCCAGCTTGG

AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG

GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG

GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT

GCTCGTCAGGGGGGCGGAGCCTATGGAAA

SEQ ID NO:55 Short-Hc-Rho1-E145G in AAV vector nucleic acid sequence (6479nt)

AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGC

AGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCC

GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGC

AC GC GT G AAT T CC AC GGGGT T AAT C GAATAAATAAATAAATAAATAATATAAATAATAAATGTCCAGGAA

TCAGAGCTCAAACTCAGATCCTTAGTCTTAAACTCCAGTCCCTTTTCTTCCTAACTCCAAGACCTTGGAG

TAAGATCTTGTGGCTGTAGGTATGGCTGATGCCCTGAAGAGTTGAAGTTGGCAGGGAAGGTGCCCAGAAA

ATTTTGGATTGAAGATTTCATGGCAAGTCTCTGGCCAGTGGCCTAGCCCGGGTAAGCCATGCTATGCTCA

CCTCCCCACAGCCCCCTCTCGCCTTTTTTTTTTTTTTTTTTTTTACCTTGACTGGAAGCACAAGCAGAAA

CTGGGACATGAGCACCAGGAGACCAGATTTCCATGGTCCCGTTGGGGGCATGGGGTTGGGGAGAGGTTGC

AGAGGAGGGCTCTGGAGGGGAGCAACTGTCACAGCTGTGAGAGGTGGGGGTGAGCAGGCAGTCAGGGCTG

TTCCCTCCAGAATCCTGGGGTGTCCTCTGCACTTCTGCGCCAAGCTGGAGTGCTAGTGTGATGGACAAGG

TGGTAAGAGAGCTGAAAGAGCACGAGCATAACAAGAAAGACAGAGGCAGAAGCAAAAAAAAAAAAAAAAA

AAAAACAGAGGGCAACAGAGAGACAGTTACAGAGACTACAGTGATCCACAGAGGGAGAGCCATCCCTGTG

AATTAGCCATCATTTCCCTGTAAACCTTAGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTC

TCTAGAGATGAAGTTGCCAGGGTAACTGCATCCTGTCATTCGTTCCTGGGGACCATCCGGAATGCGGCAC

CCACTGGCTGTTACCATGGCAACTGCCTTTTTGCCCCACTTAATCCCATCCCGTCTGCTACAAGGGCCCC

ACAGTTGGAGGTGGGGGAGGTGGGAAGAGAAAAGATCACTTGTGGACAAAGTTTGCTCTATTCCACCTCC

TCCAGGCCCTCCTTGGGTCCATCACCCCAGGGGTGCTGGGTCCATCCCACCCCCAGGCCCACACAGGCTT

GCAGTATTGTGTGCGGTATGGTCAGGGCGTCCGAGAGCAGGTTTCGCAGTGGAAGGCAGGCAGGTGTTGG

GGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTTGGAGGTGGTTGCCATGGGGACCTGGATGCTGAC

GAAGGCTCGCGAGGCTGTGAGCAGCCACAGTGCCCTGCTCAGAAGCCCCGGGCTCGTCAGTCAAACCGGT

TCTCTGTTTGCACTCGGCAGCACGGGCAGGCAAGTGGTCCCTAGGTTCGGGAGCAGAGCAGCAGCGCCGG

ATCC . :TGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGG

AAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGAT

TAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTC

AAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGG

CTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGA

ATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACA TCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTA

TCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATC

TCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAA

GGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAA

ACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGC

TCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGC

CGTGCCTGCCAGAGTCCCCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAAT

GCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGT

TCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCT

GCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGT

GATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAGCCCGACAGGATGATGGTGC

AGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCAGCTATGTGAGCATGAG

AATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGCATACATTTTATTCAATTTA

ATATACTGGTCTATTTTCTCCTAGGATATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTG

AAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT

GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTT

TATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCA

CTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGAACTTTCGCTTTCCCCCTCCCTATTGCCAC

GGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC

GTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCG

GGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGC

TCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCG

CATCGATACCGAGCGCTGCTCGA^^

GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCT

GACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGA

CAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGC

AATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGC

ATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCT

CCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCAC

■ T ■G ■CT ■C ■C ■CTT ■C ■C ■CT ■G ■T ■CCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAG

TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG

ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATG

CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCC

CTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC

CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA

GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACG

TTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATT TATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA

TTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT

GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA

ATAGCACCTAGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGT

TCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG

CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCT

GAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTG

CTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGT

CATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGA

TCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC

GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGC

TCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCAT

GGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGAC

ATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT

ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTAT

TAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGG

TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT

CCGCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA

AAGG AT C T T CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC

AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG

CAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC

CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG

GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGGTCGTGCACACAG

CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC

TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA

GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA

TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

Key: non-coding.seguence; AAV2 ITR; hCAMKII: Kozak; H.c. GluCI glutamate binding portion; GABA-

Rho1 transmembrane portion; P295; WPRE; MGHpA; F1 origin of replication: NeoR/KanR: Origin of replication

SEQ ID NO:56 Short-Hc-Rho1-E145G-P295G in AAV vector nucleic acid sequence (6479nt)

AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGC

AGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCC

GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGC

AC GC GT G AAT T CC AC GGGGT T AAT C GAATAAATAAATAAATAAATAATATAAATAATAAATGTCCAGGAA

TCAGAGCTCAAACTCAGATCCTTAGTCTTAAACTCCAGTCCCTTTTCTTCCTAACTCCAAGACCTTGGAG

TAAGATCTTGTGGCTGTAGGTATGGCTGATGCCCTGAAGAGTTGAAGTTGGCAGGGAAGGTGCCCAGAAA

ATTTTGGATTGAAGATTTCATGGCAAGTCTCTGGCCAGTGGCCTAGCCCGGGTAAGCCATGCTATGCTCA CCTCCCCACAGCCCCCTCTCGCCTTTTTTTTTTTTTTTTTTTTTACCTTGACTGGAAGCACAAGCAGAAA

CTGGGACATGAGCACCAGGAGACCAGATTTCCATGGTCCCGTTGGGGGCATGGGGTTGGGGAGAGGTTGC

AGAGGAGGGCTCTGGAGGGGAGCAACTGTCACAGCTGTGAGAGGTGGGGGTGAGCAGGCAGTCAGGGCTG

TTCCCTCCAGAATCCTGGGGTGTCCTCTGCACTTCTGCGCCAAGCTGGAGTGCTAGTGTGATGGACAAGG

TGGTAAGAGAGCTGAAAGAGCACGAGCATAACAAGAAAGACAGAGGCAGAAGCAAAAAAAAAAAAAAAAA

AAAAACAGAGGGCAACAGAGAGACAGTTACAGAGACTACAGTGATCCACAGAGGGAGAGCCATCCCTGTG

AATTAGCCATCATTTCCCTGTAAACCTTAGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTC

TCTAGAGATGAAGTTGCCAGGGTAACTGCATCCTGTCATTCGTTCCTGGGGACCATCCGGAATGCGGCAC

CCACTGGCTGTTACCATGGCAACTGCCTTTTTGCCCCACTTAATCCCATCCCGTCTGCTACAAGGGCCCC

ACAGTTGGAGGTGGGGGAGGTGGGAAGAGAAAAGATCACTTGTGGACAAAGTTTGCTCTATTCCACCTCC

TCCAGGCCCTCCTTGGGTCCATCACCCCAGGGGTGCTGGGTCCATCCCACCCCCAGGCCCACACAGGCTT

GCAGTATTGTGTGCGGTATGGTCAGGGCGTCCGAGAGCAGGTTTCGCAGTGGAAGGCAGGCAGGTGTTGG

GGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTTGGAGGTGGTTGCCATGGGGACCTGGATGCTGAC

GAAGGCTCGCGAGGCTGTGAGCAGCCACAGTGCCCTGCTCAGAAGCCCCGGGCTCGTCAGTCAAACCGGT

ATCCGCCGCCACCATGCTGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGG

AAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGAT

TAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTC

AAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGG

CTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGA

ATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACA

TCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTA

TCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATC

TCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAA

GGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAA

ACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGC

TCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGC

CGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAAT

GCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGT

TCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCT

GCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGT

GATGGGGAGGTGAATGACCTGGACAACTACATGCCAGAGAATGGAGAGAAGCCCGACAGGATGATGGTGC

AGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAGTCAGAGAAGCAGCTATGTGAGCATGAG

AATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTTCCAGCAGCATACATTTTATTCAATTTA

ATATACTGGTCTATTTTCTCCTAGGATATC^GCTTATCGAT^T_CAACCTCT_GGATTACAAAATTTGT_G

AAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT

GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTT

TATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCA CTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGAACTTTCGCTTTCCCCCTCCCTATTGCCAC

GGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC

GTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCG

GGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGC

TCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCG

CATCGATACCGAGCGCTGCTCGA^^

GGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCT

GACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGA

CAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGC

AATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGC

ATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCT

CCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCAC

■ T ■G ■CT ■C ■C ■CTT ■C ■C ■CT ■G ■T ■CCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAG

TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG

ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATG

CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCC

CTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC

CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC

TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA

GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACG

TTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATT

TATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA

TTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT

GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA

ATAGCACCTAGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGT

TCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATG

CCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCT

GAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTG

CTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGT

CATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGA

TCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC

GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGC

TCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCAT

GGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGAC

ATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT

ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTAT

TAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGG

TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCA

AAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG CAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGGTCGTGCACACAG CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

Key: non-codinaseguence; AAV2 ITR; hCAMKII: Kozak; H.c. GluCI glutamate binding portion; GABA- Rho1 transmembrane portion; P295G; WPRE; hGHpA; F1 origin of replication: NeoR/KanR: Origin of replication

SEQ ID NO:57 Short-Hc-Rho1-E145G-P295G-395HA in AAV vector nucleic acid sequence (6506nt)

.^CGCCAGC^CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT

AGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCC GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGC

ACGCGTGAATTCCACGGGGTTAATCGAATAAATAAATAAATAAATAATATAAATAATAAATGTCCAGGAA TCAGAGCTCAAACTCAGATCCTTAGTCTTAAACTCCAGTCCCTTTTCTTCCTAACTCCAAGACCTTGGAG TAAGATCTTGTGGCTGTAGGTATGGCTGATGCCCTGAAGAGTTGAAGTTGGCAGGGAAGGTGCCCAGAAA

ATTTTGGATTGAAGATTTCATGGCAAGTCTCTGGCCAGTGGCCTAGCCCGGGTAAGCCATGCTATGCTCA

CCTCCCCACAGCCCCCTCTCGCCTTTTTTTTTTTTTTTTTTTTTACCTTGACTGGAAGCACAAGCAGAAA

CTGGGACATGAGCACCAGGAGACCAGATTTCCATGGTCCCGTTGGGGGCATGGGGTTGGGGAGAGGTTGC

AGAGGAGGGCTCTGGAGGGGAGCAACTGTCACAGCTGTGAGAGGTGGGGGTGAGCAGGCAGTCAGGGCTG

TTCCCTCCAGAATCCTGGGGTGTCCTCTGCACTTCTGCGCCAAGCTGGAGTGCTAGTGTGATGGACAAGG

TGGTAAGAGAGCTGAAAGAGCACGAGCATAACAAGAAAGACAGAGGCAGAAGCAAAAAAAAAAAAAAAAA

AAAAACAGAGGGCAACAGAGAGACAGTTACAGAGACTACAGTGATCCACAGAGGGAGAGCCATCCCTGTG

AATTAGCCATCATTTCCCTGTAAACCTTAGAACCCAGCTGTTGCCAGGGCAACGGGGCAATACCTGTCTC

TCTAGAGATGAAGTTGCCAGGGTAACTGCATCCTGTCATTCGTTCCTGGGGACCATCCGGAATGCGGCAC

CCACTGGCTGTTACCATGGCAACTGCCTTTTTGCCCCACTTAATCCCATCCCGTCTGCTACAAGGGCCCC

ACAGTTGGAGGTGGGGGAGGTGGGAAGAGAAAAGATCACTTGTGGACAAAGTTTGCTCTATTCCACCTCC

TCCAGGCCCTCCTTGGGTCCATCACCCCAGGGGTGCTGGGTCCATCCCACCCCCAGGCCCACACAGGCTT

GCAGTATTGTGTGCGGTATGGTCAGGGCGTCCGAGAGCAGGTTTCGCAGTGGAAGGCAGGCAGGTGTTGG

GGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTTGGAGGTGGTTGCCATGGGGACCTGGATGCTGAC

GAAGGCTCGCGAGGCTGTGAGCAGCCACAGTGCCCTGCTCAGAAGCCCCGGGCTCGTCAGTCAAACCGGT TCTCTGTTTGCACTCGGCAGCACGGGCAGGCAAGTGGTCCCTAGGTTCGGGAGCAGAGCAGCAGCGCCGG

ATi :TGGCCCTCATCTGTACTGTCAGCACAATTATGTCCGCAGTAGAGGCCAAGAGG

AAACTCAAAGAACAGGAGATTATCCAACGTATTCTCAATAATTACGATTGGAGAGTCAGGCCGAGGGGAT TAAATGCTTCCTGGCCAGATACTGGTGGTCCTGTGCTGGTGACGGTAAACATCTATTTGCGTTCAATTTC

AAAAATTGACGACGTTAATATGGAGTACAGTGCTCAGTTTACTTTTCGAGAAGAATGGGTGGATGCTAGG

CTTGCCTACGGCCGTTTCGAGGACGAATCCACGGAGGTGCCGCCGTTCGTAGTGTTGGCGACCAGCGAGA

ATGCCGACCAGTCACAACAGATTTGGATGCCGGACACATTCTTCCAAAATGAAAAAGGGGCACGACGACA

TCTCATAGACAAGCCGAACGTGCTCATTCGAATTCACAAGGACGGCTCGATCCTTTACAGCGTTAGGTTA

TCTCTGGTGCTGTCCTGCCCCATGTCATTGGAGTTCTACCCGTTGGATCGACAGAACTGCCTTATCGATC

TCGCATCATATGCGTACACGACGCAGGACATCAAGTACGAATGGAAGGAGCAGAATCCGGTCCAGCAGAA

GGACGGCTTACGTCAGTCATTGCCAAGTTTCGAATTGCAAGATGTCGTCACCAAGTACTGCACCAGTAAA

ACCAATACCGGAGAATACAGTTGTGCTCGGGTCAAACTTCTCTTGCGAAGACACATCTTCTTCTTCTTGC

TCCAAACTTATTTCCCCGCTACCCTGATGGTCATGCTGTCCTGGGTGTCCTTCTGGATCGACCGCAGAGC

CGTGCCTGCCAGAGTCGGCTTAGGTATCACAACGGTGCTGACCATGTCCACCATCATCACGGGCGTGAAT

GCCTCCATGCCGCGCGTCTCCTACATCAAGGCCGTGGACATCTACCTCTGGGTCAGCTTTGTGTTCGTGT

TCCTCTCGGTGCTGGAGTATGCGGCCGTCAACTACCTGACCACTGTGCAGGAGAGGAAGGAACAGAAGCT

GCGGGAGAAGCTTCCCTGCACCAGCGGATTACCTCCGCCCCGCACTGCAATGCTGGACGGCAACTACAGT

GAT GGGG AGGT GAAT GACC T GGAC AAC TAG AT GC C ATACCCATACGATGTTCCAGA^ G

GAGAGAAGCCCGACAGGATGATGGTGCAGCTGACCCTGGCCTCAGAGAGGAGCTCCCCACAGAGGAAAAG

TCAGAGAAGCAGCTATGTGAGCATGAGAATCGACACCCACGCCATTGATAAATACTCCAGGATCATCTTT

CCAGCAGCATACATTTTATTCAATTTAATATACTGGTCTATTTTCTCCTAGGATATCAAGCTTATCGATA

ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCT

ATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCC

TTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGT

GCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGAAC

TTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGG

GCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCG

CCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGA

CCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGT

CTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTA

ATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGG

TATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAG

TGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCT

CCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGG

GTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAA

GGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC

TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCG

CGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCA

TACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA

CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG

GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT

TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA

ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA

GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTC

GGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG

ACAATAACCCTGATAAATGCTTCAATAATAGCACCTAGATCAAGAGACAGGATGAGGATCGTTTCGCATG

ATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGG

CACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTT

TGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCC

ACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGG

GCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGA

TGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATC

GAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGC

TCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCA

TGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGG

CTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG

AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCG

CCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCAT

CTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTA

TTTTTCTAAATACATTCAAATATGTATCCGCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT

GAGC GT C AG AC CC CGT AGAAAAG AT C AAAGG AT C T T CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG

CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT

CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCC

ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC

CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG

GGCTGAACGGGGGGGTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC

AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG

GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG

TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

Key: npn-c0din3.segu.ence; AAV2 ITR; hCAMKII: Kozak; H.c. GluCI glutamate binding portion; GABA-

Rho1 transmembrane portion; P295G; HA; WPRE; hGHpA; F1 origin of replication: NeoR/KanR; Origin of replication

SEQ ID NO:58 T2A linker protein sequence (18aa)

EGRGSLLTCGDVEENPGP

SEQ ID NO:59 T2A linker nucleic acid sequence (54nt) GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA

SEQ ID NQ:60 P2A linker protein sequence (19aa)

ATNFSLLKQAGDVEENPGP

SEQ ID NO:61 P2A linker nucleic acid sequence (57nt)

GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

SEQ ID NO:62 E2A linker protein sequence (20aa)

QCTNYALLKLAGDVESNPGP

SEQ ID NO:63 E2A linker nucleic acid sequence (60nt)

CAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAACCCAGGTCCC

SEQ ID NO:64 F2A linker protein sequence (22aa)

VKQTLNFDLLKLAGDVESNPGP

SEQ ID NO:65 F2A linker nucleic acid sequence (66nt)

GTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCT

SEQ ID NO:66 GSG linker protein sequence (3aa)

GSG

SEQ ID NO:67 GSG linker nucleic acid sequence Ont)

GGATCAGGA

SEQ ID NO:68 IRES nucleic acid sequence (568nt)

TCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATG

TTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGA

GCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGT

TCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCT

GGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGT

GCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCT

GAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGT

GTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACAC

GATGATAA

SEQ ID NO:69 Hexahistidine protein sequence (6aa)

HHHHHH References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein by reference.

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Claims

Claims: What is claimed is:

1 . A chimeric fusion protein, comprising:

(i) a transmembrane domain; and

(ii) a glutamate binding domain; wherein (i) and (ii) are heterologous.

2. The chimeric fusion protein of claim 1 , wherein the transmembrane domain is a transmembrane portion of a Homo sapiens chloride channel subunit.

3. The chimeric fusion protein of claim 1 or claim 2, wherein the transmembrane domain is a transmembrane portion of a gamma-aminobutyric acid (GABA) receptor Rho1 subunit.

4. The chimeric fusion protein of claim 3, wherein the transmembrane domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

5. The chimeric fusion protein of claim 3 or claim 4, wherein the transmembrane domain comprises an amino acid substitution at position P295, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1.

6. The chimeric fusion protein of claim 5, wherein the substitution is a Proline (P) Glycine (G) substitution (P295G).

7. The engineered GluCI of claim 6, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:7.

8. The chimeric fusion protein of any one of claims 1 to 7, wherein the chimeric fusion protein further comprises:

(iii) a peptide tag.

9. The chimeric fusion protein of claim 8, wherein the peptide tag comprises a haemagglutinin (HA) epitope tag; optionally wherein the HA epitope tag comprises the amino acid sequence set forth in SEQ ID NO:13.

10. The chimeric fusion protein of claim 8 or claim 9, wherein the peptide tag is joined to the transmembrane domain; optionally wherein the amino acid sequence of the peptide tag interrupts the amino acid sequence of the transmembrane domain.

11. The chimeric fusion protein of claim 10, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:14.

12. The chimeric fusion protein of any one of claims 1 to 11 , wherein the glutamate binding domain is a glutamate binding portion of a glutamate-gated chloride channel (GluCI) subunit.

13. The chimeric fusion protein of claim 12, wherein the glutamate binding domain is a glutamate binding portion of a Haemonchus contortus glutamate-gated chloride channel (GluCI) subunit.

14. The chimeric fusion protein of claim 13, wherein the glutamate binding domain comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:20.

15. The chimeric fusion protein of claim 14, wherein the glutamate binding domain comprises an amino acid substitution at position E145, as numbered with reference to the amino acid sequence set forth in SEQ ID NO:1.

16. The chimeric fusion protein of claim 15, wherein the substitution is a Glutamic acid (E) Glycine (G) substitution (E145G).

17. The chimeric fusion protein of claim 16, wherein the glutamate binding domain comprises the amino acid sequence set forth in:

SEQ ID NO:16; or

SEQ ID NO:18.

18. The chimeric fusion protein of any one of claims 1 to 17, comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .

19. The chimeric fusion protein of claim 18, comprising the amino acid sequence set forth in:

SEQ ID NO:2; or

SEQ ID NO:3; or

SEQ ID NO:4; or

SEQ ID NO:21 ; or

SEQ ID NO:22.

***

20. An engineered glutamate-gated chloride channel (GluCI) comprising two or more subunits, wherein at least one subunit comprises the chimeric fusion protein of any one of claims 1 to 19.

21. The engineered GluCI of claim 20, having a half-maximal effective concentration (EC50) for glutamate of between 1 and 100 μM.

22. The engineered GluCI of claim 21 , having an EC50 for glutamate of between 10 and 20 μM.

23. A nucleic acid encoding the chimeric fusion protein of any one of claims 1 to 19.

24. The nucleic acid of claim 23, comprising at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:23.

25. The nucleic acid of claim 24, comprising the nucleic acid sequence set forth in:

SEQ ID NO:24; or

SEQ ID NO:25; or

SEQ ID NO:26; or

SEQ ID NO:44; or

SEQ ID NO:45.

26. An expression vector, comprising the nucleic acid of any one of claims 23 to 25, operably linked to a promoter.

27. The expression vector of claim 26, wherein the promoter comprises a human calcium-calmodulin (CaM)-dependent protein kinase II (hCaMKII) promoter; optionally comprising the nucleic acid sequence set forth in SEQ ID NO:46.

28. The expression vector of any one of claims 26 to 27, wherein the expression vector is a viral expression vector.

29. The expression vector of claim 28, wherein the viral expression vector is an AAV vector; optionally 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.

30. The expression vector of any one of claims 26 to 29, further comprising one or more of the following elements:

(a) an AAV2 inverted terminal repeat (ITR) sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:47 or SEQ ID NO:48; and/or

(b) a Kozak sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:49; and/or

(c) a woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (WPRE) sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NQ:50; and/or

(d) a human growth hormone polyadenylation signal (hGHpA) sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:51 ;

(e) an F1 origin of replication sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:52; and/or

(f) a neomycin or kanamycin resistance gene (NeoR/KanR) sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:53; and/or

(g) an origin of replication sequence, optionally comprising the nucleic acid sequence set forth in SEQ ID NO:54; and/or

(h) one or more non-coding sequences.

31. The expression vector of any one of claims 26 to 30, comprising at least 80% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:55.

32. The expression vector claim 31 , comprising the nucleic acid sequence set forth in:

SEQ ID NO:56; or

SEQ ID NO:57.

33. The expression vector of any one of claims 26 to 32, encapsidated into a recombinant virus particle.

34. A recombinant virus particle, comprising the expression vector of any one of claims 26 to 33.

35. The recombinant virus particle of claim 34, wherein the recombinant virus particle is a recombinant AAV particle; optionally an AAV 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.

36. An in vitro method of preparing a recombinant virus particle, the method comprising:

(i) transducing a cell with the expression vector of any one of claims 26 to 33;

(ii) and expressing the viral packaging and/or envelope proteins necessary for the formation of a recombinant virus particle in the cell; and

(iii) culturing the cell in a culture medium, such that the cell produces the recombinant virus particle.

37. The in vitro method of claim 36, further comprising 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.

38. The in vitro method of any one of claims 36 to 37, further comprising recovering recombinant virus particles from the cell culture medium and/or concentrating the recombinant virus particles.

39. An engineered cell, comprising one or more of the following elements:

(a) the chimeric fusion protein of any one of claims 1 to 19;

(b) the engineered GluCI of any one of claims 20 to 22;

(c) the nucleic acid of any one of claims 23 to 25;

(d) the expression vector of any one of claims 26 to 33; and/or

(e) the recombinant virus particle of any one of claims 34 to 35.

40. The cell of claim 39, wherein the engineered cell is a neuronal cell; optionally a CA1 , CA2 or CA3 pyramidal cell; or an inhibitory interneuron cell.

41. A method of treating a disease in a subject in need thereof, the method comprising: administering the chimeric fusion protein of any one of claims 1 to 19; the engineered GluCI of any one of claims 20 to 22; the nucleic acid of any one of claims 23 to 25; the expression vector of any one of claims 26 to 33, the recombinant virus particle of any one of claims 34 to 35, or the engineered cell of any one of claims 39 to 40 to the subject; optionally wherein the disease is an epilepsy; an epilepsy-related neurological disorder; a neurological disorder characterised by pathological neuronal overactivity; or a neuropsychiatric disorder characterised by pathological neuronal overactivity.

42. The chimeric fusion protein of any one of claims 1 to 19; the engineered GluCI of any one of claims 20 to 22; the nucleic acid of any one of claims 23 to 25; the expression vector of any one of claims 26 to 33, the recombinant virus particle of any one of claims 34 to 35, or the engineered cell of any one of claims 39 to 40; for use in a method of treating a disease in a subject in need thereof; the method comprising: administering the chimeric fusion protein; the engineered GluCI; the nucleic acid; the expression vector; the recombinant virus particle, or the engineered cell to the subject; optionally wherein the disease is an epilepsy; an epilepsy-related neurological disorder; a neurological disorder characterised by pathological neuronal overactivity; or a neuropsychiatric disorder characterised by pathological neuronal overactivity.

43. Use of the chimeric fusion protein of any one of claims 1 to 19; the engineered GluCI of any one of claims 20 to 22; the nucleic acid of any one of claims 23 to 25; the expression vector of any one of claims 26 to 33, the recombinant virus particle of any one of claims 34 to 37, or the cell according to any one of claims 39 to 40 in the preparation of a medicament for the treatment of a disease in a subject in need thereof; optionally wherein the disease is an epilepsy; an epilepsy-related neurological disorder; a neurological disorder characterised by pathological neuronal overactivity; or a neuropsychiatric disorder characterised by pathological neuronal overactivity.

44. An in vitro method of expressing a chimeric fusion protein in a cell, the method comprising:

(i) transfecting the cell with the nucleic acid of any one of claims 23 to 25; the expression vector of any one of claims 26 to 33; or the recombinant virus particle of any one of claims 34 to 35 and

(ii) culturing the cell in a culture medium, such that the cell expresses the chimeric fusion protein.

45. The in vitro method of claim 44, wherein the cell is a neuronal cell; optionally a CA1 , CA2 or CA3 pyramidal cell; or an inhibitory interneuron cell.