Synthetic Receptors

Abstract

The present invention relates to modified G protein-coupled receptors (GPCRs) that (i) have reduced responsiveness to endogenous activating ligands and (ii) can be activated by exogenous agonists, which can be relatively benign over-the-counter drugs such as antihistamines. The modifications include mutations at specific amino acid positions relative to the unmodified GPCR. The invention also provides methods of use, including administration of the modified GPCRs, for example, in the treatment of neural circuit disorders.

Description

[Technical field]

[0001] The present invention relates generally to synthetic G protein-coupled receptors for therapeutic uses, and methods and materials relating thereto. [Background technology]

[0002] Gene therapy using synthetic G protein-coupled receptors (GPCRs) holds great promise for sensitizing neurons to exogenous agents to achieve controllable manipulation of neural circuits. For example, the inhibitory human M4 (hM4) muscarinic receptor has been mutated to be insensitive to its endogenous ligand, acetylcholine, but sensitive to a range of molecules including the antipsychotics clozapine and olanzapine.

[0003] Receptors modified in this way are referred to in the literature as DREADDs (designer receptors exclusively activated by designer drugs) and RASSLs (receptors simply activated by synthetic ligands).

[0004] Expression of hM4 DREADD (hm4D) using adeno-associated viral (AAV) vectors in the brains of experimental animals is well tolerated and has no effect in the absence of exogenous ligand.

[0005] International Patent Application Publication No. WO2015 / 136247 (UCL Business Limited) describes how hM4-derived DREADD (hM4D(Gi)), when expressed in epileptogenic regions of the rodent brain, was able to suppress seizures on demand upon administration of clozapine, olanzapine or clozapine-N-oxide, a metabolite of clozapine. Other DREADDs have also been described.

[0006] International Patent Application Publication No. WO2018 / 045178A1 (Rutgers, State University) relates to use in treating a disease or disorder of the nervous system in a subject.

[0007] International Patent Application Publication No. WO2018 / 175443 (University of Pittsburgh-Commonwealth System of Higher Education) relates to modified ligand-gated ion channel proteins for use in excitable or secretory cells for the treatment of nervous system related diseases or disorders.

[0008] Further developments in the technology can be found in: Avaliani N et al., "DREADDs suppress seizure-like activity in a mouse model of pharmacoresistant epileptic brain tissue.", Gene therapy 23.10(2016):760-766l; Evan Wicker and Patrick A Forcelli, "Chemogenetic silencing of the midline and intralaminar thalamus blocks amygdala-kindled seizures.", Experimental neurology 283(2016):404-412; Fredrik Berglind, My Andersson, Merab Kokaia, "Dynamic interaction of local and transhemispheric networks is necessary for progressive intensification of hippocampal seizures.", Scientific reports 8.1(2018):1-15; Jana Desloovere et al., "Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy.", Epilepsia, 60.11(2019):2314-2324; described by Mikail Weston, "Olanzapine: a potent agonist at the hM4D (Gi) DREADD amenable to clinical translation of chemogenetics," Science advances 5.4(2019):eaaw1567.

[0009] Given their therapeutic utility, it is recognized that the provision of novel DREADDs with activation properties distinct from those known in the art would provide a valuable technical contribution in this field. Summary of the Invention

[0010] The inventors have observed that drugs used to activate known DREADDs may be suboptimal in some situations. For example, both olanzapine and clozapine have multiple pharmacological targets, including histaminergic, muscarinic, and dopaminergic receptors, that contribute to their antipsychotic effects. They are both prescription-only drugs.

[0011] Olanzapine is relatively well tolerated but is mildly sedating and is associated with mild weight gain, antimuscarinic side effects, eosinophilia, and sexual dysfunction. Clozapine may also be epileptogenic and is associated with white blood cell abnormalities requiring frequent blood testing, making it less attractive as an activating ligand. ***

[0012] The inventors used an innovative approach to define residues in GPCRs that alter their activation properties, making them activatable by different types of drugs than those used in prior art DREADDs: these new DREADDs can be activated by ligands that are relatively benign over-the-counter drugs, such as antihistamines.

[0013] As a non-limiting example, the inventors altered residues in the hM4D(Gi) receptor so that it can be activated by the well-tolerated antihistamine diphenhydramine.

[0014] The inventors have further identified residues in other G protein-coupled receptors that correspondingly modify pharmacology.

[0015] These modifications provide a desirable combination of properties, namely, higher potency and higher efficacy with associated drugs, and lower basal activity in the absence of ligand. These designer receptors that can be activated by commercially available drugs may be referred to herein, purely for brevity, as "GRANPA" (G Protein-Coupled Receptors Activated by Non-Prescription Drugs).

[0016] GRANPA can be used to affect or induce G protein-mediated cellular responses in target cells, such as neurons, of a subject. For example, a cell population can be transformed with a vector encoding GRANPA. Thus, GRANPA has utility in a wide range of indications, particularly in the treatment of neural circuit disorders.

[0017] More specifically, as described below, the inventors further modified the muscarinic type 4 DREADD (hM4D), derived from the M4 receptor (CHRM4) acetylcholine receptor, also known as the cholinergic receptor, by incorporation of the known Y113C and A203G substitutions. The wild-type amino acid sequence of the M4 receptor is shown in SEQ ID NO: 1. Unless otherwise stated, all numbering refers to this M4 receptor sequence.

[0018] Extensive mutagenesis of hM4D and extensive screening of activity with novel "off the shelf" ligands has identified several novel mutants that confer beneficial properties.

[0019] As with known DREADDs, in GRANPA, (i) responsiveness to endogenous activating ligands is reduced and (ii) responsiveness to exogenous agonists of the type described herein is maintained or enhanced, where "responsiveness" as used herein relates to the potency and / or efficacy of the ligand or exogenous agonist.

[0020] For example, S85V and Y416F showed improved efficacy with the ligand diphenhydramine, whereas V120I showed improved response.

[0021] "Potency" as used herein is the concentration of drug required for half-maximal effect (EC50) on the protein investigated.

[0022] "Efficacy" as used herein is the maximum effect (Emax) that can be achieved with a drug on the investigated protein compared to a control compound.

[0023] The combination of these mutations had (at least) additive effects. Mutations at these positions, and in preferred embodiments, these specific substitutions (S85V, Y416F, and V120I), individually and in combination, constitute aspects of the invention.

[0024] In the preferred hM4D GRANPA, the sequence S85V+Y113C+V120I+A203G+Y416F is incorporated.

[0025] Other mutations that confer beneficial properties are also described herein, such as L123T, L123C, L123S, L123V, or L123I, F128I, F128L, or F128V, M121F, A200T, F204Y, W413L, and I410V.

[0026] Mutations at these positions, and in preferred embodiments, these specific substitutions, individually and in combination, also constitute aspects of the invention.

[0027] Unless otherwise stated, any of these novel modifications (ie, not including the Y113C and A203G mutations) may be referred to herein for brevity as "modifications of the invention."

[0028] For example, in the further referenced preferred hM4D GRANPA, L123T is incorporated in combination with S85V, Y416F, and / or V120I.

[0029] GRANPA, derived from hM4D, is i Alpha subunit (or G i / G0, or Gi protein) and activates G protein-coupled inwardly rectifying potassium channels (GIRKs).

[0030] As mentioned above, the inventors have identified corresponding residues in other G protein-coupled receptors, and therefore their pharmacology may be modified as well. Thus, the present invention has broad applicability to GPCRs. Unless otherwise stated, whenever the modifications of the present invention are described with reference to the M4 receptor sequence, it will be understood that the disclosure applies mutatis mutandis to the corresponding modifications in the GPCRs discussed herein. The identification of "corresponding" positions and modifications will be described in detail below, and therefore such corresponding modifications should also be understood as "modifications of the present invention".

[0031] Thus, in one aspect of the invention there is provided an engineered G protein-coupled receptor (GPCR), the engineered GPCR comprising: Compared to the parent native GPCR, (i) reduced responsiveness to endogenous activating ligands; (ii) preserved or enhanced responsiveness to an exogenous agonist; The modified GPCR comprises one, more preferably two, three or four modifications relative to the parent GPCR at positions selected from: (i)85 (ii)416 (iii)120 (iv)123 In addition, optionally, it comprises one or more modifications, relative to the parent GPCR, at a position selected from the following: (v)128 (vi)121 (vii)200 (viii)204 (viii)410 (vix)413

[0032] In all cases herein (unless the context requires otherwise), amino acid positions given for modified GPCRs are numbered relative to the amino acid sequence of SEQ ID NO: 1 (i.e. the amino acid positions correspond to the numbering of SEQ ID NO: 1). As described in more detail below, the actual amino acid numbering may therefore differ in the GPCR in question compared to SEQ ID NO: 1.

[0033] As explained above, the modified GPCR may comprise the following residues at the following positions: (a) 113C or 113N, and (b)203G

[0034] The modified GPCR may comprise one or more of the following substitutions at the following positions: (a) Y113C or Y113N, and (b) A203G

[0035] The modified GPCR may comprise one or more of the following residues at the following positions: (i) 85V or 85C (ii) 120I (iii) 416F (iv) 123C, 123I, 123T, or 123S

[0036] The modified GPCR may comprise one or more of the following substitutions at the following positions: (i) S85V or S85C, most preferably S85V (ii) V120I (iii) Y416F (iv) L123C, L123I, L123T, or L123S

[0037] The modified GPCR may comprise, in addition to residues (a) and (b) or modifications (a) and (b), the residues or substitutions (i); (i) and (ii); (i), (ii), and (iii); (i), (ii), (iii), and (iv) above.

[0038] In addition to the residues or modifications described above, the modified GPCR may include one or more of the following residues at the following positions: (v) 128I, 128L, or 128V (vi)121F (vii) 200T (viii) 204Y(viii) 410V (ix)413L

[0039] The modified GPCR may comprise one or more of the following substitutions at the following positions: (v) F128I, F128L, or F128V (vi) M121F (vii) A200T (viii)F204Y (viii) I410V (ix) W413L

[0040] In one embodiment, the modified GPCR comprises Y113C+A203G+S85V+L123T+V120I+Y416F.

[0041] In one embodiment, the modified GPCR comprises Y113C+A203G+S85V+Y416F.

[0042] In some embodiments, the modified GPCR is Y113C+A203G, Y113C+A203G+S85V+Y416F, Y113C+A203G+S85V+Y416F+V120I, Y113C+A203G+S85V+Y416F+L123T, Y113C+A203G+S85V+Y416F+V120I+L123T, Y113C+A203G+S85V+Y416F+L123C, Y113C+A203G+S85V+Y416F+V120I+L123C, Y113C+A203G+S85V+Y416F+L123S, Y113C+A203G+S85V+Y416F+V120I+L123S, Y113C+A203G+S85V+Y416F+L123I, Y113C+A203G+S85V+Y416F+V120I+L123I, or Includes Y113C+A203G+S85V+Y416F+L123V. ***

[0043] In another aspect, a method of increasing the potency and / or efficacy of an exogenous agonist that is an antihistamine for a parent modified G protein-coupled receptor (GPCR), wherein the parent modified GPCR comprises modified residues at the following positions: (a) 113, and (b)203 making further modifications at one or more further positions in the parent modified GPCR, as described above; The amino acid positions of the parent modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0044] In one embodiment, the exogenous agonist (or exogenous ligand) is diphenhydramine or an analog thereof. In another embodiment, the exogenous agonist or ligand is selected from Table 1 or Table 2, e.g., diphenhydramine, cyproheptadine, diphenylpyraline, desloratadine, benzatropine. ***

[0045] GPCR As used herein, "GPCR" refers to a receptor that transmits a G protein-mediated signal that results in a cellular response upon binding of its natural ligand and activation of the receptor. GPCRs form a large family of evolutionarily related proteins (see International Patent Application Publication No. WO97 / 035478). Proteins that are members of the GPCR family are structurally related and generally consist of seven putative transmembrane domains.

[0046] As explained at https: / / www.addgene.org / guides / chemogenetics / (the contents of which are incorporated herein by cross-reference), GPCRs are the largest group of membrane receptors in eukaryotes and the largest class of signaling molecules in the brain. GPCRs are cell surface receptors that can intercept a variety of extracellular signals, including light, peptides, sugars, and lipids, and relay the signal to intracellular G proteins. Intracellular G proteins that bind to GPCRs are composed of three subunits, namely alpha, beta, and gamma subunits. In the resting state, heterotrimeric G proteins bind to GPCRs, specifically the alpha subunit in its inactive, GDP-bound state. When a signal is received by the GPCR, it undergoes a conformational shift that activates the G protein, causing an exchange of GTP for GDP. The trimeric G protein now dissociates into two parts, the active, GTP-bound alpha subunit and the beta-gamma dimeric complex, which can then both diffuse laterally (still bound to the plasma membrane) and transmit the signal to other membrane proteins.

[0047] Activated G proteins can signal a variety of other proteins and activate the production of second messengers.

[0048] Each protein subunit functions because it has different versions with different binding partners. In mammals, five beta subunits, eleven gamma subunits, and twenty alpha subunits have been identified. Some of these G proteins activate targets, others may have inhibitory effects, and the combination of different G protein subunits to compose G proteins results in a diverse repertoire of G protein and GPCR signaling in organisms. For example, the alpha subunit αs (Gαs) activates adenylate cyclase, causing the production of a common second messenger. In neurons, elevated cAMP activates neuronal firing, whereas in smooth muscle, elevated cAMP causes muscle relaxation. Alternatively, the alpha subunit αi (Gαi) may inhibit adenylate cyclase, resulting in opposing effects (neuronal inhibition and smooth muscle contraction, respectively). Different alpha subunits may have similar phenotypic outcomes. For example, the alpha subunit αq (Gαq) also causes smooth muscle contraction, but through activation of phospholipase C. Thus, G proteins can activate diverse signaling pathways, leading to a variety of cellular responses.

[0049] The present invention has utility for both inhibitory and excitatory GPCRs.

[0050] GPCRs usually prefer one G protein subtype but can couple to multiple subtypes. For example, the human muscarinic receptor M1 primarily activates Gαq but has also been shown to couple to the Gαi and Gαs pathways. However, the human muscarinic receptor M3 has only been shown to couple to Gαq.

[0051] Non-limiting examples of DREADDs and their activity in neurons are provided at https: / / www.addgene.org / guides / chemogenetics / .

[0052] [Table 1]

[0053] Another mechanism by which GPCRs can modify neuronal excitability and thus neurotransmission is through G protein-mediated binding to G protein-coupled inwardly rectifying potassium channels (GIRKs), where the G protein-coupled cellular response is thus membrane hyperpolarization and neuronal inhibition.

[0054] As used herein, "G protein-coupled cellular response" refers to a cellular response or signaling pathway that occurs upon ligand binding by a GPCR. Such protein-coupled cellular responses in the context of the present invention are those that modify neuronal excitability and thus neurotransmission. One response is an inhibitory response, in which activation of a receptor by a ligand causes synaptic silencing or inhibition.

[0055] As described herein, the inventors used a variety of assays, including arrestin recruitment, Gi cascade (to verify the ability to inhibit cAMP production), and electrophysiology assays (to examine G protein-dependent opening of Kir3.1 and Kir3.2 GIRKs).

[0056] In one embodiment, the GPCR is a Gi-coupled GPCR.

[0057] In one embodiment, the GPCR is coupled via a G protein to an ion channel, which is optionally inwardly rectifying, and / or the ion channel is optionally a potassium channel, preferably a protein-coupled inwardly rectifying potassium channel.

[0058] In one embodiment, the GPCR is a Gq- or Gs-coupled GPCR.

[0059] In one embodiment, the GPCR is selected from a cholinergic receptor muscarinic receptor (CHRM), a histamine receptor (HRH), a 5-hydroxytryptamine (serotonin) receptor (HTR), a dopamine receptor (DRD), an alpha adrenergic receptor (ADRA), a beta adrenergic receptor (β1-4 adrenoceptor) (ADRB).

[0060] In one embodiment, the GPCR is selected from CHRM4, CHRM3, CHRM1, CHRM2, CHRM5, HRH1, HRH2, HRH3, HRH4, 5HTR-1A, 5HTR-1B, 5HTR-1D, 5HTR-1E, 5HTR-1F, 5HTR-2A, 5HTR-2B, 5HTR-2C, 5HTR-4, 5HTR-5A, 5HTR-6, 5HTR-7, DRD-1, DRD-2, DRD-3, DRD-4, DRD-5, ADRA-1A, ADRA-1B, ADRA-1D, ADRA-2A, ADRA-2B, ADRA-2C, ADRB-1, ADRB-2, ADRB-3.

[0061] In one embodiment, the GPCR is selected from the GPCRs identified in Table 3 below. ***

[0062] Where the present invention is utilized with non-hM4 GPCRs, residues that "correspond" to those numbered for the M4 receptor (CHRM4) can be readily identified based on the disclosure herein.

[0063] Example 13 provides an alignment of a GPCR to clearly show the corresponding positions of the mutations of the invention.

[0064] It will be understood that the native residues in these GPCRs may not necessarily be identical to those given in CHRM4 used for reference numbering, nevertheless, due to the high degree of conservation among aminergic GPCRs (see, e.g., Example 13, Figures 23 and 24), the present invention may be applied to these other GPCRs as well.

[0065] By way of example, the naturally occurring residues corresponding to the amino acids of CHRM4 in selected GPCRs are as follows: S85V: CHRM(S), HRH3(C), HRH4(S). Thus, again by way of example, the corresponding substitution in HRH3 is C→V. The corresponding position is 87. Thus, this is C87V in HRH3. V120I: CHRM(V), HRH3(A), HRH4(V). Thus, by way of example, the corresponding substitution in HRH3 is A→I. The corresponding position is 122. Thus, this is A122I.

[0066] Additionally, the corresponding positions and native residues are listed in Example 13. ***

[0067] Alternatively, in any aspect or embodiment of the invention, the "Ballesteros-Weinstein numbering system" may be used to identify "corresponding" positions and residues. This class A GPCR residue numbering system (Ballesteros JA, Weinstein H, "Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors." Methods in neurosciences. 1995;25:366-428) is well understood in the art, with over 1100 citations by 2015 (see Vignir Isberg et al., "Generic GPCR residue numbers-aligning topology maps while minding the gaps." Trends in pharmacological sciences 36.1(2015):22-31).

[0068] Briefly, the Ballesteros-Weinstein numbering system is based on the presence of highly conserved residues in each of the seven transmembrane (TM) helices of GPCRs. It consists of two numbers, the first number indicating the helix, 1-7, and the second number indicating the residue position relative to the most conserved residue, defined as number 50. For example, 5.42 indicates a residue located in TM5, eight residues before the most conserved residue, Pro5.50.

[0069] Using this system, the residues described herein with reference to CHRM4 have the following Ballesteros-Weinstein numbering: S85-(2.57) Y113-(3.33) V120-(3.40) M121-(3.41) L123-(3.43) F128-(3.48) A200-(5.43) A203-(5.46) F204-(5.47) I410-(6.45) W413-(6.48) Y416-(6.51) ***

[0070] The GRANPAs of the present invention are themselves functional mutants of DREADDs or naturally occurring GPCRs, however, one of skill in the art will appreciate that further mutants derived from the GRANPAs described herein may be similarly employed in the present invention.

[0071] For example, GRANPA may contain further modifications (relative to wild type) that nevertheless do not substantially affect their activity or usefulness. According to the present invention, preferred further changes in the drugs are generally known as "conservative" or "safe" substitutions. Conservative amino acid substitutions are substitutions with amino acids of sufficiently similar chemical properties to preserve the structure and biological function of the drug. It is clear that insertions and deletions of amino acids can also occur in the above defined sequences without change in function, especially if the insertions or deletions only involve a few amino acids, e.g., less than 10, preferably less than 5, and do not remove or replace amino acids that are important for the functional validation of the drug (e.g., agonist binding pocket). The literature provides many models in which the selection of conservative amino acid substitutions can be performed based on statistical and physicochemical studies of the sequence and / or structure of natural proteins. In such cases, GRANPA retains the properties of the above defined terms, such as, for example, target cell activation in the presence of an agonist but not a natural ligand.

[0072] Furthermore, due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence can be altered or modified to provide functional variants thereof without substantially affecting the sequence of the pharmaceutical protein encoded thereby. Suitable nucleotide variants are those that have a sequence altered by the substitution of different codons that code for the same amino acid within the sequence, thus producing a silent change. ***

[0073] Modification of GPCR and expression of GRANPA can be performed by one of skill in the art in light of the present disclosure through conventional molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning: Cold Spring Harbor Laboratory Press). Exemplary vectors and promoters are described below.

[0074] Embodiments of the invention are further directed to a nucleic acid or isolated nucleic acid encoding GRANPA as described herein. Further embodiments are directed to an expression vector comprising a nucleic acid or isolated nucleic acid as described herein operably linked to a regulatory sequence.

[0075] Further embodiments are also directed to host cells comprising an expression vector described herein, or a nucleic acid encoding GRANPA as described herein.

[0076] Yet another embodiment is directed to a method for producing GRANPA as described herein, comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding GRANPA, culturing the transformed host cell under appropriate conditions to produce GRANPA, and regenerating GRANPA.

[0077] In some embodiments, the host cell is a bacterial cell or a fungal cell, which may be useful, for example, for structural analysis or for producing GRANPA proteins to raise antibodies.

[0078] In some embodiments, the host cell is a mammalian cell (e.g., a subject to be treated by the methods of the invention) or a stem cell. Suitable vectors for this purpose are described below.

[0079] For stable expression of GRANPA proteins, suitable expression hosts are bacterial expression host genera including Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Pseudomonas fluorescens or Pseudomonas stutzerei), Proteus (e.g., Proteus mirabilis), Ralstonia (e.g., Ralstonia eutropha), Streptomyces, Staphylococcus (e.g., Staphylococcus carnosus), Lactococcus (e.g., Lactococcus lactis), or Bacillus (Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, etc.). Also particularly suitable are yeast expression hosts such as Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis, or Pichia pastoris.

[0080] Also suitable are mammalian expression hosts such as mouse (e.g., NS0), Chinese hamster ovary (CHO), HEK, or baby hamster kidney (BHK) cell lines. Other eukaryotic hosts such as insect cells or viral expression systems (e.g., bacteriophages such as M13, T7 phage, or lambda, or viruses such as baculovirus) are also suitable for producing recombinant polypeptides such as GRANPA. ***

[0081] GRANPAs are mutant polypeptides that may be "substantially similar" to the wild-type reference GPCR or DREADD from which they are derived and may have at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the reference GPCR from which they are derived.

[0082] For example, they may be substantially similar to any one of the polypeptide sequences identified in Table 8, SEQ ID NOs: 1-35.

[0083] For example, in one embodiment, the modified GPCR has at least 70% sequence identity to a naturally occurring parent GPCR of any one of SEQ ID NOs: 1-35 in Table 8.

[0084] The modified GPCR may comprise a sequence as shown in any one of Tables 3 to 6, including the modifications described above.

[0085] The term "variant polynucleotide" refers to a polynucleotide that encodes GRANPA and has a particular degree of homology / identity with a parent polynucleotide or hybridizes under stringent conditions to a parent polynucleotide or its complement. For example, a variant polynucleotide has at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity with the parent polynucleotide.

[0086] For example, they may be substantially similar to any one of the polynucleotide sequences identified in Table 8, SEQ ID NOs: 36-70.

[0087] For example, the invention provides a polynucleotide comprising a nucleic acid sequence encoding a modified GPCR as described herein.

[0088] The nucleic acid may have at least 70% sequence identity to its naturally occurring parent GPCR of any one of SEQ ID NOs: 36-70 in Table 8.

[0089] Calculation of the percentage of identity between different amino acid / polypeptide / nucleic acid sequences can be performed as follows: First, a multiple alignment is generated by the ClustalX program (pairwise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delayed divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix Gonnet series, DNA weight IUB; protein gap parameters, residue specific penalty on, hydrophilic penalty on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off). Then, the percentage of identity is calculated from the multiple alignment as (N / T)*100, where N is the number of positions where the two sequences share the same residue, and T is the total number of positions compared. Alternatively, the percentage of identity can be calculated as (N / S)*100, where S is the length of the shorter sequence to be compared. The amino acid / polypeptide / nucleic acid sequences may be de novo synthesized or may be naturally occurring amino acid / polypeptide / nucleic acid sequences, or derivatives thereof.

[0090] Exogenous agonists As described above, the inventors used an innovative approach to define residues in G protein-coupled receptors (GPCRs) that alter the activation properties, making them activatable by different types of drugs than those used in prior art DREADDs, such as potentially commercially available drugs, such as antihistamines.

[0091] Thus, GRANPA is activated by the presence of an exogenous agonist. An exogenous agonist (or "drug", ligand, or small molecule, the terms are generally used interchangeably herein) is one that can be delivered directly or indirectly to a target cell that expresses GRANPA. Modes of administration are discussed in more detail below. Ligands are exogenous in that they are generally not present in target cells or are present at a basal concentration low enough that they do not activate GRANPA.

[0092] Suitable target cells in which GRANPA can be expressed are discussed in more detail below.

[0093] In one embodiment, the target cells are in the brain, and the agonist can be administered directly or can penetrate the blood-brain barrier either passively or via active transport. Usually, molecules that pass the blood-brain barrier are less charged than peptide molecules. Synthetic drugs can be made to pass or not pass the blood-brain barrier depending on the number of charged groups on the molecule (see, for example, Freidinger, 1993, Prog.Drug Res.40:33-98). Smaller molecules, for example, molecules less than 4000 Da, are also more likely to pass the blood-brain barrier.

[0094] The ligand may be a natural product, but preferably the ligand is synthetic, i.e., not naturally occurring. Preferred ligands are those that have minimal or benign biological activity other than GRANPA activation. Preferably, the ligand is a "commercially available" drug as described herein, e.g., an antihistamine or structural analog thereof. Any of these ligands described herein may be referred to as "agonists of the invention" for brevity.

[0095] One preferred ligand is the antihistamine diphenhydramine (DPH), which has been used as the active ingredient or component of several over-the-counter medications used as mildly sedating anti-allergy or anti-motion sickness treatments available in the UK, including Nytol Original, Nytol One-a-Night, Sleepeaze, Benylin Chesty Coughs, Covonia Night Time Formula, and Histergan. In the US, it is sold as Benadryl and Nytol, among other popular brands, and is a component of many other non-prescription medications, including Excedrin, Sudafed, Motrin PE, and Robitussin Night Time Cough and Cold.

[0096] DPH offers several advantages as a ligand for DREADDs, including: (i) It is well tolerated and safe. (ii) its pharmacology is well known and provides a good separation between the concentrations at which it acts as an H1 antagonist and those at which it acts at different receptors; (iii) The fact that it is effective as a mild sedative, including for use in motion sickness, is confirmation that it penetrates the human central nervous system. Many native GPCRs exhibit relatively low binding affinity for DPH compared to clozapine, which is used in existing DREADDs.

[0097] [Table 2]

[0098] The modifications described herein can be used to enhance the potency and / or efficacy of DPH or related compounds, thereby making them effective agonists for therapeutic GRANPA.

[0099] The alternative ligand may be selected from: Table 1 - Examples of drugs and their salts that have chemical similarity to diphenhydramine (-)-Cetirizine, alprazolam, amitriptyline, amoxapine, antazoline, benzatropine, bibenzonium bromide, biperiden, bromazine, bromodiphenhydramine, bromodiphenhydramine hydrochloride, brompheniramine, buclizine, butorphanol, butriptyline, captodiam, carbinoxamine, carbinoxamine maleate, cetirizine, chlorcyclizine, chlorphenamine, chlorphenoxamine, cinnarizine, cinoxacin, clomipramine, cloperastine, cyamemazine, cyclizine, cyclobenzaprine, cyclopentolate, cyclimine, demexiptyline, desipramine, desvenlafaxine, dexbrompheniramine, dimenhydrinate, dimethindene, diphenhydramine citrate, diphenhydramine hydrochloride, methyldiphenhydramine bromide, Diphenhydramine salicylate, diphenylpyraline, doxepin, doxylamine, embramine, emedastine, felbamate, fendiline, fluconazole, flumexadol, flunarizine, fluorescein, fosphenytoin, glycopyrrolate, hydroxyzine, imipramine, levallorphan, levocetirizine, mazindol, meclizine, mepyramine, mequitazine, methdilazine, methimazole Xene, mianserin, moxastine, nefopam, nortriptyline, olanzapine, orphenadrine, orphenadrine citrate, orphenadrine hydrochloride, oxytriptyline, paroxetine, perhexiline, phenindamine, pheniramine, procyclidine, progabide, protriptyline, rotoxamine, trihexyphenidyl, trimipramine, tripelennamine, triprolidine, tymazoline.

[0100] Table 2 - Other antihistamines Acrivastine, Alimemazine, Alimemazine Tartrate, Antazoline, Astemizole, Azatadine, Azelastine, Bepotastine, Bilastine, Bromazine, Bromodiphenhydramine, Brompheniramine, Buclizine, Carbinoxamine, Cetirizine, Chlorcyclizine, Chlordiphenhydramine, Chlorpyramine, Chlorphenamine, Chlorpheniramine, Cinnarizine, Clemastine, Chlofedanol, Cyclizine, Cyproheptadine, Desloratadine, Dexbrompheniramine, Dexchlorpheniramine, Dexchlorpheniramine Maleate, Dextromethorphan, Dimenhydrinate, Dimethindene Maleate, Dimethindene, Diphenyl Pyraline, Dosulepin, Doxylamine, Ebastine, Emblamine, Emedastine, Epinastine, Fexofenadine, Hydroxyzine, Ketotifen, Levocabastine, Levocetirizine, Loratadine, Meclizine, Mepyramine, Mirtazapine, Mizolastine, Naphazoline, Olopatadine, Orphenadrine, Phenindamine, Pheniramine, Phenylpropanolamine, Phenyltoloxamine, Pizotifen, Promethazine, Propiomazine, Pseudoephedrine, Pyrilamine, Quetiapine, Quifenadine, Rupatadine, Sertraline, Terfenadine, Thonzylamine, Trazodone, Trimeprazine, Tripelennamine, Triprolidine, Xylomeazoline. ***

[0101] It is understood that GRANPA as used herein is modified with respect to their corresponding native GPCR in that GRANPA shows reduced, preferably substantially reduced, more preferably substantially eliminated binding to selected natural ligands, based on the binding of natural ligands by their corresponding native GPCR.Thus, GRANPA activity is relatively unaffected by the natural fluctuations of selected natural ligands (e.g., acetylcholine).Preferably, GRANPA binding of selected natural ligands is reduced by at least 5-fold, preferably 10-fold, more preferably 50-fold, even more preferably 75-fold, and may be reduced by 100-fold or more, based on the binding of GRANPA by its corresponding native G protein-coupled receptor.

[0102] GRANPAs may also be characterized by the ratio of synthetic ligand (e.g., antihistamines or other agents as described above) binding affinity to the binding affinity of the selected natural ligand. Preferably, the GRANPAs of the invention exhibit a high synthetic ligand to selected natural ligand binding ratio, exhibiting a synthetic ligand:selected natural ligand binding ratio of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, preferably at least 1.0, more preferably at least 5, even more preferably 10, even more preferably 100 or more.

[0103] Preferably, GRANPA exhibits a synthetic ligand:selected natural ligand binding ratio that is 2-fold greater, preferably 5-fold greater, more preferably 10-fold greater, and even more preferably 50-100-fold greater than the synthetic ligand:selected natural ligand binding ratio of a native G protein-coupled receptor.

[0104] GRANPA may also be characterized by the ratio of the level of activation upon exposure to a synthetic ligand to the level of activation upon exposure to a selected natural ligand ("activation ratio"). The activation level may be measured as described in the Examples herein. Preferably, the GRANPA of the present invention exhibits a ratio of synthetic ligand activation to selected natural ligand activation, exhibiting a synthetic ligand:selected natural ligand activation ratio of at least 0.8, preferably at least 1.0, more preferably at least 5, even more preferably 10, even more preferably 100 or more. Preferably, the GRANPA exhibits an activation ratio that is 2-fold greater, preferably 5-fold greater, more preferably 10-fold greater, even more preferably 50-100-fold greater than the synthetic molecule ligand:selected natural ligand activation ratio of a natural G protein-coupled receptor. ***

[0105] The invention provides methods for making mutant or modified GPCRs by modifying peptides, or the nucleic acids encoding same, with one or more of the inventive amino acid modifications described herein, which may be used to increase the potency and / or efficacy of exogenous agonists (such as antihistamines).

[0106] The invention provides methods for producing the GRANPAs described herein by expression from nucleic acid encoding same.

[0107] The present invention provides a method for increasing the potency and / or efficacy of an exogenous agonist (such as an antihistamine) for an engineered G protein-coupled receptor (GPCR), comprising: The modified GPCR comprises modified residues at the following positions: (a) 113, and (b)203 Methods are provided which involve making additional modifications of the invention at one or more further positions in the modified GPCR, which may typically be carried out by modification of the nucleic acid encoding it.

[0108] The present invention provides the use of the variants of the invention described herein to achieve novel technical effects, such modifications being one or more amino acids introduced into a modified G protein-coupled receptor (GPCR), the modified GPCR comprising modified residues at the following positions: (a) 113, and (b)203 Increasing the potency and / or efficacy of exogenous agonists (such as antihistamines). This use may involve modification of the nucleic acid encoding it.

[0109] Thus, in one aspect, there is provided a process for generating an engineered G protein-coupled receptor (GPCR) having an altered responsiveness to an exogenous agonist, comprising modifying the parent GPCR relative to the parent GPCR by two, three, or four modifications at positions selected from: (i)85 (ii)416 (iii)120 (IV)123 and optionally comprising, relative to the parent GPCR, one or more modifications at a position selected from: (v)128 (vi)121 (vii)200 (viii)204 (viii)410 (ix)413 A process is provided in which the amino acid positions of the modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0110] This process may be to improve the potency and / or efficacy of the exogenous agonist at the engineered GPCR compared to the parent GPCR.

[0111] The parent GPCR to be modified may be derived from a naturally occurring GPCR that contains particular residues or substitutions described above (eg at 113 and / or 203).

[0112] Also provided is a modified GPCR obtained or obtainable by the processes or methods described herein.

[0113] Another aspect of the invention provides the use of a modification as described herein, which is an additional amino acid modification introduced into a parent modified G protein-coupled receptor (GPCR), for increasing the potency and / or efficacy of an exogenous agonist, such as an antihistamine, wherein the parent modified GPCR comprises modified residues at the following positions: (a) 113 and (b) 203, the amino acid positions of the modified GPCR being numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0114] Usefulness and Usage In one aspect, the invention provides a method of selectively altering activation of a G protein or activating a G protein in a cell of a subject or organism, comprising: (i) expressing GRANPA in a cell; (ii) administering to the subject or organism an agonist of the invention against expressed GRANPA.

[0115] GRANPA is normally expressed in cells prior to administration of an agonist. Such methods can be used to alter G protein activation in cells in a region- and time-specific manner.

[0116] Thus, the subject or organism may have been previously administered a polynucleotide prior to the performance of the method, in which case a polynucleotide comprising a nucleic acid sequence encoding a heterologous GRANPA is already present within the cells of the subject or organism.

[0117] Once activated, the G protein can then inhibit or stimulate further signaling pathways and cellular processes or responses that can affect, for example, the excitability or other characteristics of a cell, tissue, subject, or organism (see the discussion of Gαs, Gαi, and Gαq proteins and corresponding modified GPCRs above).

[0118] Subject or organism The mammal may be a human subject.

[0119] The mammal may be a non-human mammal, for example a test animal such as a rodent (e.g., mouse, rat) or a primate. The mammal may be a transgenic mammal.

[0120] The subject or organism may be a bird, fish, reptile, or amphibian.

[0121] Such test animals (non-human) form a further aspect of the present invention.

[0122] Target cell type As described herein, the methods are useful in a wide variety of target cell types, and the method or mode of expression (e.g., cell-specific expression) and administration will be adopted depending on the subject and the desired target cell type.

[0123] Preferably, the cell is an "excitable cell" such as a neuron of the central or peripheral nervous system, a muscle cell, including striated and smooth muscle, or an endocrine cell.

[0124] GRANPA may be useful for manipulating the autonomic nervous system and heart, as hM3D(Gq) has been used in this way previously (Agulhon C, Boyt KM, Xie AX, Friocourt F, Roth BL, McCarthy KD, "Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo." J Physiol. 2013 Nov 15;591.22:5599-609, doi:10.1113 / jphysiol.2013.261289, Epub 2013 Sep 16, PMID:24042499, PMCID:PMC3853498; Kaiser E, Tian Q, Wagner M, Barth M, Xian W, Schroeder L, Ruppenthal S, Kaestner L, Boehm U, Wartenberg P, Lu H, McMillin SM, Bone DBJ, Wess J, Lipp P, "DREADD technology reveals major impact of Gq signaling on cardiac electrophysiology.", Cardiovasc Res.2019 May 1;115(6):1052-1066, doi:10.1093 / cvr / cvy251, PMID:30321287, PMCID:PMC6736079).

[0125] GRANPA may have utility in altering pancreatic function, as hM4D(Gi) has previously been used in this way to manipulate pancreatic alpha cells (Zhu L, Dattaroy D, Pham J, Wang L, Barella LF, Cui Y, Wilkins KJ, Roth BL, Hochgeschwender U, Matschinsky FM, Kaestner KH, Doliba NM, Wess J, "Intra-islet glucagon signaling is critical for maintaining glucose homeostasis." JCI Insight. 2019 Apr 23;5(10):e127994, doi:10.1172 / jci.insight.127994, PMID:31012868, PMCID:PMC6542600).

[0126] In other embodiments, the cell is a "non-excitable" cell, for example a hepatocyte. For example, activation of hM4D(Gi) in hepatocytes is thought to worsen glucose regulation, whereas loss of Gi in hepatocytes is thought to improve glucose regulation (Rossi M, Zhu L, McMillin SM, Pydi SP, Jain S, Wang L, Cui Y, Lee RJ, Cohen AH, Kaneto H, Birnbaum MJ, Ma Y, Rotman Y, Liu J, Cyphert TJ, Finkel T, McGuinness OP, Wess J. Hepatic Gi signaling regulates whole-body glucose homeostasis., J Clin Invest.2018 Feb 1;128(2):746-759, doi:10.1172 / JCI94505, Epub 2018 Jan 16, PMID:29337301, PMCID:PMC5785257). Therefore, Gs-linked GRANPA may have utility in improving glucose control. ***

[0127] In one aspect of the invention, there is provided a method for selectively altering neuronal excitability in a region- and time-specific manner in a mammalian central nervous system (e.g., brain), comprising: a. administering to a subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding GRANPA; b. expressing GRANPA in step (a) prior to administration of an agonist against GRANPA; c. administering to the subject an agonist for expressed GRANPA.

[0128] As explained above, in a preferred embodiment, activation of GRANPA alters the excitability of neurons in the subject's nervous system.

[0129] Typically, GRANPA is expressed in the central nervous system (brain or spinal cord). Suitable vectors and promoters for this purpose are described below.

[0130] Activation of GRANPA can inhibit neurotransmission by excitatory neurons or activate inhibitory neurons, the activation of which can result in inhibitory responses such as synaptic silencing or inhibition.

[0131] In other embodiments, activation of GRANPA can activate excitatory neurons.

[0132] In other embodiments, activation of GRANPA can inhibit excitatory neurons.

[0133] In other embodiments, activation of GRANPA can inhibit inhibitory neurons.

[0134] Thus, in one aspect, the invention provides a method for selectively altering neuronal excitability in a region- and time-specific manner in a mammalian central nervous system (e.g., brain), comprising administering to a subject: (i) expressing GRANPA prior to administration of an agonist against GRANPA; (ii) administering to the subject an agonist of the invention against expressed GRANPA.

[0135] Mode of administration Exogenous agonists may be administered by any suitable method known in the art, provided that it results in distribution to target cells that contain GRANPA.

[0136] Non-limiting routes of administration include the following: a) Oral b) Parenterally, by intravenous or intramuscular route c) Subcutaneous injection (which may be preferred in palliative care, where GRANPA is used, for example, to relieve pain, nausea, or anxiety) d) Sublingual, buccal, intranasal, rectal, for rapid absorption, routes used for benzodiazepines in the treatment of acute seizures in adults or children (e.g. rectal diazepam, buccal or nasal midazolam). e) via inhaler or nebulizer (which may be preferred in the treatment of chronic obstructive airways disease, cystic fibrosis, emphysema, bronchiolitis, or bronchiectasis due to its similarity to salbutamol, a beta-2 adrenergic agonist used to treat COAD). f) Topical: May be preferred for treating eye diseases such as glaucoma, due to its similarity to timolol (an alpha-2 adrenergic agonist) eye drops used to treat open-angle glaucoma. g) Topical: on the skin (transdermal), due to its similarity to topical clonidine gel (an alpha-2 adrenergic agonist) used to treat neuropathic pain h) Other local routes: ear, intrathecal, intravaginal, intravesical

[0137] Systemic modes of administration may be preferred.

[0138] Exogenous agonists may be adapted for route of administration according to methods known in the art, for example, oral, injectable, and topical formulations of diphenhydramine are known in the art. ***

[0139] The novel GRANPAs described herein may be useful, for example, in gene therapy for a wide range of diseases or disorders of the nervous system, such as neural circuit disorders. These include neuropsychiatric disorders, neurodegenerative diseases, chronic pain, cerebrovascular accidents (CVA), or stroke. Examples of diseases for which GRANPAs may be useful are provided below.

[0140] In certain embodiments, GRANPA is based on hM4D(Gi) (human M4 muscarinic cholinergic Gi-coupled DREADD). In certain embodiments, the DREADD is the human muscarinic acetylcholine receptor M4, including the inventive modifications described herein.

[0141] As non-limiting examples, controlled inhibition of activated neurotransmission (e.g., by inhibition of excitatory neurons via Gi-coupled receptors or activation of inhibitory neurons) has utility in epilepsy and other disorders characterized by episodes of abnormal cellular activity, such as migraine, cluster headache, trigeminal neuralgia, post-herpetic neuralgia, paroxysmal movement disorders, and unipolar or bipolar affective disorders.

[0142] In certain embodiments, GRANPA is Gq-coupled. In certain embodiments, GRANPA is based on the Gq-coupled human M3 muscarinic receptor (hM3Dq), including the inventive variations described herein (see, e.g., Alexander et al., (2009) Neuron 63(1):27-39; Armbruster et al., (2007) Proc. Natl. Acad. Set, 104(12):5163-5168).

[0143] Activation of excitatory (eg, Gs- and Gq-coupled) GPCRs may be useful in other mental health disorders, such as Parkinson's disease and other diseases in which some neural circuits are thought to be underactive. ***

[0144] In one aspect of the invention, there is provided a method of treating a disease or disorder in a subject, comprising: (i) expressing GRANPA in a target cell or organ of a subject; (ii) administering to the subject an agonist for expressed GRANPA.

[0145] In one aspect of the invention, there is provided a method of treating a disease or disorder in a subject, comprising: (a) administering to a subject, for example by direct injection, an effective amount of a polynucleotide or vector described herein encoding GRANPA; (b) expressing GRANPA of step (a) in a target cell or organ of the subject; (c) administering to the subject an exogenous agonist for expressed GRANPA.

[0146] In one aspect of the invention, there is provided a method of treating a disease or disorder of the nervous system in a subject, comprising: a. administering to a subject an effective amount of a polynucleotide comprising a nucleic acid sequence encoding GRANPA; b. expressing GRANPA in step (a) prior to administration of an agonist against GRANPA; c. administering to the subject an agonist for expressed GRANPA.

[0147] As explained above, GRANPA is normally expressed in the central nervous system (brain or spinal cord).

[0148] In one aspect, there is provided a method of treating a disease or disorder of the nervous system in a subject, comprising: (i) expressing GRANPA prior to administration of an agonist against GRANPA; (ii) administering to the subject an agonist for expressed GRANPA. ***

[0149] As described in International Patent Application Publication No. WO2015 / 136247, the use of DREADDs potentially allows fine tuning of therapeutic effects, so that optimal regulation of circuit function can be achieved with minimal off-target effects on normal brain function. Therapy can be targeted to both the brain region where the viral vector is introduced and the cell types within that region, effectively localizing the effect when the ligand is delivered. Without the ligand, one would not expect any effect on brain function. Thus, therapy is target-limited and time-limited. However, the major inhibitory DREADD hM4D(Gi) is limited by the side effect profile of the activating ligand.

[0150] Thus, in one aspect, there is provided a method of treating a seizure disorder in a patient suffering from said disorder, comprising: (a) administering to the patient a vector encoding GRANPA, wherein GRANPA is expressed neurons in a seizure focus in the patient's brain; (b) administering an exogenous agonist to the patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, Methods are provided in which activation of GRANPA reversibly alters the excitability of neurons in the seizure focus.

[0151] 1. A method of treating a seizure disorder in a patient suffering from said disorder, comprising: the patient has previously been administered a vector encoding GRANPA, where GRANPA is expressed neurons in a seizure focus in the patient's brain; administering an exogenous agonist to the patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, Methods are provided in which activation of GRANPA reversibly alters the excitability of neurons in the seizure focus.

[0152] In the context of the present invention, GRANPA can be used to treat seizure disorders in subjects suffering from the above-mentioned diseases.In such treatment, the presence of agonist in the brain of the patient activates GRANPA, thereby reversibly changing, preferably inhibiting, the excitability of neurons in seizure focus.

[0153] For example, activation of GRANPA (i) reversibly inhibits the excitability and thereby the neurotransmission of excitatory neurons in the seizure focus, or (ii) reversibly excites inhibitory neurons in the seizure focus.

[0154] In a preferred embodiment, the seizure disorder is epilepsy, such as idiopathic, symptomatic, and cryptogenic epilepsy. The methods described herein may be used to suppress or block epileptogenic activity. These methods may be used to increase the seizure threshold in the brain or neural tissue of a patient in need thereof, or to reduce epileptogenic bursts in the patient's brain cells.

[0155] The combined chemical-genetic (known as chemogenetic) approach of the present invention can be used to treat epilepsy through the suppression of seizures in a region- and time-specific manner.

[0156] In one embodiment, epilepsy is generalized epilepsy. It has been reported that silencing inhibitory interneurons can exacerbate seizures (Wicker, Evan and Patrick A. Forcelli, "Chemogenetic silencing of the midline and intralaminar thalamus blocks amygdala-kindled seizures." Experimental neurology 283 (2016):404-412), suggesting that other manipulations (silencing thalamocortical excitatory cells) can achieve on-demand seizure suppression. In one embodiment, the epilepsy is human focal epilepsy.

[0157] The patient may be diagnosed with well-defined focal epilepsy, affecting a single region of the neocortex of the brain. Focal epilepsy may arise, for example, from developmental abnormalities or following stroke, tumor, penetrating brain injury or infection.

[0158] However, the present invention may also be used to treat multiple epileptic foci simultaneously by direct injection into multiple identified locations.

[0159] Patients may also be those who have been diagnosed with drug-resistant or medically refractory epilepsy, which means that epileptic seizures continue despite appropriate administration of anti-epileptic drugs.

[0160] The patient may be under existing treatment with an anti-epileptic drug and the method aims to discontinue the existing treatment or to reduce drug therapy.

[0161] The patient may be one who has been diagnosed with epilepsy partialis continua.

[0162] The treatment of the present invention is particularly useful when permanent reduction of neuronal excitability (such as can be achieved by overexpression of potassium channels) is undesirable, for example because the risk for normal brain function is too high. Even if the epileptogenic area is in the cortical area responsible for language or motor functions, these functions will not be affected except when the ligand is administered. Patients with refractory focal epilepsy are likely to consider this a tolerable side effect.

[0163] The present invention is particularly useful for seizure disorders characterized by focal onset, such as temporal lobe epilepsy and focal neocortical epilepsy, but may also be applied to more generalized forms of epilepsy, especially as a secondary treatment indication. In these cases, the target of delivery is appropriately selected depending on the condition. For example, it may be delivered bilaterally to the thalamus. Other disorders to which the present invention may be applied therefore include infantile spasms, myoclonic and "little motor" seizures, as well as tonic-clonic and complex partial seizures.

[0164] Furthermore, the present invention may in principle be used prophylactically in some circumstances by inducing continuous changes in neuronal excitability for a period of time with the aim of "resetting" epileptogenic circuits, resulting in a sustained reduction in seizures that outlasts administration of the ligand.

[0165] Thus, in a different embodiment of the present invention, (i) the seizure disorder is epilepsy, optionally focal or generalized epilepsy; and / or (ii) the subject has been diagnosed with drug-resistant or medically refractory epilepsy; and / or (iii) the subject is under existing treatment with an antiepileptic drug and the method has the purpose of allowing the existing treatment to be discontinued or the drug therapy to be reduced; and / or (iv) the subject has been diagnosed with partial epilepsy continuum; and / or (v) the exogenous ligand is administered to the subject before the patient suffers from an epileptic seizure; and / or (vi) the exogenous ligand is administered to the subject during an epileptic seizure; and / or (vii) the exogenous ligand is administered to the subject after suffering an epileptic seizure; and / or (viii) the exogenous ligand is administered to the subject within 30 minutes before or within 24 hours after the human suffers an epileptic seizure; and / or (ix) the exogenous ligand is administered to the subject automatically, either (i) by a device coupled to an automated seizure detection mechanism, or (ii) in response to a predicted seizure by EEG analysis; and / or (x) The exogenous ligand is administered in combination therapy with one or more other agents for treating a seizure disorder. ***

[0166] In a study of diphenhydramine overdosage, the most common adverse events were tachycardia, hallucinations, somnolence, agitation, and mydriasis, and the incidence of seizures was much lower (Palmer et al., 2019, https: / / doi.org / 10.1080 / 15563650.2019.1609683). The effect of lowering the seizure threshold is a rare side effect, similar to other antihistamines, but is generally thought to be related to the antimuscarinic effect. Our pharmacological studies suggest that diphenhydramine activates GRANPA at lower concentrations than it blocks muscarinic receptors. ***

[0167] In addition to the diseases discussed above, DREADDs (and thus GRANPA) have potential in other disorders. For example, in rodent models, DREADDs have been shown to regulate neuronal activity to improve Parkinson's disease. 6 , Down Syndrome 7 , and autism 10 DREADD-based approaches have also demonstrated the ability to ameliorate disease phenotypes in a variety of conditions, such as addiction. 11,12 ,sleep 13 , aggression 14 , breathing 15 , and feeding 16-18 DREADDs also have been used to enhance and silence learned memories and to create artificial memories. 19-21 .

[0168] Other central nervous system applications of DREADD-based therapeutics suggested in the art include psychostimulant drug abuse (Ferguson et al., 2011) and ethanol (Pleil et al., 2015), depression (Urban et al., 2015), post-traumatic stress disorder (Zhu et al., 2014), refractory seizures (Katzel et al., 2014), and many other disorders (English and Roth, 2015).

[0169] In addition to the well-established effects of DREADDs on the nervous system, a number of studies have also identified potential therapeutic strategies using DREADDs in other organs. Animal models of such diseases / disorders that are treated by DREADDs include diabetes (Jain, S. et al., "Chronic activation of a designer G(q)-coupled receptor improves β cell function," J Clin Invest. 2013;123:1750-1762), metabolic disorders (Li, J. et al., "A novel experimental strategy to assess the metabolic effects of selective activation of a G(q)-coupled receptor in hepatocytes in vivo," Endocrinology 2013;154:3539-3551), inflammatory disorders (Park, J. et al., "Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal," Proc Natl Acad Sci USA 2014;111:5896-5901), and respiratory disorders (Curado, T. et al., "DREADD approach to sleep disordered breathing," Am J Respir Crit Care 2014;111:5896-5901). Med., ahead of print 10.1164 / rccm.202002-0321OC), but are not limited to these.

[0170] Specific examples of target circuits that can be manipulated by GRANPA for therapeutic benefit are described in the following studies using preclinical models of neurological and neuropsychiatric circuit disorders. Pain (Weir et al., PMID: 28969375) Spasticity and spinal cord injury (Asboth et al., PMID: 29556028; Chen et al., PMID: 30033363) Parkinson's disease, dystonia, chorea, Huntington's disease (Assaf and Schiller, PMID: 30536778) Alzheimer's disease and frontotemporal degeneration (Rorabaugh et al., PMID: 29053824; Yuan et al., PMID: 26758850) Amyotrophic lateral sclerosis (Khademullah et al., PMID: 32203578; Alami et al., PMID: 32900826) Attention-deficit / hyperactivity disorder, obsessive-compulsive disorder, impulse control disorder, Tourette's syndrome, and autism spectrum disorder (Rapanelli et al., PMID: 28584117) Schizophrenia (Boekhoudt et al., PMID: 27712862; Katzel et al., PMID: 33178010; Katzel and Kullmann, "Optogenetic and Chemogenetic Tools for Drug Discovery in Schizophrenia", https: / / doi.org / 10.1039 / 9781782622499-00234) Major depressive disorder, bipolar disorder (Biselli et al., PMID: 31633833; Muir et al., PMID: 30555161) Anxiety and generalized anxiety disorder (Hirschberg et al., PMID: 29027903; Jiang et al., PMID: 29410218) Insomnia and sleep disorders (Sasaki et al., PMID: 21647372) Anorexia, bulimia, and other eating disorders (Krashes, NBK453150; Xu et al., PMID: 29670283)

[0171] In other embodiments of the invention, the disease or disorder is a non-central nervous system and / or a non-peripheral nervous system disorder.

[0172] vector As explained above, the GRANPA of the present invention is usually expressed in vivo to provide its medical benefits. This is achieved by using a polynucleotide comprising a nucleic acid sequence encoding GRANPA, operably linked to a suitable promoter. Usually, the polynucleotide is in the form of or is included in a genetic construct comprising an open reading frame encoding GRANPA, for example under the transcriptional control of a transcriptional control element that governs cell-specific expression in central nervous system neurons or other excitable cells.

[0173] Target central nervous system neurons include spinal cord cells, such as dorsal horn cells and / or brain cells, including, but not limited to, excitatory or inhibitory cell populations of the brainstem, hindbrain, midbrain, or forebrain.

[0174] In the method of delivering a nucleic acid encoding GRANPA to a cell or patient according to any embodiment described herein, the nucleic acid may be delivered by any useful method and in any useful form, as will be appreciated by those skilled in the art of gene therapy. The nucleic acid may be a naked nucleic acid, such as a plasmid or part of a recombinant viral genome, deposited by colloidal drug delivery methods, such as, but not limited to, liposomes, e.g., cationic liposomes, or nanoparticles, as is commonly known.

[0175] In general, those skilled in the art are fully capable of constructing vectors and designing protocols for recombinant gene expression. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, marker genes, and other sequences as necessary, in addition to the elements of the invention described above. For further details, see, for example, Molecular Cloning: a Laboratory Manual, 2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, or Current Protocols in Molecular Biology, 2nd Edition, Ausubel et al., eds., John Wiley and Sons (1995, and supplemented periodically).

[0176] For example, the polynucleotide can be in the form of or contained within a viral vector that contains a nucleic acid sequence encoding GRANPA, and optionally a promoter operably linked to the 3' untranslated region.

[0177] According to the present invention, any of a variety of vectors can be used to generate GRANPA expressing cells. The vectors for use in the treatment of the present invention are suitable for in vivo gene therapy protocols. The vectors can be stable integrating vectors or stable non-integrating vectors. The preferred vectors are viral vectors, such as lentiviral vectors or AAV (adeno-associated virus) vectors.

[0178] The use of both of these types of viral vectors is well known in the field of gene therapy. By way of example only, International Patent Application Publication No. WO2008 / 011381 describes the use of these and other vectors to express receptors in subjects. The contents of that application are specifically incorporated herein by reference for its description of the preparation and characteristics of AAV and lentiviral vectors.

[0179] Briefly, as described in International Patent Application Publication No. WO2008 / 011381, AAV is a defective parvovirus, capable of infecting many cell types and non-pathogenic to humans, and is therefore a preferred vector. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably insert into chromosome 19. Another type of AAV vector comprises a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter directing cell-specific expression operably linked to a heterologous gene (here, GRANPA). Further information can be found in U.S. Patent No. 6,261,834. AAV vectors are discussed in International Patent Application Publication No. WO2018 / 175443.

[0180] Viral vectors are well known in the art and are commercially available, for example, from Viralgen, Parque Cientifico y Tecnologico de Gipuzkoa, Paseo Mikeletegi 83, 20009 San Sebastian, Spain.

[0181] Lentiviral vectors are a special type of retroviral vectors, and are usually characterized by a long latency period for infection. Furthermore, lentiviral vectors can infect non-dividing cells. Lentiviral vectors are based on the nucleic acid backbone of a virus from the lentivirus family of viruses. Usually, lentiviral vectors contain the 5' and 3' LTR regions of lentiviruses, such as SIV and HIV. Lentiviral vectors also usually contain the Rev response element (RRE) of lentiviruses, such as SIV and HIV. Examples of lentiviral vectors include those in Dull, T. et al., "A Third-generation lentivirus vector with a conditional packaging system," J. Virol 72(11):8463-71 (1998). ***

[0182] The vectors described herein can be delivered locally to target cells by a variety of access methods known in the art.

[0183] For example, liposomes or nanoparticles containing nucleic acid can be injected at a desired site, such as within or adjacent to a specific neuronal tissue. In other embodiments, recombinant viral particles (transduction particles) are delivered, e.g., injected, at a desired site, such as within or adjacent to a specific neuronal tissue, or targeted to it. The nucleic acid can be injected once or more than once to establish sufficient expression of GRANPA in the target neuron.

[0184] In particular, delivery may be via direct injection into the brain using known methods such as direct interstitial injection, burr hole craniotomy, and stereotactic injection (see, e.g., "Stereotactic and Functional Neurosurgery," Editors: Nikkhah and Pinsker, Acta Neurochirurgica Supplement, Vol. 117, 2013).

[0185] For the treatment of seizure disorders, infusions are targeted to a defined seizure focus (e.g., in focal epilepsy) or, more generally, to areas of the brain suspected of overactivity in other seizure disorders.

[0186] Vectors may be used to effect permanent transformation or may only be expressed transiently in the brain.

[0187] Therefore, in one embodiment, an expression vector is provided that comprises the polynucleotide of the present invention described above.The vector can be a virus vector, for example, an adenovirus vector and / or an adeno-associated vector (AAV), and optionally selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof.Alternatively, the vector can be a herpes virus vector, a retrovirus vector, or a lentivirus vector. ***

[0188] Preferably, the DNA construct comprises a promoter to facilitate expression of the DNA encoding GRANPA in the target cell.

[0189] The promoter may be any known in the art suitable for gene therapy. See, for example, Papadakis ED et al., "Promoters and control elements: designing expression cassettes for gene therapy," Current gene therapy, 4.1(2004):89-113, and Joshi CR, Labhasetwar V, Ghorpade A, "Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery." J Neuroimmune Pharmacol. 2017;12(1):51-83. The promoter may be a naturally occurring nucleotide sequence or a synthetic combination of a minimal promoter sequence with other regulatory elements such as enhancers. Commonly used promoters include hSyn, mdl,CBA, Ef1a, TH, CMV, mDlx5 / 6, DRD2, Drd1a.

[0190] However, specificity can only be achieved by local and cell type specific expression of the receptor, for example using tissue or region specific promoters.

[0191] For example, the promoter may direct cell-specific expression in central nervous system neurons, such as dorsal horn neurons, spinal cord cells, or brain cells, or in inhibitory or neural cells.

[0192] A promoter is "specific" for particular cells (e.g., excitable or secretory cells) if it causes gene expression in those cells to a degree sufficient to produce a useful or therapeutically effective amount of the GRANPA in those cells, but causes meaningless expression elsewhere in the context of a use, e.g., a therapeutic use.

[0193] One example is the Camk2a (alpha CaM kinase II gene) promoter, which drives expression relatively specifically in the forebrain. See, e.g., Sakurada et al. (2005) "Neuronal cell type-specific promoter of the alpha CaM kinase II gene is activated by Zic2, a Zic family zinc finger protein." Neurosci Res. 2005 Nov;53(3):323-30, Epub 2005 Sep 12.

[0194] Other neural cell type specific promoters include the NSE promoter (Liu H et al., Journal of Neuroscience, 23(18):7143-54, 2003), the tyrosine hydroxylase promoter (Kessler MA et al., Brain Research. Molecular Brain Research, 112(1-2):8-23, 2003), the myelin basic protein promoter (Kessler MA et al., Biochemical & Biophysical Research Communications, 288(4):809-18, 2001), the glial fibrillary acidic protein promoter (Nolte C et al., GLIA, 33(1):72-86, 2001), and the neurofilament gene (heavy, medium, and light chain) promoter (Yaworsky PJ et al., Journal of Biological Chemistry. 272(40):25112-20, 1997). (All of which are incorporated herein by reference at least for the promoter sequences and related sequences.) The NSE promoter is disclosed in Peel AL et al., Gene Therapy 4(1):16-24, 1997 (SEQ ID NO:69) (pTR-NT3myc, Powell Gene Therapy Center, University of Florida, Gainesville FL). A further suitable promoter is the synapsin 1 promoter (see Kugler et al., "Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area." Gene Ther. 2003 Feb:10(4):337-47). A further suitable promoter is the cd68 promoter, which is expressed in microglia. Suitable promoters for general expression include the EF1a promoter or the CAG promoter.

[0195] In one embodiment, a vector encoding GRANPA may contain any of these promoters.

[0196] In one embodiment, the nucleic acid encoding the modified GPCR is operably linked to a tissue or cell specific promoter, e.g., a neural cell type specific promoter. In one embodiment, the promoter is the CaMk2A promoter.

[0197] In yet another embodiment, the neuron-specific promoter is the preprotachykinin-1 promoter (TAC-1). ***

[0198] While it is possible for the ligand to be used (eg, administered) alone, it is often preferable to present it as a composition or formulation, for example with a pharma- ceutically acceptable carrier or diluent.

[0199] The term "pharmacologically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject in the subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0200] In some embodiments, the composition is a pharmaceutical composition (e.g., formulation, dosage form, drug product) that comprises, consists primarily of, or consists of as the sole active ingredient a ligand described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0201] In gene therapy embodiments employing viral delivery of GRANPA, as described in International Patent Application Publication No. WO2008 / 096268, a unit dose may be calculated in terms of the dose of viral particles being administered. A viral dose includes a specific number of viral particles or plaque forming units (pfu). For embodiments involving AAV, a specific unit dose is 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 Due to the presence of infectiously defective particles, including pfu or vector genomes, the particle dose may be somewhat higher (10-100 fold).

[0202] In one embodiment, the vector is 5×10 11 500 microliters of vg / ml suspension (=2.5 x 10 11 The virus is injected as a virion (viral genome). ***

[0203] For ligands used with GRANPA in the treatment of disease, appropriate dosages may be utilized based on half-life, as well as other pharmacokinetic and pharmacodynamic parameters. For example, for DPH, based on a half-life of up to about 9 hours, this would preferably mean dosing 3-4 times per day. A typical dosage based on comparable affinity for H1 and GRANPA would be about 25-50 mg (orally) 3-4 times per day for adults. However, other dosages are envisioned based on the physician's discretion.

[0204] The dosage form may be an extended or delayed release (see, e.g., Krowczynski, Laezek, “Extended-release dosage forms”, CRC press, 2020), or an immediate release form (see, e.g., Nyol, Sandeep, and MMGupta, “Immediate drug release dosage forms: A review”, Journal of Drug Delivery and Therapeutics, 3.2 (2013)).

[0205] In one embodiment, diphenhydramine is administered at 50-100 mg per day in divided doses. ***

[0206] In some embodiments, the methods or treatments of the invention may be combined with other therapies, whether symptom or disease improving.

[0207] The term "treatment" includes combination treatments and therapies, where two or more treatments or therapies are combined, for example sequentially or simultaneously.

[0208] For example, it may be beneficial to combine treatment with the compounds described herein with one or more (eg, 1, 2, 3, 4) other agents or therapies.

[0209] Suitable examples of co-treatment will be known to those skilled in the art based on the disclosure herein. In general, co-treatment can be any known in the art that is believed to provide therapeutic benefit in treating the diseases described herein, subject to the diagnosis of the individual being treated. For example, epilepsy can sometimes be improved by directly treating the underlying etiology, but anticonvulsants such as phenytoin, gabapentin, lamotrigine, levetiracetam, carbamazepine, and clobazam, as well as topiramate, which suppress abnormal discharges and seizures, are the mainstay of conventional treatment (Rho and Sankar, 1999, Epilepsia 40:1471-1483).

[0210] The particular combination is at the discretion of the physician, who will also select dosages using their common general knowledge and dosing regimens known to skilled medical practitioners.

[0211] The agents (i.e., GRANPA and the ligand, and one or more other agents) may be administered simultaneously or sequentially, with individually varying dose schedules and via different routes. For example, when administered sequentially, the agents may be administered closely spaced (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 hours or more apart, or even longer if necessary), with the exact dosage regimen depending on the nature of the therapeutic agent. ***

[0212] Where an aspect of the invention includes a method of treating a disease or disorder through the use of GRANPA, or a polynucleotide comprising a nucleic acid sequence encoding GRANPA, and / or an agonist, the following is also provided. (i) GRANPA, or a polynucleotide comprising a nucleic acid sequence encoding GRANPA (e.g., a vector as described herein), and / or an agonist, for use in such methods; (ii) the use of GRANPA, or a polynucleotide comprising a nucleic acid sequence encoding GRANPA (e.g., a vector described herein), and / or an agonist, in the preparation of a medicament for such treatment. ***

[0213] The invention also provides a vector encoding GRANPA and an exogenous agonist for the receptor for use in a method of treating a seizure disorder in a patient suffering from the disorder, the treatment comprising: (a) administering to a patient a vector in which GRANPA is expressed in neurons of a seizure focus in the patient's brain; (b) administering an exogenous agonist to the patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, Methods are provided in which activation of GRANPA reversibly alters the excitability of neurons in the seizure focus.

[0214] The invention also provides a vector encoding GRANPA for use in a method of treating a seizure disorder in a patient suffering from said disorder, the treatment comprising: (a) administering to a patient a vector in which GRANPA is expressed in neurons of a seizure focus in the patient's brain; (b) administering an exogenous agonist to the patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, Activation of GRANPA reversibly alters neuronal excitability in the seizure focus.

[0215] The invention also provides an exogenous agonist for use in a method of treating a seizure disorder in a patient suffering from said disorder, the treatment comprising: (a) administering to the patient a vector encoding GRANPA, which is expressed in neurons of a seizure focus in the patient's brain; (b) administering an exogenous agonist to the patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, Activation of GRANPA reversibly alters neuronal excitability in the seizure focus.

[0216] The invention also provides an exogenous agonist for use in a method for the treatment of a seizure disorder in a patient suffering from said disorder, The patient has previously been administered a vector encoding GRANPA, which is expressed in neurons in the seizure focus of the patient's brain; The treatment includes administering an exogenous agonist to a patient; Thereby, the presence of the agonist in the patient's brain activates GRANPA, GRANPA activation thereby reversibly alters neuronal excitability in the seizure focus.

[0217] The invention also provides vectors and / or agonists defined for use in these methods of treating seizure disorders.

[0218] The present invention also provides the use of GRANPA and / or a vector and / or a polynucleotide and / or an agonist as described herein in the preparation of a medicament for use in a treatment or method of treatment as described herein. ***

[0219] The present invention also provides kits comprising one or more components, including but not limited to a viral vector, a promoter, and GRANPA, together with one or more additional components, including but not limited to a pharma- ceutically acceptable carrier and a GRANPA agonist, are also contemplated.

[0220] The viral vector, promoter, GRANPA composition, and / or GRANPA agonist may be formulated into a pharmaceutical composition either as a pure composition or in combination with a pharma- ceutically acceptable carrier.

[0221] The kits may also contain primers, buffers, and probes along with instructions for use in the methods described herein.

[0222] In one embodiment, the kit contains a viral vector, a promoter, a GRANPA composition of the invention, or a pharmaceutical composition thereof in one container, and a GRANPA agonist or a pharmaceutical composition thereof in another container (e.g., a sterile glass or plastic vial).

[0223] If the kit includes a pharmaceutical composition for parenteral administration to a subject, it may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices.

[0224] Common expressions As used herein, a "polypeptide" refers to a molecule that comprises multiple amino acids linked via peptide bonds. The terms "polypeptide", "peptide" and "protein" are used interchangeably. Proteins can optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, and sulfonated) to add function. Conventional one-letter or three-letter codes for amino acid residues are used, and amino acid sequences are displayed in the standard amino-terminal to carboxy-terminal direction (i.e., N→C).

[0225] The term "polynucleotide" includes DNA, RNA, heteroduplexes, and synthetic molecules that can code for a polypeptide. Nucleic acids can be single-stranded or double-stranded and can have chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid, and the present compositions and methods encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0226] As used herein, the term "wild-type", "natural" or "reference" refers to a polypeptide or polynucleotide found in nature. With respect to a polypeptide, the term refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion or deletion at one or more amino acid positions. With respect to a polynucleotide, the term refers to a naturally occurring polynucleotide that does not contain an artificial substitution, insertion or deletion at one or more nucleosides. However, it should be noted that a polynucleotide encoding a wild-type or natural or reference polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes the polypeptide.

[0227] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," and "made from," and generally indicates that a particular material is derived from, or has characteristics that can be described with reference to, another particular material (which may be referred to as a "reference" or "parent"). GRANPAs herein are derived from a reference or parent sequence, which may be a wild-type GPCR or DREADD of the prior art.

[0228] The term "hybridization", as known in the art, refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing. The term "hybridization conditions" refers to the conditions under which a hybridization reaction is carried out. These conditions are usually classified by the degree of "stringency" of the conditions under which hybridization is measured. The degree of stringency can be determined, for example, by the melting temperature (T m For example, "maximum stringency" may be based on approximately T m -5°C (T m 5°C below T m "High stringency": Approximately 5-10 °C below the T of the probe m "Intermediate stringency" at approximately 10-20°C below T m"Low stringency" occurs at about 20-25°C. Alternatively or additionally, hybridization conditions can be based on the salt or ionic strength conditions of hybridization and / or one or more stringency washes. For example, 6X saline sodium citrate (SSC) = very low stringency, 3X SSC = low to medium stringency, 1X SSC = medium stringency, and 0.5X SSC = high stringency. Functionally, maximum stringency conditions may be used to identify nucleic acid sequences that have exact or close identity to the hybridization probe, while high stringency conditions are used to identify nucleic acid sequences that have about 80% or more sequence identity to the probe. For applications requiring high selectivity, it is usually desirable to use relatively stringent conditions to form hybrids (e.g., relatively low salt and / or high temperature conditions are used).

[0229] The terms "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides means that the polynucleotides or polypeptides include either a sequence having at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a parent or reference sequence, or a sequence that contains amino acid substitutions, insertions, deletions, or modifications made solely to avoid the current type without adding function.

[0230] The term "expression vector" refers to a DNA construct that contains a DNA sequence encoding a particular polypeptide and is operably linked to suitable control sequences that can affect the expression of the polypeptide in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may be integrated into the genome itself.

[0231] The term "recombinant" refers to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells containing such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating a coding sequence to create an altered polypeptide, fusing the coding sequence to that of another gene, placing the gene under the control of a different promoter, expressing the gene in a heterologous organism, expressing the gene at reduced or elevated concentrations, expressing the gene conditionally or constitutively in a manner that differs from the natural expression profile, etc. Generally, recombinant nucleic acids, polypeptides, and cells based thereon are not identical to the related nucleic acids, polypeptides, and cells found in nature, because they have been manipulated by man.

[0232] "Receptor-ligand binding," "ligand binding," and "binding" are used interchangeably herein and refer to the physical interaction between a receptor (e.g., a naturally occurring GPCR or GRANPA) and a ligand (e.g., a natural ligand, (e.g., a peptide ligand) or a synthetic ligand (e.g., a synthetic small molecule ligand)). Ligand binding can be measured by a variety of methods known in the art (e.g., detecting binding with a radiolabeled ligand).

[0233] "Signaling" refers to the production of a biochemical or physiological response as a result of ligand binding (eg, as a result of a synthetic ligand binding to GRANPA).

[0234] "Receptor activation," "GRANPA activation," and "GPCR activation" refer to the binding of a ligand (e.g., a natural or synthetic ligand) to a receptor in a manner that induces G protein-mediated signaling and a physiological or biochemical response associated with G protein-mediated signaling. Activation can be measured by measuring a biological signal associated with a G protein-associated signal (e.g., using electrophysiology or other assays described herein).

[0235] "Targeted cell activation" and "target cell activation" are used interchangeably herein and refer to GRANPA-mediated activation of a specific G protein-mediated physiological response in a target cell, which occurs by binding of a synthetic small molecule to GRANPA. As used herein, cell activation includes (but is not limited to) inhibitory responses such as synaptic silencing or inhibition, and activation of G proteins in both inhibitory and stimulatory cells.

[0236] "Natural ligand," and "naturally occurring ligand," and "endogenous ligand," are used interchangeably herein, to refer to a biomolecule endogenous to a mammalian host, which binds to a native GPCR and elicits a G protein-coupled cellular response. One example is acetylcholine.

[0237] "Synthetic small molecule," "synthetic small molecule ligand," "synthetic ligand," and "synthetic agonist," etc., are used interchangeably herein and are capable of binding within the transmembrane domain of a GPCR or modified GPCR (i.e., GRANPA) and promoting receptor activation and receptor-mediated responses.

[0238] The terms "transfect", "transfection", "transfected", and the like, in this case refer to the introduction of genes into eukaryotic cells, such as neurons or keratinocytes, and include "transduction", which is viral-mediated gene transfer, for example, by use of recombinant AAV, adenovirus (Ad), retrovirus (e.g., lentivirus), or any other applicable viral-mediated gene transfer platform.

[0239] "Transformation" refers to a transient or permanent genetic change induced in a cell following the incorporation of new DNA (i.e., DNA exogenous to the cell). If the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.

[0240] "Promoter" refers to a minimal DNA sequence sufficient to direct transcription of an operably linked DNA sequence. "Promoter" is also meant to encompass those promoter elements sufficient for regulatable promoter-dependent gene expression in a cell type-specific, tissue-specific, or inducible by an external signal or agent, and such elements may be located in the 5' or 3' regions of the native gene.

[0241] A "subject" can be a human or an animal, such as a vertebrate or mammal, including rats, mice, rabbits, pigs, monkeys, chimpanzees, cats, dogs, horses, goats, guinea pigs, and birds. A subject can be a "patient."

[0242] The term "treatment", as used herein in the context of treating a condition, generally relates to treatments and therapies in which some desired therapeutic effect is achieved, whether in a human or animal (e.g., in vertebrate applications), such as inhibiting progression of the condition, including reducing the rate of progression (prolonging survival), halting the rate of progression, reversing the condition, ameliorating the condition, and curing the condition.

[0243] The term "therapeutically effective amount" as used herein relates to an amount of a compound of the invention, or a material, composition or dosage form containing the compound, that is effective to produce some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio when administered in accordance with a desired treatment regimen.

[0244] The present invention also encompasses treatment, where "treating" is understood to include prophylactic measures. Prophylactic treatment utilizes a "prophylactically effective amount," which, as used herein, refers to an amount of an agent that is effective to produce some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio when administered in accordance with a desired treatment regimen.

[0245] "Prophylaxis" in the context of this specification should not be understood to define complete success, i.e., complete protection or complete prevention. Rather, prophylaxis in this context refers to measures taken prior to detection of symptoms with the goal of preserving health by helping to delay, alleviate, or avoid a symptomatic condition. ***

[0246] Whenever a method of treatment employing an agent is described herein, it will be understood that the agent is also described for use in that method, as well as for use in the manufacture of a medicament for treating the relevant disease.

[0247] It will be understood that whenever a composition is described herein, the same composition is also contemplated for use in the therapeutic methods (including prophylactic methods) described herein, as well as compositions for use in the manufacture of a medicament for treating the relevant disease.

[0248] Numerous patents and publications are cited herein in order to more fully describe and disclose the present invention and the state of the art to which it pertains. Each of these references is incorporated by reference in its entirety into this disclosure to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0249] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a stated integer, or step, or group of integers or steps, but not to the exclusion of any other integer, or step, or group of integers or steps.

[0250] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0251] Ranges are often 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, using the antecedent "about," it will be understood that the particular value forms another embodiment.

[0252] Any subheadings herein are included for convenience only and should not be construed as limiting the disclosure in any way.

[0253] The invention will now be further described with reference to the following non-limiting figures and examples, in the light of which other embodiments of the invention will occur to those skilled in the art.

[0254] The disclosures of all references cited herein, as well as those that may be used to practice the invention by those skilled in the art, are specifically incorporated herein by cross-reference. [Brief description of the drawings]

[0255] [Figure 1] A) Comparison of the binding modes of diphenhydramine (DPH, blue) and iperoside (orange, PDB entry 4MQS (Kruse, Ring et al. 2013) (iperoside bound to the M2 muscarinic receptor)) suggests that the tyrosine is involved in hydrophobic interactions with iperoside but clashes with DPH. B) The methyl group (highlighted by the dotted circle) of methysergide (red stick) clashes with A225 (gold stick) in the active 5-HT2B conformation (gold, PDB entry 6DRY (McCorvy et al., Nat. Struct. Mol. Biol. 2018, 25, 787-796, https: / / doi.org / 10.1038 / s41594-018-0116-7)) but fits in the A225G mutant (green, PDB entry 6DRZ (McCorvy et al., Nat. Struct. Mol. Biol. 2018, 25, 787-796, https: / / doi.org / 10.1038 / s41594-018-0116-7)). Note that residues 180-198 and residues 132-138 are not shown to improve visibility of the binding site. [Diagram 2] A) Activation of wild-type hM4 (WT CHRM4) by DPH. Y113C in combination with A203G, but not A203N, converts DPH from an antagonist to a low-potency agonist. B) Y113N can replace Y113C, but several other Y113 substitutions reduce DPH sensitivity without substantially changing potency. C) Still other Y113C substitutions abolish DPH-dependent activation. For reference, the double mutant A203G+Y113C (hM4D, first reported by Armbruster et al., 2007) is shown in blue. [Diagram 3] S85 participates in an interaction network with D112, Y443, and S116 in the active (left) conformation, and with D112 and Y443 in the inactive (right) conformation. [Figure 4] Effect of the S85 mutation on DPH-dependent activation in combination with A203G+Y113C. The double mutant A203G+Y113C is shown in blue for comparison. [Diagram 5] Y416 is involved in hydrogen-bonding interactions with olanzapine (left panel), but creates a polar binding site that is less compatible with the binding of the hydrophobic DPH phenyl ring (right panel). The Y416F mutation renders the binding site in this region hydrophobic. [Figure 6] A) Effect of the Y416 mutation on DPH-dependent activation in combination with A203G+Y113C. The double mutant A203G+Y113C is shown in blue for reference. B) Effect of Y416F in combination with S85V, as well as A203G+Y113C. [Figure 7] Alignment models of olanzapine (green) and DPH (blue) bound to hM4D(Gi) suggest that the methyl group of Ile compensates for the loss of the methyl group of olanzapine that is missing in DPH. [Figure 8] A) Effect of V120 mutation on DPH-dependent activation in combination with A203G+Y113C. The double mutant A203G+Y113C is shown in blue for reference. B) V120I also increases DPH efficacy in combination with Y113C+A203G+S85V+Y416F as described in Figure 6. For reference, the construct Y113C+A203G+S85V+Y416F is shown in green. [Figure 9] Mutation of L123 can stabilize a network of interactions that exists in the active conformation (left panel) but cannot form in the inactive conformation (right panel, PDB entry 5DSG (hM4 bound to tiotropium) (Thal et al., Nature 2016, 531:335-340, DOI:10.1038 / nature17188)). [Figure 10]A) Effect of the L123 mutation on DPH-dependent activation in combination with A203G+Y113C. The double mutant A203G+Y113C is shown in blue for reference. B) The addition of the L123 mutation leads to increased efficacy / potency of DPH, also in additional constructs. For reference, construct Y113C+A203G+S85V+Y416F is shown in green and construct Y113C+A203G+S85V+Y416F+V120I is shown in red. [Figure 11] The addition of the F128 mutation also results in increased efficacy / potency of DPH in additional constructs. For reference, the construct Y113C+A203G+S85V+Y416F is shown in green and the double mutant A203G+Y113C is shown in blue. [Figure 12] The addition of the M121F mutation results in an increase in the potency of DPH. For reference, the double mutant A203G+Y113C is shown in blue. [Figure 13] The A200T mutation introduces a hydrogen bond with N417, thus stabilizing the active conformation of the receptor. [Figure 14] The addition of the A200T mutation results in an increase in the potency of DPH. For reference, the double mutant A203G+Y113C is shown in blue. [Figure 15] The F204Y mutation stabilizes the active conformation of the receptor because it introduces a new hydrogen bond with T414. [Figure 16] The addition of the F204Y mutation results in an increase in the potency of DPH. For reference, the double mutant A203G+Y113C is shown in blue. [Figure 17A] The addition of the W413L mutation results in increased potency of DPH. For reference, the double mutant A203G+Y113C is shown in blue. [Figure 17B] The addition of the I410V mutation results in increased efficacy in DPH. For reference, the double mutant A203G+Y113C is shown in blue. [Figure 18]Gi-coupled activity of hM4D(Gi)(Y113C+A203G) measured by GloSensor assay. Addition of the mutations S85V, V120I, and Y416F shifts the EC50 of DPH from 1.4 μM to 3.7 nM. [Figure 19] Gi-coupled activity of hM4D(Gi)(Y113C+A203G) and mutants measured by GloSensor assay. [Figure 20] hM4D(Gi) and mutants were tested for their effect on basal cAMP concentrations using the GloSensor assay. [Figure 21]Electrophysiology-based screening of hM4D(Gi) activation. A) Left: Representative traces of Kir3.1 and Kir3.2 currents with (+DPH, 100 nM, bottom) and without (baseline, top) agonist application in cells expressing Y113C+A203G+S85V+V120I+Y416F+L123T. Middle: Mean currents measured during the time indicated by the grey area in the left panel plotted against holding voltage. Red line shows calculation of membrane leak conductance obtained from a linear fit between 0 and +50 mV. Right: Leak-subtracted Kir3.1 / Kir3.2-mediated currents together with a linear fit to the currents at negative potentials (blue). The slope (k) of the current-voltage relationship was used for subsequent analysis of mutant activation. B) Left: DPH acts as a potent agonist for both the Y113C+A203G+S85V+V120I+Y416F and Y113C+A203G+S85V+V120I+Y416F+L123T mutants. Right: EC50 (nM) for the Y113C+A203G+S85V+V120I+Y416F and Y113C+A203G+S85V+V120I+Y416F+L123T mutants. C) In the absence of agonist application, the Y113C+A203G+S85V+V120I+Y416F mutant shows significant basal activity compared to transfection of GFP alone and the Y113C+A203G+S85V+V120I+Y416F+L123T mutant (GFP vs. Y113C+A203G+S85V+V120I+Y416F p=0.008, GFP vs. Y113C+A203G+S85V+V120I+Y416F+L123T p=0.777, one-way ANOVA with Bonferroni post-hoc test). D) In ​​contrast to FIG. 10B, the Y113C+A203G+S85V+V120I+Y416F+L123T variant does not show lower DPH efficacy than Y113C+A203G+S85V+V120I+Y416F in Kir3.1 and Kir3.2 activation (p=0.513, unpaired t-test). [Figure 22]Other drugs can be used as activating ligands: A) Other drugs usually show weaker activation of hM4D(Gi) compared to DPH; B) their potency and / or efficacy is improved upon addition of the mutations S85V, V120I, and Y416F. [Diagram 23] Alignments of closely related GPCR family members show high structural conservation. Alignment of all structures (a) and pairwise alignment of CHRM4 (red, PDB entry 5DSG (Thal et al., Nature 2016, 531:335-340)) with (b) CHRM2 (orange, PDB entry 5ZKC (Suno et al., Nat Chem Biol 2018, 14:1150-1158)), (c) ADRB2 (cyan, PDB entry 3PDS (Rosenbaum et al., Nature 2011, 469:236-240)), and (d) HRH1 (blue, PDB entry 3RZE (Shimamura et al., Nature 2011, 475:65-70)). Note the high structural similarity between diphenhydramine and the co-crystallized ligand doxepin. (e) DRD3 (purple, PDB entry 3PBL (Chien et al., Science 2010, 330:1091-1095)) and (f) HTR2A (dark blue, PDB entry 6A93 (Kimura et al., Nat Struct Mol Biol 2019, 26:121-128)). Diagrams and RMSD values ​​were obtained using the default alignment function of the PyMOL molecular graphics system, version 1.8.0.0 (Schroedinger, LLC). [Figure 24]CHRM4 (red, PDB entry 5DSG (Thal et al., Nature 2016, 531:335-340)), (a) CHRM2 (orange, PDB entry 5ZKC (Suno et al., Nat Chem Biol 2018, 14:1150-1158)), (b) ADRB2 (cyan, PDB entry 3PDS (Rosenbaum et al., Nature 2011, 469:236-240)), (c) HRH1 (blue, PDB entry 3RZE (Shimamura et al., Nature 2011, 475:65-70)), (d) DRD3 (purple, PDB entry 3PBL (Chien et al., Science 2010, 330:1091-1095)), and (e) 5-HT2A (dark blue, PDB entry 6A93 (Kimura et al., Nat Comparison of GRANPA residues between CHRM4 (bold) and HTR2A (e). Residues are denoted according to the numbering of CHRM3 (a), ADRB2 (b), HRH1 (c), DRD3 (d), and HTR2A (e). Note that L119W and M164W mutations were introduced into the crystallized constructs of DRD3 (PDB entry 6PBL) and HTR2A (PDB entry 6A93). These were mutated back to the wild-type residues using PyMOL version 1.8.0.0. [Diagram 25] GRANPA activation by diphenhydramine in vivo alters behavior. EXAMPLES

[0256] method For comparison of the hM4D(Gi) DREADD to related beta-adrenergic and histamine receptors, PDB entries 3PDS (Rosenbaum, Zhang et al. 2011) (beta2-adrenergic receptor complexed with an irreversible agonist) and 3RZE (Shimamura, Shiroishi et al. 2011) (H1-histamine receptor complexed with doxepin) were aligned with the olanzapine-DREADD model reported in Weston et al. (Weston, Kaserer et al. 2019) and manually scanned using PyMOL version 0.99rc6 (2014).

[0257] A raw diphenhydramine (DPH)-DREADD bound model was generated by aligning DPH to the olanzapine bound pose (Masson, Ellis et al. 1992; Young, Fong et al. 2014) using the RDKit Open 3D alignment node in KNIME version 4.0.0 (Berthold, Cebron et al. 2007). For refinement, torsion angles were adjusted and the model was minimized in UCSF-Chimera 1.13.1 (Pettersen, Goddard et al. 2004). The DPH-DREADD model was aligned with the M4 muscarinic receptor in complex with tiotropium (PDB entry 5DSG (Thal, Sun et al., 2016) to compare the active and inactive states, and the structure was manually analyzed using PyMOL version 1.8.0.0 (Hausser, 2014). Polypeptide alignments of monoamine neurotransmitter receptors to identify non-consensus amino acids in CHRM4 were performed using MEGA X version 10.1.8. 11 This was carried out using

[0258] Generation of mutants The Gi-coupled human muscarinic receptor "hM4D(Gi)" is rendered sensitive to clozapine N-oxide (CNO), an orally bioavailable and normally inactive metabolite of clozapine. This modified GPCR contains the following mutations: Y113C / A203G.

[0259] The engineered receptor hM4D(Gi) was originally described in BN Armbruster, X. Li, MH Pausch, S. Herlitze, BL Roth, "Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand", Proc. Natl. Acad. Sci. USA 104, 5163-5168 (2007). The authors describe a general, validated and unbiased approach to generate GPCRs with defined ligand specificity that was utilized to create a family of muscarinic ACh receptor (mAChR) DREADDs. The contents of that publication are specifically incorporated herein by reference for its description of the preparation and characteristics of these DREADDs.

[0260] The preparation of human M4 DREADDs is also described in V. Nawaratne, K. Leach, N. Suratman, RE Loiacono, C. C. Felder, BN Armbruster, and A. Christopoulos, (2008). “New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD(designer receptor exclusively activated by a designer drug)”. Molecular pharmacology, 74(4), 1119-1131.

[0261] A plasmid encoding hM4D(Gi) is commercially available from Addgene (Cambridge, MA02139) as plasmid45548:pcDNA5 / FRT-HA-hM4D(Gi) (http: / / www.addgene.org / 45548 / ).

[0262] A plasmid encoding hM3Dq is also commercially available from Addgene (https: / / www.addgene.org / 44361 / ). This receptor is sensitive to perlapin (27).

[0263] DNA manipulations were performed by conventional molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning: Cold Spring Harbor Laboratory Press) or with commercially available kits according to the manufacturer's protocols. Site-directed mutagenesis was performed using the QuikChange II XL kit (Agilent). Other modifications of DNA were performed using the Q5 Site-Directed Mutagenesis and Hi-Fi Assembly Kit (New England Biolabs). Plasmid DNA was purified using Monarch plasmid miniprep (New England Biolabs) or NucleoBond Xtra midi kits (Thermo Fisher). DNA was quantified by absorption spectrophotometry using a Nanodrop 1000 (Thermo Fisher) and subjected to Sanger sequencing by Source BioScience Limited (UK).

[0264] The substitutions were as follows: 253-255 - S85: TCC V - GTT / GTC / GTA / GTG 337-339 - Y113 TAC C - TGT / TGC N - AAT / AAC 358-360 - V120 GTC I - ATT / ATC / ATA 361-363 - M121 ATG F - TTT / TTC 367-369 - L123 CTT C - TGT / TGC I - ATT / ATC / ACA S - TCT / TCC / TCA / TCG / AGT / AGC T - ACT / ACC / ACA / ACG V - GTT / GTC / GTA / GTG 382-384 - F128 TTT I - ATT / ATC / ATA L - TTA / TTG / CTT / CTC / CTA / CTG V - GTT / GTC / GTA / GTG 598-600 - A200 GCC T - ACT / ACC / ACA / ACG 607-609 - A203 GCC G - GGT / GGC / GGA / GGG 610-612 - F204 TTC Y - TAT / TAC 1228-1230 - I410 ATC V - GTT / GTC / GTA / GTG 1237-1239 - W413 TGG L - TTA / TTG / CTT / CTC / CTA / CTG 1246-1248 - Y416 TAC F - TTT / TTC

[0265] Cell culture and assay All mammalian cells were maintained in DMEM (10% FBS plus relevant antibiotics at 37°C and 5% CO2). Selected mutants were engineered into CHRM4-Tango (Addgene #66251) via Quikchange mutagenesis (Agilent) and DNA was obtained via Monarch mini- or midi-prep kits (New England Biolabs). 4 μg of construct DNA was mixed with 400 μL of optimem (Thermo Fisher) containing 12 μL of turbofect (Thermo Fisher) and transfected into 70% confluent HTLA cells (HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a beta-arrestin2-TEV fusion gene) in a T25 flask in 4 mL of complete medium. 12,13 After 2 days, cells were detached with citrate saline and transferred to white 1 / 2 area 96-well plates containing 40 μL of optimem (Thermo Fisher) containing different drug concentrations. Cells were left overnight and lysed by direct addition of 40 μL of GloMax / Glo lysis buffer (1:1, Promega) before luminescence was counted on a FlexStation3 (Molecular Devices) with 1 s integration per well.

[0266] The results of the selected mutants were confirmed using a GloSensor cAMP assay. Here, construct DNA was co-transfected 1:1 with 22F reporter plasmid (Promega) into HEK-293T cells in a T25 flask with turbofect as above. The next day, cells were detached with citrate saline and transferred to a white 1 / 2 area 96-well plate containing 100 μL of optimem (Thermo Fisher). After 1 day, the medium was removed and cells were washed with 100 μL of HBSS (20 mM HEPES, pH 7.4), followed by the addition of 40 μL of 5% GloSensor reagent in HBSS. After 1 hour of incubation, 10 μL of various concentrations of drugs in HBSS were added to each well. Luminescence was counted for 15 minutes on a FlexStation3 with 1 second integration per well, after which 10 μL of isoprenaline in HBSS was added to a final concentration of 200 nM. The plate was read again for 15 minutes.

[0267] For electrophysiological confirmation of Gi-dependent Kir3.1 / 3.2 Gi activation, the mutants were inserted into the hM4D(Gi)-plasmid (Addgene#45548). After transfection as described above, Gi-dependent Kir3.1 / 3.2 activation was confirmed as described by Weston et al. 3 Quantification was performed using the whole-cell patch clamp technique as described.

[0268] Methodology References 1. Rosenbaum, DM, et al. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469, 236-240 (2011). 2. Shimamura, T., et al. Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65-70 (2011). 3. Weston, M., et al. Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics. Science Advances 5, eaaw1567 (2019). 4. The, P.O.N.E.S. Correction: Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors. PloS one 9, e110030 (2014). 5. Young, D., et al. Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy. Gene therapy (2014). 6. Masson, N., Ellis, M., Goodbourn, S. & Lee, K.A. Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter. Molecular and cellular biology 12, 1096-1106 (1992). 7. Berthold, M.R., et al. KNIME: The Konstanz Information Miner. in Studies in Classification, Data Analysis, and Knowledge Organization (GfKL 2007) (Springer, 2007). 8. Pettersen, EF, et al. UCSF Chimera-A visualization system for exploratory research and analysis. Journal of Computational Chemistry 25, 1605-1612 (2004). 9. Thal, DM, et al. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335-340 (2016). 10. Hausser, M. Optogenetics: the age of light. Nature methods 11, 1012-1014 (2014). 11. Kumar, S., et al. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35, 1547-1549 (2018). 12. Barnea, G, et al. The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci US A.105, 64-69 (2008). 13. Kroeze, WK, et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol 22, 362-369 (2015).

[0269] Example 1: Structural basis of the effects of Y113 and A203 mutations These are the original residues reported by Armbruster et al., PNAS 2007, 104, 5163-5168.

[0270] The proposed mechanism of action is as follows. Y113C: Creates space for larger molecules to bind in the active conformation compared to the endogenous agonist acetylcholine, and prevents binding due to loss of hydrophobic contacts to the endogenous ligand or other agonists of similar size (e.g., iperoxo) (Figure 1A). A203G: Converts an antagonist to an agonist by removing steric bulk that inhibits movement of helix 5 upon receptor activation. The corresponding A225G mutation in the serotonergic HTR2B receptor converts the antagonist methysergide into a partial agonist but does not substantially affect the potency or efficacy of the agonist methylergonovine (McCorvy et al., Nat. Struct. Mol. Biol., 2018, 25, 787-796, https: / / doi.org / 10.1038 / s41594-018-0116-7 ). Figure 1B highlights how the methyl group of methysergide clashes with A225 in the active HTR2B conformation, but is compatible with the A225G mutant.

[0271] The experimental results are shown in Figure 2. It can be concluded that A203G provides agonist activity.

[0272] Y113 tolerates mutations to C and N, to a lesser extent A and V, and to some extent T, Q, and S.

[0273] Example 2: Effect of Mutations at Position 85 The proposed mechanism of action is as follows. S85V: As shown in Figure 3, it is part of a hydrogen bond network that includes D112, Y443, and S116 in the active (Figure 3 left) conformation, and D112 and Y442 in the inactive (Figure 3 right) conformation. Upon ligand binding, the carboxy group of D112 adopts a different rotamer. The introduction of hydrophobicity in the S85V mutation may stabilize the active conformation because it is incompatible with the D112 rotamer in the inactive state, but forms hydrophobic contacts with the C beta atom of the D112 active conformation rotamer.

[0274] The experimental results are shown in Figure 4. S85V and S85C increase DPH potency (also in combination with Y416F, see below). However, both S85V and S85C decrease efficacy.

[0275] Example 3: Effect of mutations at position 416 The proposed mechanism of action is as follows. Y416F: As shown in Figure 5, this removes the polarity of the OH group, which is otherwise in close proximity to the hydrophobic phenyl ring of DPH. The interaction with the OH group was tolerated by olanzapine (which was shown to be a full agonist of hM4D(Gi) in Weston et al., 2019, Sci Adv., 2019 Apr 17;5(4):eaaw1567) since the OH group was potentially involved in hydrogen bonding of the benzodiazepine-nitrogen of olanzapine. Comparison can be made with the crystal structure of the beta2-adrenergic receptor complexed with an irreversible agonist (3PDS; Rosenbaum et al., Nature 2011, 469:236-240).

[0276] The experimental results are shown in Figure 6. Y416F increases the potency of DPH, and Y416F in combination with S85V further increases potency.

[0277] Example 4: Effect of mutations at position 120 The proposed mechanism of action is as follows. V120I: As shown in Figure 7, this mutation may better "fill" the space in the binding site, thus increasing hydrophobic contacts. The additional methyl group of Ile compared to Val compensates for the loss of the olanzapine-methyl group. The importance of residue 3.40 and its contact on TM6 has been illustrated in NMR spectroscopic studies of the M2 receptor (Xu et al., Molecular Cell, 2019, 75:53-65).

[0278] The experimental results are shown in Figure 8. The V120I mutation enhances the efficacy of DPH.

[0279] Example 5: Effect of mutations at position 123 The proposed mechanism of action is as follows. L123T / S: As shown in FIG. 9, this modification may stabilize the network of interactions formed upon receptor activation, but absent in the inactive conformation.

[0280] The experimental results are shown in Figure 10. Mutation of L123 increases the potency and / or efficacy of DPH. Some of the other L123 substitutions lead to constitutively active receptors. Figure 10B suggests that DPH is less effective with Y113C+A203G+S85V+Y416F+V120I+L123T compared to Y113C+A203G+S85V+Y416F+V120I. However, this assay measures the recruitment of β-arrestins, and the lower efficacy is due to the G of GIRK (Kir3.1 and Kir3.2) conductance. β / γ Note that the reduced recruitment of β-arrestin is evident in assays measuring G protein-dependent activation (FIG. 21D), thus indicating reduced recruitment of β-arrestin while maintaining G protein signaling.

[0281] Example 6: Effect of mutations at position 128 As shown in FIG. 11, mutation of F128 to I, L, or V, but not to Y, increases the efficacy and / or potency of DPH.

[0282] Example 7: Effect of mutations at position 121 As shown in FIG. 12, mutation of M121 to F, but not to L, increases the potency of DPH.

[0283] Example 8: Effect of mutations at positions 200 and 204 The proposed mechanism of action is as follows. A200T: As shown in FIG. 13, this modification apparently stabilizes the active conformation of the receptor by introducing a hydrogen bond to N417 and potentially introducing new hydrophobic contacts.

[0284] The experimental results are shown in Figure 14. Mutation of A200 to T, but not to G or S, increases the potency of DPH, albeit at the cost of a smaller effect and reduced efficacy.

[0285] With respect to position 204, the proposed mechanism of action is as follows: F204Y: As shown in FIG. 15, this modification apparently stabilizes the active conformation of the receptor by introducing a hydrogen bond to T414.

[0286] The experimental results are shown in Figure 16. Mutation of F204 to Y, but not to H, increases the potency of DPH.

[0287] Example 9: Effect of mutations at position 413 As shown in FIG. 17A, the W413L mutation increases the efficacy of DPH, but at the cost of reduced response.

[0288] As shown in FIG. 17B, the I410V mutation confers increased efficacy of DPH.

[0289] Example 10: Providing alternative ligands Alternative ligands were provided by two different research strategies. Search strategy 1 was based on chemical similarity and included: a) Approved drugs similar to diphenhydramine by Shape-IT using the Swiss Similarity web server (http: / / www.swisssimilarity.ch / ; Zoete V., Daina, A., Bovigny C., and Michielin, O., SwissSimilarity: A Web Tool for Low to Ultra High Throughput Ligand-Based Virtual Screening., J. Chem. Inf. Model., 2016, 56(8), 1399-1404.). b) Compounds with a similarity of 0.5 to diphenhydramine using DataWarrior and FragFP (Thomas Sander, Joel Freyss, Modest von Korff, Christian Rufener, DataWarrior: An Open-Source Program For Chemistry Aware Data Visualization And Analysis., J Chem Inf Model, 2015, 55, 460-473, doi 10.1021 / ci500588j). c) Approved drugs with a similarity of 0.5 to diphenhydramine using the ChEMBL web server (https: / / www.ebi.ac.uk / chembl / ; A. Gaulton, L. Bellis, J. Chambers, M. Davies, A. Hersey, Y. Light, S. McGlinchey, R. Akhtar, A. P. Bento, B. Al-Lazikani, D. Michalovich, and J. P. Overington, (2012), "ChEMBL: A Large-scale Bioactivity Database For Chemical Biology and Drug Discovery", Nucleic Acids Res., Database Issue, 40 D1100-1107., DOI:10.1093 / nar / gkr777, PMID:21948594;AP Bento, A. Gaulton, A. Hersey, L. J. Bellis, J. Chambers, M. Davies, FAKruger, Y.Light, L.Mak, S.McGlinchey, M.Nowotka, G.Papadatos, R.Santos and JPOverington, (2014), “The "ChEMBL bioactivity database: an update", Nucleic Acids Res., Database Issue, 42 1083-1090., DOI:10.1093 / nar / gkt103, PMID:24214965).

[0290] The results are listed in Table 1.

[0291] Search strategy 2 was based on functional and therefore structural similarities and included: 2.) An approved antihistamine that would be expected to be an antihistamine that binds to GRANPA (e.g., diphenhydramine).

[0292] The results are listed in Table 2.

[0293] Example 11: Further analysis of GRANPA mutations by different assays FIG. 18 shows the combinations of mutations of the present invention that were used to validate their ability to inhibit cAMP production, as measured by the GloSensor assay of the Gi cascade.

[0294] FIG. 19 shows the results of Gi-coupled activity of hM4D(Gi)(Y113C+A203G) and mutants measured by GloSensor assay. Addition of S85V+Y416F±V120I±L123(T / C / S / I / V) increased the potency of activation by DPH. Data was normalized as the percentage of inhibition of cAMP measured by light output compared to control hM4D(Gi) treated with 20 μM DPH. EC50 and span of the constructs are as follows: Y113C+A203G (717nM; 94%) Y113C+A203G+S85V+Y416F(1.50nM;116%) Y113C+A203G+S85V+Y416F+V120I(1.80nM;111%) Y113C+A203G+S85V+Y416F+L123T(0.949nM;129%) Y113C+A203G+S85V+Y416F+V120I+L123T(0.956nM;129%) Y113C+A203G+S85V+Y416F+L123C(0.366nM;115%) Y113C+A203G+S85V+Y416F+V120I+L123C(0.252nM;101%) Y113C+A203G+S85V+Y416F+L123S(0.528nM;83%) Y113C+A203G+S85V+Y416F+V120I+L123S(0.321nM;110%) Y113C+A203G+S85V+Y416F+L123I(1.41nM;98%) Y113C+A203G+S85V+Y416F+V120I+L123I(0.880nM;119%) Y113C+A203G+S85V+Y416F+L123V(1.15nM;109%)

[0295] Figure 20 shows the results of hM4D(Gi) and mutants tested for their effect on basal cAMP concentration using the GloSensor assay. Data were normalized to hM4D(Gi)(Y113C+A203G). A decrease in basal luminescence without drug treatment indicates an increase in constitutive activity. Y113C+A203G (100%) was not significantly different from Y113C+A203G+S85V+Y416F (93%) and Y113C+A203G+S85V+Y416F+V120I (95%), while the other mutant combinations were different (One-way ANOVA, P<0.05).

[0296] FIG. 21 shows the G protein-coupled inward rectifying potassium channel (GIRK) of Kir3.1 and Kir3.2. β / γ 1 shows the results of an electrophysiology assay testing for addictive enhancement.

[0297] GIRK assays confirm that both Y113C+A203G+S85V+V120I+Y416F and Y113C+A203G+S85V+V120I+Y416F+L123T are potently activated by DPH. In contrast, Y113C+A203G+S85V+V120I+Y416F+L123T showed less recruitment in the β-arrestin assay data in Figure 10B. However, although they show similar efficacy in GIRK assays, Y113C+A203G+S85V+V120I+Y416F, but not Y113C+A203G+S85V+V120I+Y416F+L123T, shows some basal activity.

[0298] Therefore, Y113C+A203G+S85V+V120I+Y416F+L123T is the preferred GRANPA.

[0299] Example 12: Other Activating Ligands FIG. 22 confirms that other drugs, including other antihistamines and antimuscarinics, may be used as activating ligands, and in particular their potency and / or efficacy may be improved by the use of the modifications of the present invention, such as, for example, S85V, V120I, and Y416F.

[0300] Example 13 - Summary of Preferred Embodiments and Amino Acid Sequence Numbering The mAChR4 numbering is referenced, with Ballesteros-Weinstein numbering in parentheses. The original DREADD: Y113-C / N (3.33) A203-G (5.46) Mutations that substantially increased DPH efficacy / response: S85-V (2.57) V120-I (3.40) L123-C / I / S / T / V (3.43) F128-I / L / V (3.48) Y416-F (6.51) No substantial increase in DPH potency: M121-F (3.41) A200-T (5.43) F204-Y (5.47) I410-V (6.45) W413-L (6.48)

[0301] Alignment of closely related GPCR family members shows high structural conservation (Figure 23), and residues identified in CHRM4 can be readily aligned to corresponding residues in other aminergic GPCRs (Figure 24).

[0302] Corresponding residues in other aminergic GPCRs are shown below. FASTA peptide sequence alignments were performed using MEGA X 10.1.8 (Kumar S. et al., "MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms", Molecular Biology and Evolution 35, 1547-1549 (2018)) with the MUSCLE algorithm (standard settings: Gap Open=-2.9, Gap Extent=0.0, Hydrophobicity Multiplier=1.2, Max Iterations=16, Cluster Method=UPGMA, Min Diag Length=24).

[0303] Example 14 - GRANPA activation by diphenhydramine in vivo alters behavior Recombinant adeno-associated virus pseudotyped with AAV9 capsid expressing GRANPA (Y113C+A203G+S85V+V120I+Y416F+L123T) under the hCaMKII promoter (titer: 6.7×10∧14 vg / mL) was injected into the right substantia nigra (volume: 300 nanoliters) of anesthetized mice. Three weeks later, mice received an intraperitoneal injection of amphetamine (3 mg / kg) alone or together with diphenhydramine (1 mg / kg) to enhance locomotor activity. Then, whole-body left and right rotations were counted using a machine vision tool. Diphenhydramine led to a significant increase in left rotations (n=9, p=0.037, Student's paired t-test, FIG. 25).

[0304] [Table 3]

[0305] ACM4 S85 (residue 2.57) corresponds to ACM1 S78, ACM2 S76, ACM3 S121, ACM5 S83, HRH3 C87, HRH4 S68, and ADA2B I65.

[0306] [Table 4]

[0307] ACM4 V120 (residue 3.40) corresponds to ACM1 V113, ACM2 V111, ACM3 V156, ACM5 V118, HRH3 A122, and HRH4 V102.

[0308] ACM4 M121 (residue 3.41) is a member of ACM1 M114, ACM2 M112, ACM3 M157, ACM5 M119, HRH1 F116, HRH2 L108, HRH3 F123, HRH4 Y103, 5HT1F L112, 5HT1E L111, 5HT1D L127, 5HT1B L138, 5HT5A W130, 5HT7 M171, 5HT1A L125, 5HT2A M164, DRD3 L119, DRD2 L123, DRD4 F124, ADA1B L134, ADA1D L185, ADA1A M115, ADA2B V101, ADA2A V137, ADA2C V140, 5HT6 L115, 5HT2B M144, 5HT2C Corresponds to M143, DRD1B L129, DRD1A L112, 5HT4 F109, ADRB3 E126, ADRB1 E147, and ADRB2 E122.

[0309] ACM4 L123 (residue 3.43) is ACM1 L116, ACM2 L114, ACM3 L159, ACM5 L121, HRH1 V118, HRH2 L110, HRH3 I125, HRH4 I105, 5HT1F L114, 5HT1E L113, 5HT1D L129, 5HT1B L140, 5HT5A V132, 5HT7 L173, 5HT1A L127, 5HT2A L166, DRD3 L121, DRD2 L125, DRD4 L126, ADA1B L136, ADA1D L187, ADA1A L117, ADA2B L103, ADA2A L139, ADA2C L142, 5HT6 L117, 5HT2B L146, 5HT2C L151, ADA2C L152, ADA2C L153, ​​ADA2C L154, ADA2C L156, ADA2C L159, ADA2C L160, ADA2C L161, ADA2C L162, ADA2C L163, ADA2C L164, ADA2C L166, ADA2C L166, ADA2C L161, ADA2C L162, ADA2C L163, ADA2C L164, ADA2C L165, ADA2C L166, ADA2C L166, ADA2C L167, ADA2C L169, ADA2C L170, ADA2C L171, ADA2C L172, ADA2C L173, ADA2C L174, ADA2C L176, Corresponds to L145, DRD1B L131, DRD1A L114, 5HT4 L111, ADRB3 L128, ADRB1 L149, and ADRB2 L124.

[0310] ACM4 F128 (residue 3.48) is associated with ACM1 F121, ACM2 F119, ACM3 F164, ACM5 F126, HRH1 I123, HRH2 L115, HRH3 Y130, HRH4 Y110, 5HT1F L119, 5HT1E L118, 5HT1D L134, 5HT1B L145, 5HT5A L137, 5HT7 I178, 5HT1A L132, 5HT2A L171, DRD3 I126, DRD2 I130, DRD4 V131, ADA1B I141, ADA1D V192, ADA1A I122, ADA2B L108, ADA2A L144, ADA2C L147, 5HT6 L122, 5HT2B V151, 5HT2C L152, 5HT2D L151, 5HT2E L151, 5HT2F L151, 5HT2F L152 ...1, 5HT2F L151, 5HT2F L151, 5HT2F L151, 5HT2F L151, 5HT2F L151, 5HT2F L151, 5HT2F L151, 5HT2F L151, Compatible with L150, DRD1B V136, DRD1A V119, 5HT4 L116, ADRB3 V133, ADRB1 L154, and ADRB2 V129.

[0311] [Table 5]

[0312] ACM4 A200 (residue 5.43) corresponds to ACM1 A193, ACM2 A191, ACM3 A236, ACM5 A198, HRH1 A195, HRH2 G187, HRH3 S203, HRH4 S179, 5HT2A S239, DRD3 S193, DRD2 S194, DRD4 S197, ADA1B S208, ADA1D S259, ADA1A A189, ADA2B S177, ADA2A C216, ADA2C C215, 5HT6 S193, 5HT2B S222, 5HT2C S219, DRD1B S230, DRD1A S199, 5HT4 S197, ADRB3 S209, ADRB1 S229, and ADRB2 S204.

[0313] ACM4 F204 (residue 5.47) is ACM1 F197, ACM2 F195, ACM3 F240, ACM5 F202, HRH1 F199, HRH2 F191, HRH3 F207, HRH4 F183, 5HT1F F190, 5HT1E F191, 5HT1D F206, 5HT1B F217, 5HT5A 209, 5HT7 248, 5HT1A F204, 5HT2A F243, DRD3 F197, DRD2 F198, DRD4 F201, ADA1B F212, ADA1D F263, ADA1A F193, ADA2B F181, ADA2A F220, ADA2C F219, 5HT6 F197, 5HT2B F226, 5HT2C F227, 5HT2C F229, 5HT2C F228, 5HT2C F229 ... Compatible with F223, DRD1B F234, DRD1A F203, 5HT4 F201, ADRB3 F213, ADRB1 F233, and ADRB2 F208.

[0314] [Table 6]

[0315] ACM4 I410 (residue 6.45) corresponds to ACM1 I375, ACM2 I397, ACM3 I501, ACM5 I452, HRH1 I425, HRH2 I244, HRH3 G368, HRH4 A313, 5HT1E I301, 5HT1D I311, 5HT1B I324, 5HT7 T337, 5HT1A I355, DRD3 I339, DRD2 I383, DRD4 L356, ADA1B I304, 5HT6 F278, 5HT2B L334, 5HT2C L321, 5HT4 C269, ADRB3 T302, ADRB1 T334, and ADRB2 T283.

[0316] ACM4 W413 (residue 6.48) is a nucleotide analog of ACM1 W378, ACM2 W400, ACM3 W504, ACM5 W455, HRH1 W428, HRH2 W247, HRH3 W371, HRH4 W316, 5HT1F W306, 5HT1E W304, 5HT1D W314, 5HT1B W327, 5HT5A W298, 5HT7 W340, 5HT1A W358, 5HT2A W336, DRD3 W342, DRD2 W386, DRD4 W359, ADA1B W307, ADA1D W361, ADA1A W285, ADA2B W384, ADA2A W402, ADA2C W395, 5HT6 W281, 5HT2B W337, 5HT2C W395, 5HT6 W281, 5HT2C ... Compatible with W324, DRD1B W309, DRD1A W285, 5HT4 W272, ADRB3 W305, ADRB1 W337, and ADRB2 W286.

[0317] ACM4 Y416 (residue 6.51) corresponds to ACM1 Y381, ACM2 Y403, ACM3 Y507, ACM5 Y458, HRH1 Y431, HRH2 Y250, HRH3 Y374, and HRH4 Y319.

[0318] [Table 7]

[0319] [Table 8] TIFF2024519329000009.tif242153TIFF2024519329000010.tif241134

[0320] References Berthold, M. R., N. Cebron, F. Dill, T. R. Gabriel, T. Kotter, T. Meinl, P. Ohl, C. Sieb, K. Thiel and B. Wiswedel (2007). KNIME: The Konstanz Information Miner. Studies in Classification, Data Analysis, and Knowledge Organization (GfKL 2007) , Springer. Hausser, M. (2014). "Optogenetics: the age of light." Nat Methods 11(10): 1012-1014. Kruse, A. C., A. M. Ring, A. Manglik, J. Hu, K. Hu, K. Eitel, H. Hubner, E. Pardon, C. Valant, P. M. Sexton, A. Christopoulos, C. C. Felder, P. Gmeiner, J. Steyaert, W. I. Weis, K. C. Garcia, J. Wess and B. K. Kobilka (2013). "Activation and allosteric modulation of a muscarinic acetylcholine receptor." Nature 504(7478): 101-106. Masson, N., M. Ellis, S. Goodbourn and K. A. Lee (1992). "Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter." Mol Cell Biol 12(3): 1096-1106. Pettersen, E. F., T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng and T. E. Ferrin (2004). "UCSF Chimera-A visualization system for exploratory research and analysis." Journal of Computational Chemistry 25(13): 1605-1612. Rosenbaum, D. M., C. Zhang, J. A. Lyons, R. Holl, D. Aragao, D. H. Arlow, S. G. F. Rasmussen, H.-J. Choi, B. T. DeVree, R. K. Sunahara, P. S. Chae, S. H. Gellman, R. O. Dror, D. E. Shaw, W. I. Weis, M. Caffrey, P. Gmeiner and B. K. Kobilka (2011). "Structure and function of an irreversible agonist-β2 adrenoceptor complex." Nature 469(7329): 236-240. Shimamura, T., M. Shiroishi, S. Weyand, H. Tsujimoto, G. Winter, V. Katritch, R. Abagyan, V. Cherezov, W. Liu, G. W. Han, T. Kobayashi, R. C. Stevens and S. Iwata (2011). "Structure of the human histamine H1 receptor complex with doxepin." Nature 475(7354): 65-70. Thal, DM, B. Sun, D. Feng, V. Nawaratne, K. Leach, CC Felder, MG Bures, DA Evans, WI Weis, P. Bachhawat, TS Kobilka, PM Sexton, BK Kobilka and A. Christopoulos (2016). "Crystal structures of the M1 and M4 muscarinic acetylcholine receptors." Nature 531(7594):335–340. The, PONES (2014). "Correction: Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors." PLoS One 9(9): e110030. Weston , M. , T. Kaserer , A. Wu , A. Mouravlev , JC Carpenter , A. Snowball , S. Knauss , M. von Schimmelmann , MJ During , G. Lignani , S. Schorge , D. Young , DM Kullmann and A. Lieb (2019). "Olanzapine: A potent agonist at the hM4D(Gi)DREADD amenable to clinical translation of chemogenetics." Science Advances 5(4): eaaw1567. Young, D., D. M. Fong, P. A. Lawlor, A. Wu, A. Mouravlev, M. McRae, M. Glass, M. Dragunow and M. J. During (2014). "Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy." Gene Ther .

Claims

1. 1. A modified G protein-coupled receptor (GPCR), Compared to the parent, which is a native GPCR, (i) reduced responsiveness to endogenous activating ligands; (ii) responsiveness to an exogenous agonist is preserved or enhanced; comprises one, more preferably two, three or four alterations relative to the parent GPCR at positions selected from: (i) 85 (ii) 416 (iii) 120 (iv) 123 and optionally comprising, relative to said parent GPCR, one or more alterations at a position selected from: (v) 128 (vi) 121 (vii) 200 (viii) 204 (viii) 410 (VIX) 413 The amino acid positions of the modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:

1.

2. 2. The modified GPCR of claim 1, wherein the exogenous agonist is diphenhydramine or an analog thereof.

3. The modified GPCR as claimed in claim 1 , wherein the exogenous agonist is selected from Table 1 or Table 2.

4. 2. The modified GPCR of claim 1, wherein the exogenous agonist is selected from diphenhydramine, cyproheptadine, diphenylpyraline, desloratadine, and benzatropine.

5. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises the following residues at the following positions: (a) 113C or 113N, and (b) 203G

6. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises one or more of the following substitutions at the following positions: (a) Y113C or Y113N, and (b) A203G

7. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises one or more of the following residues at the following positions: (i) 85V or 85C (ii) 120I (iii) 416F (iv) 123C, 123I, 123T, or 123S

8. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises one or more of the following substitutions at the following positions: (i) S85V or S85C, most preferably S85V (ii) V120I (iii) Y416F (iv) L123C, L123I, L123T, or L123S

9. A modified GPCR as claimed in claim 5, wherein in addition to residues (a) and (b) or modifications (a) and (b), the modified GPCR comprises residues or substitutions of (i); (";" means "or"; same below) (i) and (ii); (i), (ii), and (iii); (i), (ii), (iii), and (iv).

10. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises one or more of the following residues at the following positions: (v) 128I, 128L, or 128V (vi) 121F (vii) 200T (viii)204Y(viii)410V (ix) 413L

11. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises one or more of the following substitutions at the following positions: (v) F128I, F128L, or F128V (vi) M121F (vii) A200T (viii)F204Y (viii) I410V (ix) W413L

12. 10. The modified GPCR of claim 9, wherein the modified GPCR comprises the following residues or substitutions: (v) 128I, 128L, or 128V; (v) 128I, 128L, or 128V and (vi) 121F.

13. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises Y113C+A203G+S85V+Y416F.

14. 2. The modified GPCR of claim 1, wherein the modified GPCR comprises Y113C+A203G+S85V+L123T+V120I+Y416F.

15. 2. The modified GPCR as claimed in claim 1, wherein the GPCR is a Gi-coupled GPCR.

16. 2. The modified GPCR of claim 1, wherein (i) the GPCR is coupled to an ion channel via a G protein, the ion channel being optionally inwardly rectifying, and / or the ion channel is optionally a potassium channel, preferably a protein-coupled inwardly rectifying potassium channel, and / or (ii) the GPCR is a Gq-coupled or Gs-coupled GPCR.

17. 2. The modified GPCR of claim 1, wherein the GPCR is selected from a cholinergic receptor muscarinic receptor (CHRM), a histamine receptor (HRH), a 5-hydroxytryptamine (serotonin) receptor (HTR), a dopamine receptor (DRD), an alpha adrenergic receptor (ADRA), a beta adrenergic receptor (β1-4 adrenoceptor) (ADRB).

18. The GPCRs include CHRM4, CHRM3, CHRM1, CHRM2, CHRM5, HRH1, HRH2, HRH3, HRH4, 5HTR-1A, 5H TR-1B, 5HTR-1D, 5HTR-1E, 5HTR-1F, 5HTR-2A, 5HTR-2B, 5HTR-2C, 5HTR-4, 5HTR-5A , 5HTR-6, 5HTR-7, DRD-1, DRD-2, DRD-3, DRD-4, DRD-5, ADRA-1A, ADRA-1B, ADRA-1D, ADRA-2A, ADRA-2B, ADRA-2C, ADRB-1, ADRB-2, ADRB-3.

19. The modified GPCR as claimed in claim 1, wherein the GPCR is selected from the GPCRs identified in Table 3.

20. 2. The modified GPCR as claimed in claim 1, wherein said modified GPCR has at least 70% sequence identity with its native parent GPCR of any one of SEQ ID NOs: 1-35 in Table 8.

21. 2. The modified GPCR as claimed in claim 1, wherein said modified GPCR comprises a sequence shown in any one of Tables 3 to 6 comprising said modification.

22. 22. A polynucleotide comprising a nucleic acid sequence encoding a modified GPCR according to any one of claims 1 to 21.

23. 23. The polynucleotide of claim 22, wherein the nucleic acid has at least 70% sequence identity to its naturally occurring parent GPCR of any one of SEQ ID NOs: 36-70 in Table 8.

24. An expression vector comprising the polynucleotide of claim 22.

25. 25. The vector as claimed in claim 24 which is a viral vector.

26. 26. The vector as claimed in claim 25, which is an adenoviral vector and / or an adeno-associated vector (AAV), optionally selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof.

27. 26. The vector as claimed in claim 25, which is a herpes viral vector, a retroviral vector, or a lentiviral vector.

28. 26. The vector of claim 25, wherein the nucleic acid encoding the modified GPCR is operably linked to a tissue- or cell-specific promoter.

29. 29. The vector of claim 28, wherein the promoter is a neural cell type specific promoter.

30. 30. The vector of claim 29, wherein the promoter is a CaMk2A promoter.

31. 25. A host cell comprising, transfected with or transformed with the vector of claim 24.

32. 22. A process for producing a modified GPCR according to any one of claims 1 to 21, comprising the steps of: (a) stably transforming a host cell with an expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a modified GPCR of any one of claims 1 to 21; (b) culturing the transformed host cell under conditions suitable for the host cell to produce the modified GPCR; (c) optionally regenerating said modified GPCR.

33. 1. A method of increasing the potency and / or efficacy of an exogenous agonist that is an antihistamine for a parent modified G protein-coupled receptor (GPCR), said parent modified GPCR comprising modified residues at the following positions: (a) 113, and (b) 203 making further modifications at one or more further positions in said parent modified GPCR; The amino acid positions of the parent modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:

1.

34. (i) the antihistamine is as defined in any one of claims 2 to 4, and / or (ii) the further modification is as defined in any one of claims 7 to 13, and / or (iii) the GPCR is defined in any one of claims 14 to 21.

35. 1. A process for making an engineered G protein-coupled receptor having an engineered responsiveness to an exogenous agonist, comprising modifying a parent GPCR relative to said parent GPCR by two, three, or four modifications at positions selected from: (i) 85 (ii) 416 (iii) 120 (iv) 123 and optionally comprising, relative to said parent GPCR, one or more alterations at a position selected from: (v) 128 (vi) 121 (vii) 200 (viii) 204 (viii) 410 (ix) 413 The process wherein the amino acid positions of the modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:

1.

36. 36. The process of claim 35, wherein the potency and / or efficacy of the exogenous agonist is increased in the modified GPCR compared to the parent GPCR.

37. 36. A process as claimed in claim 35, wherein the parent GPCR to be modified is derived from a naturally occurring GPCR but contains the residues or substitutions defined in claim 5 or claim 6.

38. (i) the exogenous agonist is as defined in any one of claims 2 to 4, and / or (ii) the modification is as defined in any one of claims 7 to 14, and / or (iii) the GPCR is defined in any one of claims 15 to 21.

39. 33. A modified GPCR obtained or obtainable by the method of claim 32.

40. 1. Use of an alteration, said alteration being a further amino acid alteration introduced into a parent altered G protein-coupled receptor (GPCR), said parent altered GPCR comprising an altered residue at the following position: (a) 113, and (b) 203 Increase the potency and / or efficacy of exogenous agonists that are antihistamines; The amino acid positions of the modified GPCR are numbered corresponding to the amino acid sequence of SEQ ID NO:

1.

41. (i) the antihistamine is as defined in any one of claims 2 to 4, and / or (ii) the further modification is as defined in any one of claims 7 to 14, and / or (iii) The use as claimed in claim 40, wherein the GPCR is defined in any one of claims 15 to 21.

42. 41. The use as claimed in claim 40, wherein the modification or further modification is performed on a polynucleotide comprising a nucleic acid sequence encoding said parent GPCR.

43. (i) providing a polynucleotide comprising a nucleic acid sequence encoding said parent GPCR; (ii) modifying said codons that code for said amino acid modification or further modification.

44. 1. A method for selectively modifying activation of a G protein or activating a G protein in a cell of a subject or organism, comprising: (i) expressing in said cell a modified GPCR as defined in any one of claims 1 to 21; (ii) administering to said subject or organism an agonist for said expressed modified GPCR.

45. 45. The method as claimed in claim 44, wherein the subject is a human subject.

46. 45. The method of claim 44, wherein the subject or organism is a non-human mammal, bird, fish, reptile, or amphibian.

47. 45. The method of claim 44, wherein the cell is a non-excitable cell, optionally a hepatocyte.

48. 45. The method of claim 44, wherein the cell is an excitable cell selected from the list consisting of a neuron from the central or peripheral nervous system, a muscle cell, and an endocrine cell.

49. 49. The method of claim 48, wherein activation of the G protein alters the excitability of an excitable cell, which is optionally a neuron.

50. 50. A method as claimed in claim 49 for modifying the excitability of excitable cells, optionally mammalian neurons, in a region-specific and time-specific manner, comprising: (a) administering to the subject an effective amount of a polynucleotide or vector as defined in claim 22 encoding a modified GPCR; (b) expressing the modified GPCR of step (a); (c) administering to the mammal said exogenous agonist for said expressed modified GPCR.

51. 45. The method as claimed in claim 44, wherein the exogenous agonist is defined in claim 2.

52. 1. A method of treating a disease or disorder in a subject, comprising: (i) expressing in a target cell or organ of the subject a modified GPCR as defined in any one of claims 1 to 21; (ii) administering to the subject an agonist for the expressed modified GPCR.

53. 1. A method of treating a disease or disorder in a subject, comprising: (a) administering to the subject an effective amount of a polynucleotide as defined in claim 22 that encodes a modified GPCR; (b) expressing the modified GPCR of step (a) in a target cell or organ of the subject; (c) administering to the subject an exogenous agonist for the expressed modified GPCR.

54. 54. The method as claimed in claim 53, wherein the polynucleotide is administered to the subject by direct injection and / or the exogenous agonist is defined in claim 2.

55. 53. The method of claim 52, wherein the disease or disorder is a disease or disorder of the nervous system and the modified GPCR is expressed in the central nervous system or the peripheral nervous system.

56. 56. The method of claim 55, wherein the disease or disorder of the nervous system is a neural circuit disorder.

57. 56. The method of claim 55, wherein the disease or disorder of the nervous system is a neuropsychiatric disorder or a neurodegenerative disease.

58. 56. The method of claim 55, wherein the disease or disorder is selected from a neuropsychiatric disorder, a neurodegenerative disease, chronic pain, cerebrovascular accident (CVA), and stroke.

59. 56. The method as claimed in claim 55, wherein the disease or disorder is a seizure disorder.

60. The method of claim 59, wherein the modified GPCR is expressed in neurons of a seizure focus in the patient's brain, and activation of the modified GPCR reversibly alters the excitability of the neurons in the seizure focus.

61. The method of claim 60, wherein activation of the modified receptor (i) reversibly inhibits excitability and thereby neurotransmission of excitatory neurons in the seizure focus, or (ii) reversibly excites inhibitory neurons in the seizure focus.

62. (i) the seizure disorder is epilepsy, optionally focal or generalized epilepsy; and / or (ii) the subject has been diagnosed with drug-resistant or medically refractory epilepsy, and / or (iii) the subject is under existing treatment with an anti-epileptic drug and the method has the purpose of allowing to discontinue the existing treatment or reduce drug therapy; and / or (iv) the subject has been diagnosed with epilepsy partialis continua, and / or (v) the exogenous ligand is administered to the subject before the patient suffers from an epileptic seizure; and / or (vi) the exogenous ligand is administered to the subject during an epileptic seizure; and / or (vii) the exogenous ligand is administered to the subject after suffering an epileptic seizure; and / or (viii) the exogenous ligand is administered to the subject within 30 minutes before or within 24 hours after the human suffers an epileptic seizure; and / or (ix) the exogenous ligand is administered to the subject automatically, either (i) by a device coupled to an automated seizure detection mechanism, or (ii) in response to a predicted seizure by EEG analysis; and / or 60. The method of claim 59, wherein (x) the exogenous ligand is administered in combination therapy with one or more other agents for treating the seizure disorder.

63. 53. The method as claimed in claim 52, wherein the disease or disorder is a non-central nervous system disorder and a non-peripheral nervous system disorder.

64. 53. The method of claim 52, wherein the agonist is administered by a route selected from the list consisting of: orally; parenterally, by intravenous or intramuscular route; subcutaneous injection; sublingually, buccal, intranasal, rectal; via an inhaler or nebulizer; topically to the eye or skin; auricular, intrathecal, intravaginal, intravesical.

65. 1. A method of treating a disease or disorder, optionally in a subject, comprising: (i) expressing in a target cell or organ of the subject a modified GPCR as defined in any one of claims 1 to 21; (ii) administering to the subject an agonist for the expressed modified GPCR.

66. 1. A method of treating a disease or disorder, optionally in a subject, comprising: (i) expressing a modified GPCR as defined in claim 1 in a target cell or organ of the subject; (ii) administering to the subject an agonist for the expressed modified GPCR.

67. A method of treating a disease or disorder in a subject, comprising: (i) expressing a modified GPCR as defined in claim 1 in a target cell or organ of the subject; (ii) administering to the subject an agonist for the expressed modified GPCR, the exogenous agonist as defined in claim 2 for use in the treatment of a disease or disorder in a subject, according to a method comprising the steps of:

68. 68. Use of a polynucleotide comprising a nucleic acid sequence encoding a modified GPCR according to any one of claims 1 to 21, and / or an expression vector comprising said polynucleotide, and / or a modified GPCR as defined in claim 66, and / or an exogenous agonist as defined in claim 67, in the preparation of a medicament for use in a method for the treatment of a seizure disorder.

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