M4di variant and use thereof in treatment of epilepsy

Abstract

The present application relates to an M4Di variant and the use thereof in the treatment of epilepsy. Specifically, the present application relates to a modified human G-protein-coupled receptor (GPCR), wherein the modified GPCR has the following characteristics: (i) reduced reactivity to an endogenous ligand compared with a wild-type GPCR; and (ii) unchanged or enhanced reactivity to an exogenous ligand compared with a wild-type GPCR. Further provided in the present application is the use of the modified human G-protein-coupled receptor (GPCR) in the treatment of epilepsy.

Description

An M4Di variant and its application in the treatment of epilepsy Technical Field

[0001] This application relates to the field of biomedicine, specifically to a mutant of the hM4Di receptor. Background Technology

[0002] Currently, chemogenetics is a technique that uses engineered receptors and specific ligands to precisely control cellular activity. The hM4Di receptor is a commonly used modified muscarinic receptor. However, the therapeutic effect of hM4Di is often limited by its sensitivity to ligands, response intensity, and desensitization time; high doses of CNO may lead to nonspecific effects; the short effective time and the limitation of rapid desensitization restrict the duration of therapeutic effect, reducing the practicality and efficacy of long-term treatment. Summary of the Invention

[0003] This application modifies the hM4Di receptor using genetic engineering technology to enhance its ability to regulate neuronal excitability, aiming to reduce abnormal discharges, suppress epileptic seizures, and provide a novel and effective treatment for epilepsy patients.

[0004] This application provides a treatment method for epilepsy using the hM4Di receptor with a 399-site mutation and its application. By addressing the key shortcomings of limited ligand sensitivity and rapid desensitization, it aims to overcome the limitations of existing antiepileptic therapies, particularly the inefficiency in treating refractory epilepsy. Through molecular biology and protein engineering, the original hM4Di receptor was optimized, enhancing its ligand sensitivity to allow for lower doses and reduce non-specific effects, while the extended desensitization time ensures sustained therapeutic efficacy. By combining with excitatory neuron-specific promoters, its targeting and inhibitory efficacy against specific neuronal subpopulations are enhanced, while reducing the risk of side effects. The optimized hM4Di receptor, when activated by its specific ligand (e.g., CNO), can more effectively promote neuronal hyperpolarization, reduce abnormal electrical activity, and thereby prevent or terminate epileptic seizures.

[0005] The core of this invention lies in the optimized design of the hM4Di receptor. The optimized hM4Di receptor gene is precisely introduced into overexcited neurons in the brain via a viral vector, achieving accurate regulation. This therapy promises to provide epilepsy patients with a safer, more effective, and personalized treatment option, particularly demonstrating its unique advantages in cases where traditional medications are difficult to control. This development opens new possibilities for the application of chemogenetics in the treatment of epilepsy.

[0006] On the one hand, this application provides a modified human G protein-coupled receptor (GPCR), wherein the modified GPCR has the following characteristics

[0007] (i) Compared with wild-type GPCRs, their responsiveness to endogenous ligands is reduced; and,

[0008] (ii) Compared with wild-type GPCRs, the reactivity to at least one exogenous ligand is enhanced.

[0009] In some embodiments, the GPCR is Gi / o coupled.

[0010] In some embodiments, the wild-type GPCR is the muscarinic acetylcholine receptor M4.

[0011] In some embodiments, the modified G protein-coupled receptor (GPCR) includes A5.46G and / or Y3.33C mutations compared to the wild-type GPCR.

[0012] In some embodiments, the wild-type GPCR comprises a sequence having at least 75% homology with SEQ ID NO:1.

[0013] In some embodiments, the modified GPCR is modified at least one of the following positions compared to the wild-type GPCR: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0014] In some embodiments, the modified GPCR includes the following substitutions compared to the wild-type GPCR:

[0015] a. 113C or 113N; and / or,

[0016] b.203G.

[0017] In some embodiments, the modified GPCR comprises the following substitutions compared to the wild-type GPCR:

[0018] a. Y113C or Y113N; and / or,

[0019] b.A203G.

[0020] In some embodiments, the modified G protein-coupled receptor (GPCR) includes at least one modification at the protein kinase A (PKA) phosphorylation site compared to the wild-type GPCR.

[0021] In some embodiments, compared to the wild-type GPCR, the modified GPCR is modified at least one position: 399, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0022] In some embodiments, the modified GPCR includes the following substitution compared to the wild-type GPCR: T399.

[0023] In some embodiments, the modified GPCR includes the following substitution compared to the wild-type GPCR: T399A.

[0024] In some embodiments, the modified GPCR comprises a sequence having at least 75% homology with SEQ ID NOs:2-3.

[0025] In some embodiments, the exogenous ligand is a ligand capable of binding to a GPCR modified with an amino acid sequence as shown in SEQ ID NO:2 or 3.

[0026] In some embodiments, the exogenous ligand is selected from clozapine, clozapine oxide (CNO), olanzapine, desclozapine (DCZ), or derivatives thereof.

[0027] On the other hand, this application provides a polynucleotide sequence comprising a polynucleotide encoding a nucleic acid sequence of the modified GPCR.

[0028] In some embodiments, the polynucleotide sequence includes a sequence having at least 75% homology with SEQ ID NO:4-5.

[0029] On the other hand, this application provides an expression vector comprising the aforementioned polynucleotide.

[0030] In some embodiments, the vector is a plasmid, transposon, granule, bacterial artificial chromosome, or viral vector.

[0031] In some embodiments, the vector is an adeno-associated virus (AAV), a herpesvirus vector, a retroviral vector, a vaccinia virus vector, an adenovirus vector, or a lentiviral vector.

[0032] In some embodiments, the AAV carrier includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, Anc80, or variants thereof.

[0033] In some embodiments, the nucleic acid encoding the modified GPCR is operatively linked to a promoter.

[0034] In some embodiments, the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0035] In some embodiments, the constitutive promoters include the immediate early promoter of cytomegalovirus (CMV), simian virus 40SV40, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70 kDa (HSP5), heat shock protein 90 kDa (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin (β-KIN), and human ROSA. One or more of the following: 26 promoter, ubiquitin C promoter UBC, phosphoglycerate kinase-1PGK promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter, or β-actin promoter.

[0036] In some embodiments, the promoter is the EF1α promoter.

[0037] In some embodiments, the inducible promoter includes one of the following: a transcriptional cofactor response promoter, a tetracycline response promoter, a ecdysone response promoter, a cumate response promoter, a glucocorticoid response promoter, an estrogen response promoter, a PPAR-γ promoter, or a RU-486 response promoter.

[0038] In some embodiments, the tissue-specific promoters include neuron-specific promoters, glial cell-specific promoters, heart-specific promoters, muscle-specific promoters, lung-specific promoters, liver-specific promoters, kidney-specific promoters, pancreas-specific promoters, adipose-specific promoters, endothelial-specific promoters, retinal-specific promoters, prostate-specific promoters, skin-specific promoters, and macrophage-specific promoters.

[0039] In some embodiments, the promoter is a neuron-specific promoter, which includes: human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 promoter, neuron-specific enolase (NSE) promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, Advillin promoter, Drosophila single homologue 1 (SIM1) promoter, oxytocin (OXT) promoter, spiky mouse-associated protein (AgRP) promoter, protein kinase C-δ (PKC-δ) promoter, auxin-releasing peptide promoter, glutamate decarboxylase (GAD1 / 2) promoter, choline acetyltransferase (ChAT) promoter, and no distal homeobox (Dlx) promoter.

[0040] In some embodiments, the promoter is CaMKIIα.

[0041] In some embodiments, glial cell-specific promoters include, but are not limited to, the astrocyte fibrillary acidic protein (GFAP) promoter, the Gfabc1D promoter, the myelin basal protein (MBP) promoter, the oligodendrocyte myelin glycoprotein (MOG) promoter, the CD11b promoter, the Iba1 promoter, and the Foxj1 promoter.

[0042] On the other hand, this application provides a cell including the aforementioned expression vector.

[0043] On the other hand, this application provides a pharmaceutical composition comprising the modified GPCR, or the polynucleotide sequence, or the expression vector, or the cell, and optionally a pharmaceutically acceptable carrier.

[0044] In some embodiments, the pharmaceutical composition further comprises an exogenous ligand.

[0045] On the other hand, this application provides a method for improving the reactivity of wild-type GPCRs to exogenous ligands, comprising modifying wild-type GPCRs at at least one of the following positions: 113 and / or 203, wherein the amino acid positions of the modified GPCRs are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0046] In some embodiments, the modification includes the following substitutions:

[0047] a. 113C or 113N; and / or,

[0048] b.203G.

[0049] In some embodiments, the modification includes the following substitutions:

[0050] a. Y113C or Y113N; and / or,

[0051] b.A203G.

[0052] In some embodiments, the method includes modifying a wild-type GPCR at at least one position: 399, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0053] In some embodiments, the modification includes the following substitution: T399.

[0054] In some embodiments, the modification includes the following substitution: T399A.

[0055] In some embodiments, the modification is performed on a polynucleotide containing a nucleic acid sequence encoding a wild-type GPCR.

[0056] On the other hand, this application provides a method for improving the efficacy of exogenous ligands, comprising, the modified GPCR, or the polynucleotide sequence, or the expression vector, or the cell, or the pharmaceutical composition.

[0057] On the other hand, this application provides a modified GPCR obtained by the method described.

[0058] On the other hand, this application provides a method for regulating the activity of GPCRs in a subject, including...

[0059] (i) expressing any of the modified GPCRs described above in the target cells of the subject; and

[0060] (ii) Administering exogenous ligands to the subject.

[0061] In some embodiments, the subject is a human.

[0062] In some embodiments, the subject is a non-human mammal, bird, fish, reptile, or amphibian.

[0063] In some embodiments, the target cell is a neuron, glial cell, muscle cell, or endocrine cell.

[0064] In some embodiments, the target cell is an activating neuron.

[0065] In some embodiments, the neuronal cells are derived from the central nervous system (CNS) or the peripheral nervous system (PNS).

[0066] In some embodiments, the neuronal cells are located in the epileptic pathological focus or epileptogenic focus.

[0067] In some embodiments, the method further includes administering an effective amount of the polynucleotide or the expression vector to the subject.

[0068] In some embodiments, the polynucleotide or expression vector is administered by injection.

[0069] In some embodiments, the polynucleotide or expression vector is contacted with the target cells of the subject.

[0070] On the other hand, this application provides the modified GPCR, the polynucleotide sequence, the expression vector, the cell, the pharmaceutical composition, or the method thereof for use in improving the responsiveness of GPCR to exogenous ligands.

[0071] On the other hand, this application provides the use of the modified GPCR, the polynucleotide sequence, the expression vector, the cell, the pharmaceutical composition, or the method thereof for treating epilepsy in a subject.

[0072] On the other hand, this application provides the use of the modified GPCR, the polynucleotide sequence, the expression vector, the cell, and the pharmaceutical composition described herein for the preparation of a drug for treating epilepsy.

[0073] On the other hand, this application provides a method for producing a modified GPCR, comprising modifying a wild-type GPCR at at least one of the following positions: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0074] In some embodiments, the modification includes the following substitutions:

[0075] a. 113C or 113N; and / or,

[0076] b.203G.

[0077] In some embodiments, the modification includes the following substitutions:

[0078] a. Y113C or Y113N; and / or,

[0079] b.A203G.

[0080] In some embodiments, the wild-type GPCR is modified at at least one position: 399, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1.

[0081] In some embodiments, the modification includes the following substitution: T399.

[0082] In some embodiments, the modification includes the following substitution: T399A.

[0083] In some embodiments, the modification is performed on a polynucleotide containing a nucleic acid sequence encoding a wild-type GPCR.

[0084] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0085] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0086] Figure 1 shows the functional validation results of the hM4Di mutant provided in this application as a Gi / o coupled GPCR.

[0087] Figures 2 and 3 show the sensitivity comparison results of the hM4Di mutant provided in this application compared with hM4Di.

[0088] Figure 4 shows the desensitization time comparison results of the hM4Di mutant provided in this application compared with hM4Di.

[0089] Figure 5 shows the sensitivity of the hM4Di mutant provided in this application to CNO and its effect on inhibiting neuronal activity.

[0090] Figure 6 shows the off-target verification of the mutant provided in this application.

[0091] Figure 7 shows a schematic diagram of the epilepsy model detection provided in this application.

[0092] Figure 8 shows the regulatory effect of intraperitoneal injection of CNO to activate hM4Di WT and hM4Di T399A mutants on cortical epilepsy, as provided in this application.

[0093] Figure 9 shows the regulatory effect of intraperitoneal injection of clozapine or olanzapine, provided in this application, on cortical epilepsy by activating the hM4Di T399A mutant.

[0094] Figure 10 shows the effect of intraperitoneal injection of CNO to activate the hM4Di mutant provided in this application on open field movement in mice. Detailed Implementation

[0095] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0096] Terminology Definition

[0097] This application discloses a novel therapeutic system that uses a vector to express a modified receptor that is insensitive to endogenous neurotransmitters but sensitive to exogenous ligands.

[0098] As described above, modified receptors activated solely by artificial agonists have been well known in the art for many years and are sometimes referred to as “designer receptors exclusively activated by designer drugs” (DREADDs) or “receptors activated solely by synthetic ligands” (RASSLs). Those skilled in the art can provide such receptors using known methods and apply them to the present invention in accordance with this disclosure. Unless the context otherwise requires, the terms “modified receptor” or similar terms, DREADD, and RASSL are used interchangeably herein.

[0099] For example, WO97 / 35478 describes a method for preparing RASSLs. The description of the preparation and properties of RASSLs in that application is hereby incorporated herein by reference. WO97 / 35478 describes how to prepare RASSLs from G protein-coupled receptors (including Gi / o-coupled acetylcholine-muscarinic receptors). This includes the description in WO97 / 35478, “Construction of RASSLs.” Furthermore, certain definitions relating to RASSLs in WO97 / 35478 are used herein to maintain consistency with their technically accepted meaning. The RASSL described therein is a modified G protein-coupled receptor with reduced binding affinity to selected natural (i.e., endogenous) ligands of GPCRs (relative to the binding affinity of wild-type G protein-coupled receptors to selected ligands), but with normal, near-normal, or preferably enhanced binding affinity to exogenous (usually synthetic) small molecules. Therefore, RASSL-mediated activation of RASSL-expressing cells is not significant in vivo in the presence of natural ligands, but the activation response is significant upon exposure to exogenously introduced small molecules. In other words, exogenous ligands activate RASSL better than natural ligands (i.e., the binding of small molecule ligands can activate RASSL to a greater extent than selected natural ligands at similar concentrations).

[0100] In this application, the term "G protein-coupled receptor" (GPCR) refers to a receptor that, upon binding to a natural ligand and activation of the receptor, transmits G protein-mediated signals, thereby generating G protein-coupled cellular responses. G protein-coupled receptors are a large family of evolutionarily related proteins (see WO97 / 35478). GPCRs interact with complexes of isotriguanine nucleotide-binding proteins (G proteins) to regulate various intracellular signaling pathways, including ion channels. GPCRs can alter neuronal excitability and thus neurotransmission by coupling G proteins to G protein-coupled inward rectifier potassium channels (GIRKs).

[0101] The preferred GPCR for activating GIRKS is the muscarinic acetylcholine receptor M4, also known as the human muscarinic acetylcholine receptor subtype M4. This receptor is a protein encoded by the CHRM4 gene in humans. The human muscarinic acetylcholine receptor subtype M4 (hM4) binds to acetylcholine (ACh) and then couples with Gi / o class G protein-coupled receptors to function, participating in Gi / o signaling pathways.

[0102] When two conserved sites, A5.46G and Y3.33C, are mutated on hM4, hM4 no longer binds to acetylcholine but instead binds efficiently to exogenous ligands (such as CNO). We refer to this mutated receptor as hM4Di. This modified GPCR includes the following mutation: Y113C / A203G. Preferably, the hM4Di sequence is shown in SEQ ID NO:2.

[0103] The G protein-coupled cellular responses related to this invention are those that alter cellular excitability. A preferred response is an inhibitory response, where ligand activation of receptors triggers a series of intracellular events, such as inhibition of adenylate cyclase (AC), thereby reducing intracellular cyclic adenosine monophosphate (cAMP) production; activation of potassium channels (such as GIRK channels), leading to cellular hyperpolarization; and inhibition of cellular action potential firing. These responses work together to ultimately result in cellular inhibition.

[0104] In this application, the term "exogenous" or "exogenous" is used in this disclosure to refer to any molecule originating outside of an organism, including nucleic acids, proteins or peptides, small molecule compounds, etc.

[0105] Conversely, the term "endogenous" refers to any molecule that originates from within an organism (i.e., is naturally produced by the organism).

[0106] The term "endogenous ligand" refers to molecules within an organism that can activate GPCR signaling pathways, causing different excitatory changes in cells. For example, acetylcholine (ACh) is an endogenous ligand for hM4. Specifically, acetylcholine (ACh) binds to the human muscarinic acetylcholine receptor subtype M4 (hM4), and then couples with Gi / o class G protein-coupled receptors to exert its effects, participating in Gi / o signaling pathways.

[0107] The term "exogenous ligand" refers to molecules outside an organism that can activate and modify GPCR signaling pathways, causing different excitatory changes in cells. Exogenous ligands are neither naturally occurring nor synthetic. For example, clozapine or clozapine oxide are exogenous ligands for hM4Di. Specifically, hM4Di can bind to an exogenous ligand (e.g., CNO), then couple to Gq-type G protein-coupled receptors, and subsequently couple to Gi / o-type G protein-coupled receptors to exert its function, participating in Gi / o signaling pathways.

[0108] The term "reactivity" in this article refers to the efficacy of endogenous or exogenous ligands for the receptor. For example, the mutant hM4Di receptor, upon activation by CNO, can cause a decrease in cyclic adenosine monophosphate (cAMP), and the degree of cAMP decrease reflects the ligand's efficacy for the receptor. Similarly, changes in ERK phosphorylation levels induced by CNO activation of the hM4Di receptor can reflect the receptor's activation state and desensitization time; therefore, changes in ERK phosphorylation levels reflect the ligand's efficacy for the receptor. Furthermore, activation of Gi / o coupled receptors inhibits neuronal excitability by reducing cAMP levels and activating potassium channels, thereby reducing the firing of action potentials; therefore, the firing of action potentials can reflect the ligand's efficacy for the receptor.

[0109] The modified human G protein-coupled receptor (GPCR) is designed to be activated by exogenous ligands. In this case, the modified human G protein-coupled receptor (GPCR) may not respond to endogenous ligands or may respond very little.

[0110] In this application, the term "modified" or "modified" in the context of proteins refers to a fragment in which at least one amino acid residue within a reference molecule is substituted, deleted, or added. Similarly, in the context of nucleic acids, the term refers to a fragment in which at least one nucleic acid residue within a reference molecule is substituted, deleted, or added.

[0111] In this application, the term "excitable cell" refers to a cell that can generate an action potential when stimulated, such as neurons of the central or peripheral nervous system, muscle cells (including striated and smooth muscle), or endocrine cells.

[0112] In this application, the term "homology" is generally equivalent to sequence "identity." Homologous sequences may include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the host sequence. Typically, homologs will contain the same active sites as the host amino acid sequence. Homology can be considered based on similarity (i.e., amino acid residues having similar chemical properties / functions) or can be expressed in terms of sequence identity. In this application, a sequence having a percentage identity with any of the referenced amino acid sequences or nucleotide sequences in SEQ ID NO means a sequence having said percentage identity over the entire length of the referenced SEQ ID NO. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0113] In this application, the term "wild type" generally refers to something that is naturally occurring or of natural origin.

[0114] In this application, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" generally refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically defined, the term may include nucleic acids containing analogs of natural nucleotides, said nucleic acids having similar binding properties to a reference nucleic acid (e.g., sequence information shown) and being metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, the sequence of a nucleic acid may include variants modified in a conserved manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences.

[0115] In this application, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0116] In this application, the term “pharmaceutically acceptable” generally refers to those compounds, materials, compositions, and / or dosage forms that are commensurate with a reasonable benefit / risk ratio and suitable for use in human and animal tissue contact without excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of reasonable medical judgment.

[0117] In this application, the term "pharmaceutically acceptable carrier" generally refers to any of those carriers commonly used and is limited only by physicochemical considerations (such as solubility and lack of reactivity with active binders) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that would be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc.; their uses are well known in the art.

[0118] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients and preferably inert at the doses and concentrations used, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0119] In this application, the terms "effective amount" or "effective dose" generally refer to an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent is generally any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease remission (proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability resulting from the disease).

[0120] In this application, the terms "host cell" or "cell" generally refer to an individual cell, cell line, or cell culture that may contain or already contains a vector including the nucleic acid molecules isolated as described in this application, or that is capable of expressing the nucleic acid molecules isolated as described in this application. The host cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they need to be capable of expressing the nucleic acid molecules isolated as described in this application. The host cell can be obtained by in vitro transfection of cells using the vector described in this application. The host cell can be a prokaryotic cell (e.g., *E. coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or neuronal cells). For example, the host cell may be an *E. coli* cell. For example, the host cell may be a yeast cell. For example, the host cell may be a mammalian cell. For example, the mammalian cell may be an N2A cell.

[0121] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0122] In this application, the term "viral vector" is used broadly to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer. Nucleic acid molecules include virus-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically include various viral components and sometimes host cell components other than nucleic acids. A viral vector can refer to a virus or viral particle capable of transferring nucleic acids into cells, or the transferred nucleic acid itself.

[0123] In this application, the term "lentivirus" generally refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); viscena-maedivirus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0124] In this application, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus in which some functions are provided by co-infected helper viruses. Thirteen AAV serotypes have been characterized. General information and reviews of AAVs can be found, for example, in Carter, 1989, *Handbook of Parvoviruses*, Vol. 1, pp. 169–228, and Berns, 1990, *Virology*, pp. 1743–1764, Raven Press, (New York). However, it is entirely expected that these same principles will apply to other AAV serotypes, as the various serotypes are known to be very closely related in both structure and function, even at the genetic level. For example, all AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three associated capsid proteins, such as those expressed in AAV6. The correlation was further demonstrated by heteroduplex analysis, which revealed extensive cross-hybridization along the genome length between serotypes and the presence of similar self-annealing segments corresponding to the ends of inverted terminal repeats (ITRs). Similar infection patterns also indicated that replication function in each serotype is under similar regulatory control.

[0125] In this application, the term "AAV vector" generally refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by an AAV terminal repeat sequence (ITR). When present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products, such an AAV vector can be replicated and packaged into an infectious viral particle. The terms "AAV virion," "AAV viral particle," or "AAV vector particle" refer to a viral particle composed of at least one AAV capsid protein and a capsidated polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply "AAV vector." Therefore, the production of an AAV vector particle necessarily includes the production of an AAV vector such that the vector is contained within the AAV vector particle.

[0126] The AAV "rep" and "cap" genes refer to the genes encoding the replication protein and capsid protein, respectively. The AAV rep and cap genes have been identified in all AAV serotypes studied to date, and these genes are described in this paper and the cited references. In wild-type AAV, the rep and cap genes are generally adjacent to each other in the viral genome (i.e., they are "coupled" together to form adjacent or overlapping transcription units), and they are generally conserved among AAV serotypes. The AAV rep and cap genes can also be referred to individually or collectively as "AAV packaging genes." The AAV cap gene encodes the Cap protein, which, in the presence of rep and adenovirus helper functions, packages the AAV vector and binds to target cell receptors.

[0127] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. Promoters can be recognized by RNA polymerase, which initiates transcription to synthesize RNA. During RNA synthesis, promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). A promoter comprises a core promoter region and a regulatory region, located in the regulatory sequence controlling gene expression, upstream of the gene transcription start site (at the 5' direction of the DNA antisense strand), and does not itself have a coding function. Based on their mode of action and function, promoters are classified into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or developmentally specific), and inducible promoters (regulated by various biological, external chemical, or physical signals inside and outside the cell).

[0128] The term "tissue-specific promoter" refers to promoters that regulate and guide gene expression in a tissue-specific manner, such as in brain tissue, muscle tissue, liver tissue, and kidney tissue.

[0129] In this application, the term "operably linked" generally refers to placing a regulatory sequence necessary for the expression of a coding sequence in an appropriate position relative to the coding sequence in order to achieve the expression of the coding sequence. The term "operably linked" can also refer to the arrangement of the coding sequence and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition is sometimes also applied to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules in which hybrid nucleic acid molecules are generated.

[0130] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0131] In this application, the terms “polypeptide,” “peptide,” “protein,” and “protein protein” are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers containing modified amino acids. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeled component). The term “amino acid” includes natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0132] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0133] In this application, the term "treatment" generally refers to a clinical intervention used to alter the natural processes of an individual or cell in a clinicopathological process. It may include improving disease status, eliminating lesions, or improving prognosis.

[0134] In this application, the term "relief" refers to reducing, shortening, or delaying a symptom, disease, condition, or phenotype. The symptom, disease, condition, or phenotype may include subjective perceptions of the subject, such as pain, dizziness, or other physiological disturbances, or medically detectable indicators, such as lesions detected through medical testing.

[0135] In this application, the term "prevention" generally refers to the preventive administration of a combination to a healthy subject to prevent the occurrence of a disease or condition. It may also include the preventive administration of a combination to a patient in the pre-treatment stage of an allergic disease to be treated. "Prevention" does not require the complete elimination of the possibility of the disease or condition occurring; in other words, "prevention" generally means a reduction in the likelihood of the disease or condition occurring in the presence of said application combination.

[0136] In this application, the term "epilepsy" refers to a brain disorder in which subjects experience recurrent seizures over time, meaning that the seizures are not isolated events and will recur at least once more. Subjects with epilepsy typically experience at least two uninduced seizures with an interval of more than 24 hours. The term "epileptic seizure" refers to a physical finding or behavioral change that occurs following an episode of abnormal electrical activity in the brain. For clinical evaluation purposes, it should be understood in the art that subjects with epilepsy are typically classified according to the type of seizure. According to the latest classification of the ILAE (updated in 2017), epileptic seizures can be divided into three main categories: focal seizures, generalized seizures, and seizures of unknown origin. Focal seizures are further subdivided into focal onset aware seizures and focal onset impaired awareness seizures based on the state of consciousness during the seizure. Generalized seizures, based on clinical presentation characteristics, encompass various types, including generalized tonic-clonic seizures, absence seizures, myoclonic seizures, clonic seizures, tonic seizures, and atonic seizures. When it is impossible to determine whether a seizure is focal or generalized, it is classified as a seizure of unknown origin.

[0137] In this application, the term "epilepsy focus" is a neuropathological concept involving structural or metabolic abnormalities in a specific region of the brain that directly or indirectly trigger abnormal neuronal firing patterns, manifested as intractable discharges on an electroencephalogram (EEG), and ultimately leading to clinically visible epileptic seizures.

[0138] In this application, the term "epileptic focus" is a neurophysiological concept, specifically referring to an area identifiable on electroencephalography (EEG) that exhibits significant epileptiform discharges, directly involved in and driving the occurrence of epileptic seizures. An epileptogenic focus may consist of single or multiple clusters of pathological neurons whose abnormal firing patterns are the direct source of epileptic seizures. Epileptogenic focuses are often located in the peripheral regions of structural lesions, especially in the presence of a single, clearly defined pathological focus, such as a brain tumor or vascular malformation. However, when faced with widespread or diffuse pathological changes, such as hippocampal sclerosis in the medial temporal lobe or extensive scar tissue after trauma, the epileptogenic focus may be distributed throughout the entire pathological focus, or even extend to adjacent or contralateral brain regions.

[0139] In this application, the term "lesional functional epileptogenic complex" refers to a comprehensive description of the epileptic pathological focus and the epileptogenic focus. This complex may be visible on imaging examinations such as computed tomography (CT) or magnetic resonance imaging (MRI), such as brain tumors, encephalomalacia, vascular malformations, or post-traumatic brain scars.

[0140] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0141] Invention Details

[0142] Modified human GPCR

[0143] On the one hand, this application provides a modified human G protein-coupled receptor (GPCR), wherein the modified GPCR has the following characteristics

[0144] (i) Compared with wild-type GPCRs, the reactivity to endogenous ligands is reduced; and (ii) Compared with wild-type GPCRs, the reactivity to exogenous ligands remains unchanged or is enhanced.

[0145] The aforementioned wild-type GPCRs can be wild-type human GPCRs. Human GPCRs can be classified according to the signaling proteins they interact with. GPCRs regulate cellular function and physiological responses by coupling with specific G proteins, converting extracellular signals into intracellular signals. G proteins can be classified into several main types based on the type of their α subunit, including excitatory (e.g., Gs, Gq / 11, G12 / 13) and inhibitory (e.g., Gi / o). In some cases, activation of GPCRs coupled to downstream G proteins can alter the electrophysiological activity of excitable cells (e.g., muscle cells, neurons). The functional diversity of GPCRs depends on their coupling with different types of G proteins, which determines the characteristics of GPCR signaling and downstream effects. In a specific example, an inhibitory GPCR (e.g., coupled with Gi / o) can be selected to treat epilepsy.

[0146] Examples of wild-type human GPCRs may include hM4, the sequence of which is shown in SEQ ID NO: 1.

[0147] When two conserved sites, A5.46G and Y3.33C, are mutated on hM4, hM4 no longer binds to acetylcholine but instead binds efficiently to exogenous CNO. The mutated receptor is called hM4Di. Compared to the wild-type GPCR, the modified GPCR (e.g., hM4Di) includes the following substitutions: a. 113C or 113N; and / or, b. 203G. Further, compared to the wild-type GPCR, the modified GPCR (e.g., hM4Di) includes the following substitutions: a. Y113C or Y113N; and / or, b. A203G.

[0148] Preferably, the sequence of hM4Di is shown in SEQ ID NO:2, and the nucleotide sequence encoding hM4Di is shown in SEQ ID NO:4.

[0149] In some embodiments, the modified GPCR is unresponsive or substantially unresponsive to endogenous ligands, and is therefore primarily activated by exogenous ligands.

[0150] In some embodiments, the therapeutic effect of hM4Di is often limited by its sensitivity to ligands, the intensity of the response, and the time required for desensitization. High doses of CNO may lead to nonspecific effects. The shorter effective time and the limitation of rapid desensitization restrict the duration of the therapeutic effect, reducing the practicality and effectiveness of long-term treatment.

[0151] Therefore, this application provides hM4Di with a mutation at the protein kinase A (PKA) phosphorylation site. The PKA phosphorylation site is a threonine residue on the intracellular loop 3 of hM4Di, which is phosphorylated by PKC upon receptor activation. Mutating this site to other amino acids (e.g., alanine) significantly reduces the receptor's ability to internalize and maintain sustained activation of G proteins in the presence of an agonist, thus prolonging receptor activation time.

[0152] Compared to the modified GPCR (e.g., hM4Di), it further includes at least one modification at the protein kinase A (PKA) phosphorylation site.

[0153] Mutations in the hM4Di receptor at PKA phosphorylation sites (e.g., site 399) address key limitations in ligand sensitivity and rapid desensitization, providing a more effective and practical solution for long-term chemogenetic therapy. Enhanced sensitivity to CNO allows for lower doses and reduced nonspecific effects, while extended desensitization time ensures sustained therapeutic efficacy.

[0154] In some embodiments, hM4Di with a PKA phosphorylation site mutation (e.g., site 399) is unresponsive or substantially unresponsive to endogenous ligands and, compared to hM4Di, enhances receptor sensitivity, response strength, and optimizes desensitization to exogenous ligands, including but not limited to clozapine-N-oxide (CNO), thereby improving therapeutic efficacy in chemogenetic applications. The sequence of hM4Di with the 399 mutation is shown in SEQ ID NO: 3, and the nucleotide sequence encoding hM4Di with the 399 mutation is shown in SEQ ID NO: 5.

[0155] In some embodiments, the mutation at position 399 can be a mutation of the amino acid at position 399 from the original amino acid to any one of the following: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. For example, the mutation at position 399 can be a mutation from threonine to any one of the following: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0156] In some embodiments, the exogenous ligands mentioned above include: CNO, clozapine, piperapine, olanzapine, desclozapine, Compound 21, JHU37152, JHU37160, etc.

[0157] In some embodiments, the exogenous ligand comprises an N4'-alkyl-substituted CNO analogue, including 3-chloro-6-(4-ethylpiperazin-1-yl)-5H-benzo[b][1,4]benzodiazepine, 4-(8-chloro-5H-dibenzo[b,e][1,4]diazepine-11-yl)-1,1-dimethylpiperazin-1-onium iodide, 3-chloro-6-(piperazin-1-yl)-5H -Benzo[b][1,4]benzodiazepine, 8-chloro-11-[4-(1,1-dideuterated ethyl)piperazin-1-yl]-5H-dibenzo[b,e][1,4]diazepine, 11-(piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine, and 11-(4-ethylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine.

[0158] In some embodiments, GPCRs may include one or more of the following: CHRM1, GNRHR, GPR73, GPR45, PTHR1, CHRM2, GNRHR2, GPR73, GPR63, PTHR2, CHRM3, HRH1, GPR10, GPR83, SCTR, CHRM4, HRH2, F2R, PGR15, ADCYAP1R1, CHRM5, HRH3, F2RL1, PGR15L, VIPR1, ADORA1, HRH4, F2RL2, GPR103, VIPR2, ADORA2A, FSHR 93. F2RL3, GPR103L, BAI1, ADORA2B, LHCGR, P2RY1, GRCA, BAI2, ADORA3, TSHR, P2RY2, PGR1, BAI3, P2RY12, GPR54, P2RY4, HGPCR11, CD97, GPR105, LTB4R, P2RY6, SALPR, EMR 1. GPR86, LTB4R2, P2RY11, MAS1, EMR2, GPR87, MRGX1, LGR7, GPR90, EMR3, ADRA1A, MRG X2, LGR8, P2Y5, PGR16, ADRA1B, MRGX3, RGR, GPR23, LEC1, ADRA1D, MRGX4, HTR1A, P2Y10 275. LEC2, ADRA2A, MRGD, HTR1B, FKSG79, LEC3, ADRA2B, MrgA1, HTR1D, PGR2, CELSR1, ADRA2C, MrgA2, HTR1E, PGR3, CELSR2, ADRB1, MrgA3, HTR1F, AGR9, CELSR3, ADRB2 21. MrgA4, HTR2A, CMKLR1, GPR64, ADRB3, MrgA5, HTR2B, EBI2, PGR17, ADMR, MrgA6, HTR2C, GPC R150, DJ287G14, C3AR1, MrgA7, HTR4, GPR1, KIAA0758, C5R1, MrgA8, HTR5A, GPR15, PGR18, GPR 77. MrgA9, HTR5B, GPR17, PGR19, AGTR1, MrgA10, HTR6, GPR18, PGR20, AGTR2, MrgA11, HTR7, GP R19, ​​TEM5, AGTRL1, MrgA12, SSTR1, GPR20, KIAA1828, BRS3, MrgA13, SSTR2, GPR22, PGR21, GRPR 31. MrgA14, SSTR3, GPR25, ETL, NMBR, MrgA15, SSTR4, GPR30,FLJ14454、BDKRB1、MrgA16、SSTR5、GPR31、GPR56、BDKRB2、MrgA19、G2A、GPR 32、OA1、CNR1、MrgB1、GPR4、GPR33、PGR22、CNR2、MrgB2、GPR65、GPR34、PGR23 、CCR1、MrgB3、GPR68、GPR35、PGR24、CCR2、MrgB4、EDG1、GPR39、PGR25、CCR3 、MrgB5、EDG2、GPR40、PGR26、CCR4、MrgB6、EDG3、GPR44、PGR27、CCR541、MrgB 8、EDG4、GPR55、VLGR1、CCR6、MrgB10、EDG5、GPR61、CCR743、MrgB11、EDG6、G PR62、CCR8、MrgB13、EDG7、GPR75、CCR9、GPR24、EDG8、GPR80、GPR2、SLT、TACR 1、GPR82、CASR、CCRL1、MC1R、TACR2、GPR84、GABBR1、CCRL2、MC2R、TACR3、GP R88、GPR51、CCBP2、MC3R、TRHR、GPR91、GPRC5B、CMKBR1L1、MC4R、TRHR2、GPR9 2, GPRC5C, CMKBR1L2, MC5R, GPR57, GPR101, GPRC5D, CCXCR1, MTNR1A, GPR58, H963, RAI3, CX3CR1, MTNR1B, PNR, HGPCR2, GRM1, IL8RA, GPR50, TAR1, HGPCR 19、GRM2、IL8RB、GPR66、TAR2、HUMNPIIY20、GRM3、GPR9、NMU2R、TAR3、MRG、G RM4、CXCR4、NPFF1R、TAR4、MRGE、GRM5、BLR1、GPR74、GPR102、MRGF、GRM6、CXC R6, GPR7, TA7, MRGG, GRM7, CCKAR, GPR8, TA8, OPN3, GRM8, CCKBR, NPY1R, TA10, OPN4, GPRC6A, CYSLT1, NPY2R, TA11, PGR4, PGR28, CYSLT2, PPYR1, TA12, PG R5、DRD1、NPY5R、TA14、PGR6、DRD2、NPY6R、TA15、PGR7、DRD3、NTSR1、GPR14、 PGR8、DRD4、NTSR2、AVPR1A、PGR10、FZD1、DRD5、OPRD1、AVPR1B、PGR11、FZD2、FY, OPRK1, AVPR2, PGR12, FZD3, TG1019, OPRM1, OXTR, PGR13, FZD4, HM74, OPRL1, GPR48, PGR14, FZD5, GPR81, OPN1LW, GPR49, RDC1, FZD6, EDNRA, OPN1MW, LG R6, RE2, FZD7, EDNRB, OPN1SW, GPR27, RRH, FZD8, FPR1, RHO, GPR85, FZD9, FPRL1, HCRTR1, SREB3, FZD10, FPRL2, HCRTR2, GPR3, SMOH, FPR-RS1, PTAFR, GPR6, CALCR, FPR-RS2, PTGDR, GPR12, CALCRL, FPR-RS3, PTGER1, GPR21, CRHR1, FPR-RS4, PTGER2, GPR52, CRHR2, GALR1, PTGER3, GPR26, GIPR, TM7SF1, GALR2, PTG ER4, GPR78, GCGR, TM7SF1L1, GALR3, PTGFR, GPR37, GLP1R, TM7SF1L2, GHSR, PTGIR, GPR37L1, GLP2R, TM7SF3, GPR38, TBXA2R, GPR41, GHRHR, TPRA40, and GPR43. ,

[0159] In some embodiments, the reactivity of the modified GPCR to endogenous ligands (e.g., ACh) may be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 86%, less than 87%, less than 88%, less than 89%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100%.

[0160] In some embodiments, the modified GPCR is partially homologous to the wild-type GPCR. In some cases, the modified GPCR shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% amino acid homology with the wild-type GPCR.

[0161] expression carrier

[0162] On the other hand, this application provides a vector for expressing modified GPCRs.

[0163] In some embodiments, any vector suitable for introducing an expression cassette or polynucleotide encoding a modified GPCR into neuronal cells can be used. Illustrative examples of suitable vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, clomids, bacterial artificial chromosomes, and viral vectors. In some cases, the vector is a circular nucleic acid, such as a plasmid, BAC, PAC, YAC, clomid, fosmid, etc. In some cases, circular nucleic acid molecules can be used to deliver nucleic acid molecules encoding modified GPCRs to a subject. For example, a plasmid DNA molecule encoding a modified GPCR can be introduced into a subject's cells, thereby transcribing the DNA sequence encoding the modified GPCR into mRNA and translating the mRNA "information" into a protein product. Circular nucleic acid vectors will typically include regulatory elements that regulate the expression of target proteins. For example, a circular nucleic acid vector may include any number of promoters, enhancers, terminators, splicing signals, origins of replication, initiation signals, etc.

[0164] In some embodiments, the vector is a viral vector. For example, the viral vector may include a replication-defective virus. Non-limiting examples of viral vectors suitable for delivering the nucleic acid molecules of the present invention to a subject include vectors derived from adenoviruses, retroviruses (e.g., lentiviruses), adeno-associated viruses (AAVs), and herpes simplex virus-1 (HSV-1). For example, viral vectors include, but are not limited to, retroviral vectors (e.g., lentiviral vectors), herpesvirus-based vectors, and parvovirus-based vectors (e.g., adenovirus-based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors).

[0165] As used herein, the term "parvovirus" encompasses all parvoviruses, including autonomously replicating parvoviruses and virus-dependent viruses. Autonomous parvoviruses include members of the genera parvovirus, erythrovirus, lysinic virus, iteravirus, and contravirus. Exemplary autonomous parvoviruses include, but are not limited to, mouse parvovirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline leukopenia virus, feline parvovirus, goose parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, for example, Fields et al., Virology, 1996, Vol. 2, Chapter 69 (3rd edition, Lippincott-Raven Publishers).

[0166] In some embodiments, the dependent virus includes adeno-associated viruses, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV Retro, AAV DJ, AAVrh10, Anc80 and variants thereof.

[0167] In some embodiments, AAV includes poultry AAV, cattle AAV, dog AAV, horse AAV, and sheep AAV.

[0168] The adeno-associated virus vector (AAV vector) may also include a recombinant adeno-associated virus vector (rAAV vector).

[0169] In some cases, the vector may further include a restriction enzyme site downstream of the promoter to allow insertion of a polynucleotide encoding the modified GPCR, wherein the promoter and restriction enzyme site may be located downstream of the 5'AAV ITR and upstream of the 3'AAV ITR. In some cases, the vector may further include a post-transcriptional regulatory element located downstream of the restriction enzyme site and upstream of the 3'AAV ITR. In some cases, the vector may further include a polynucleotide inserted at the restriction enzyme site and operatively linked to the promoter, wherein the polynucleotide may contain the coding region of the modified GPCR. As will be appreciated by those skilled in the art, any of the AAV vectors disclosed in this application can be used as viral constructs in the methods to generate recombinant AAV.

[0170] In some embodiments, the vector includes a promoter to which a nucleic acid encoding a modified GPCR is operatively linked.

[0171] The promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0172] The constitutive promoters mentioned therein include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), simian virus 40SV40, Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1α) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70 kDa (HSP5), heat shock protein 90 kDa (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinin (β-KIN), and human ROSA. 26 promoter, ubiquitin C promoter UBC, phosphoglycerate kinase-1PGK promoter, cytomegalovirus enhancer / chicken β-actin CAG promoter or β-actin promoter.

[0173] The inducible promoters mentioned therein include, but are not limited to, transcription cofactor response promoters, antibiotic response promoters, ultraviolet-induced promoters, metallothionein promoters, tetracycline response promoters, ecdysone response promoters, cumate response promoters, glucocorticoid response promoters and estrogen response promoters, PPAR-γ promoters or RU-486 response promoters.

[0174] In some embodiments, tissue specificity may include, but is not limited to: neuron-specific promoters, heart-specific promoters (e.g., cTNT, POSTN), muscle-specific promoters (e.g., MHCK7, SM22a, ACTA1), lung-specific promoters (e.g., SP-C), liver-specific promoters (e.g., TBG), kidney-specific promoters (e.g., nphs1, nphs2), pancreas-specific promoters (e.g., padx1, insulin2), adipose-specific promoters (e.g., FABP4), endothelial-specific promoters (e.g., TIE), retinal-specific promoters (e.g., rpe65), prostate-specific promoters (e.g., DD3), skin-specific promoters (e.g., keratin14), and macrophage-specific promoters (e.g., F4 / 80).

[0175] Tissue-specific promoters include neuron-specific promoters or glial cell-specific promoters. Neuron-specific promoters include, but are not limited to, human synaptic protein-1 (SYN-1) promoter, calcium-calmodulin-dependent protein kinase IIα (CaMKIIα) promoter, tubulin α1 promoter, neuron-specific enolase (NSE) promoter, derivatized growth factor β chain promoter (PDGFB), TRPV1 promoter, Nav1.7 promoter, Nav1.8 promoter, Nav1.9 promoter, Advillin promoter, Drosophila single homologue 1 (SIM1) promoter, oxytocin (OXT) promoter, spiky mouse-associated protein (AgRP) promoter, protein kinase C-δ (PKC-δ) promoter or auxin-releasing peptide promoter, glutamate decarboxylase (GAD1 / 2) promoter, choline acetyltransferase (ChAT) promoter, and Dlx promoter. Glial cell-specific promoters include, but are not limited to, the astrocyte fibrillary acidic protein (GFAP) promoter, the Gfabc1D promoter, the myelin basal protein (MBP) promoter, the oligodendrocyte myelin glycoprotein (MOG) promoter, the CD11b promoter, the Iba1 promoter, and the Foxj1 promoter.

[0176] In some embodiments, the promoter is a neuron-specific promoter, such as CaMKIIα.

[0177] In some embodiments, the viral vector may comprise the nucleotide sequences shown in SEQ ID NOs:4-5. For example, the vector may comprise a nucleotide sequence that is at least 90% homologous to the nucleotide sequences shown in SEQ ID NOs:4-5, such as any polynucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0178] cell

[0179] This application provides a cell comprising cells transfected, infected, or transduced in vivo, in vitro, or extracellularly with the aforementioned expression vector or polynucleotide. Host cells may include virus-producing cells and cells infected with a viral vector. In some embodiments, in vivo host cells are infected with a viral vector as described herein.

[0180] Compositions and Formulations

[0181] This application provides a pharmaceutical composition comprising the aforementioned modified GPCR, or the aforementioned polynucleotide sequence, or the aforementioned expression vector, or the aforementioned cell, and optionally a pharmaceutically acceptable carrier.

[0182] In some embodiments, the pharmaceutical composition may further include the exogenous ligand.

[0183] The pharmaceutical composition may comprise a liquid formulation, a solid formulation, or a combination thereof. The compositions of the present invention may further comprise any number of excipients. Excipients may include any and all solvents, coatings, flavoring agents, coloring agents, lubricants, disintegrants, preservatives, sweeteners, binders, diluents, and mediators (or carriers). Generally, the excipients are compatible with the therapeutic compositions of the present invention.

[0184] In the pharmaceutical compositions disclosed in this application, the formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, such as developing suitable dosages and treatment regimens for the use of the specific compositions described herein in a variety of therapies, including, for example, oral, parenteral, intravenous, intranasal, intramuscular, intrathecal, intracranial, intraneural, intraganglionic, and intraventricular administration.

[0185] In some embodiments, the expression vector may be, for example, about 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 120 μg, about 140 μg, about 160 μg, about 180 μg, about 200 μg, about 220 μg, about 240 μg, or about 260 μg. Approximately 280 μg, approximately 300 μg, approximately 320 μg, approximately 340 μg, approximately 360 μg, approximately 380 μg, approximately 400 μg, approximately 420 μg, approximately 440 μg, approximately 460 μg, approximately 480 μg, approximately 500 μg, approximately 520 μg, approximately 540 μg, approximately 560 μg, approximately 580 μg, approximately 600 μg, approximately 620 μg, approximately 640 μg, approximately 660 μg, approximately 680 μg, approximately 700 μg, approximately 720 μg, approximately 740 μg, approximately 760 μg, approximately 780 μg, approximately 800 μg, approximately 820 μg, approximately 840 μg, approximately 860 μg, approximately 880 μg, approximately 900 μg, approximately 920 μg Approximately 940 μg, approximately 960 μg, approximately 980 μg, approximately 1 mg, approximately 2 mg, approximately 3 mg, approximately 4 mg, approximately 5 mg, approximately 6 mg, approximately 7 mg, approximately 8 mg, approximately 9 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 120 mg, approximately 140 mg, approximately 160 mg, approximately 180 mg, approximately 200 mg, approximately 220 mg, approximately 240 mg, approximately 260 mg, approximately 280 mg, approximately 300 mg, approximately 320 mg, approximately 340 mg, approximately 360 mg, approximately 380 mg, approximately 400 mg The composition is present in amounts of about 420 mg, about 440 mg, about 460 mg, about 480 mg, about 500 mg, about 520 mg, about 540 mg, about 560 mg, about 580 mg, about 600 mg, about 620 mg, about 640 mg, about 660 mg, about 680 mg, about 700 mg, about 720 mg, about 740 mg, about 760 mg, about 780 mg, about 800 mg, about 820 mg, about 840 mg, about 860 mg, about 880 mg, about 900 mg, about 920 mg, about 940 mg, about 960 mg, about 980 mg, about 1000 mg, or greater than 1000 mg.

[0186] Regulation methods and indications

[0187] On the other hand, this application provides a method for improving the exogenous ligand potency of wild-type human GPCRs, comprising modifying the wild-type GPCR at at least one position: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1. Further, the modification comprises the following substitutions: a. Y113C or Y113N; and / or, b. A203G. In some embodiments, the method comprises modifying the wild-type GPCR at at least one position: a protein kinase A (PKA) phosphorylation site, for example, position 399. Further, the modification comprises the following substitution: T399. Further, the modification comprises the following substitution: T399A. In some embodiments, the modification is performed on a polynucleotide containing a nucleic acid sequence encoding the wild-type GPCR. Through the above method, the reactivity of the wild-type human GPCR to endogenous ligands (e.g., ACh) is reduced, while the reactivity to exogenous ligands (e.g., CNO) is increased.

[0188] On the other hand, this application describes a method for regulating the GPCR activity of a subject, including...

[0189] (i) expressing an effective dose of the aforementioned modified GPCR in the target cells of the subject; and

[0190] (ii) Administer an effective dose of exogenous ligand to the subject.

[0191] In some implementations, the subject is a human. In other implementations, the subject is a non-human mammal, bird, fish, reptile, or amphibian.

[0192] In some implementations, the target cell is an excitable cell, such as a neuron, glial cell, muscle cell, or endocrine cell.

[0193] In some implementations, the neurons are derived from the central nervous system (CNS) or the peripheral nervous system (PNS). For example, the neurons are brain neurons, and further, the neurons are excitatory neurons.

[0194] In some implementations, the neurons are neurons of the epileptic foci or epileptogenic focus.

[0195] In some embodiments, the method of modulating the GPCR activity of a subject includes administering an effective amount of the aforementioned polynucleotide or the aforementioned expression vector to the subject. In some embodiments, the method of modulating the GPCR activity of a subject includes administering an effective amount of the aforementioned polynucleotide or the aforementioned expression vector to target cells.

[0196] The methods of administration include subcutaneous administration, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, oral administration, infusion, intracranial administration, intrathecal administration, intranasal administration, intraganglionic administration, intraspinal administration, cerebellomedullary cistern administration, and intraneural administration.

[0197] In some embodiments, application may involve a liquid formulation that can be injected into a carrier.

[0198] In some embodiments, administration may involve oral delivery of a solid formulation of an exogenous ligand. In some cases, the oral formulation may be administered with food. In some embodiments, the carrier is administered to a subject via parenteral, intravenous, intramuscular, intraperitoneal, intrathecal, intraneural, intraganglionic, intraspinal, or intracardiac administration to introduce the carrier into one or more neuronal cells (e.g., target cells). In various embodiments, the carrier is an AAV.

[0199] In some embodiments, the carrier can be administered to the subject via intracranial delivery (i.e., direct entry into the brain). In a non-limiting example of intracranial delivery, the carrier of the present invention can be delivered to an epileptic focus or epileptogenic focus in the brain for treatment. In another specific case, the carrier can be administered to the subject via intraneural injection (i.e., direct injection into a nerve). The nerve can be selected based on the indications for treatment, such as injecting into excitatory neurons of the epileptic focus or epileptogenic focus to treat epilepsy.

[0200] Vector dosage can be expressed as the number of vector genome units delivered to a subject. As used herein, "vector genome unit" refers to the number of individual vector genomes administered in a dose. The size of a single vector genome typically depends on the type of viral vector used. The vector genomes of this invention can be approximately 1.0 kbps, 1.5 kbps, 2.0 kbps, 2.5 kbps, 3.0 kbps, 3.5 kbps, 4.0 kbps, 4.5 kbps, 5.0 kbps, 5.5 kbps, 6.0 kbps, 6.5 kbps, 7.0 kbps, 7.5 kbps, 8.0 kbps, 8.5 kbps, 9.0 kbps, 9.5 kbps, 10.0 kbps, or more than 10.0 kbps. Therefore, a single vector genome can contain up to or more than 10,000 base pairs of nucleotides. In some cases, the carrier dose can be approximately 1×10^6, 2×10^6, 3×10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^7, 3×10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^8, 3×10^8, 4×10^8, 5×10^8, 6× 10^8, 7×10^8, 8×10^8, 9×10^8, 1×10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^9, 1×10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^10, 1×10^11, 2×10^11, 3 ×10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8×10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^12, 8×10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×1 0^13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6×10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^15, 6×10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^16,3×10^16, 4×10^16, 5×10^16, 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10^17, 5×10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18, 4×10^18, 5×10^18, 6×10^18, 7 Vector genome units of ×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10^20, 3×10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20, 9×10^20, or higher.

[0201] In specific embodiments, the vectors considered herein have a density of at least approximately 1×10^9 genome particles / mL, at least approximately 1×10^10 genome particles / mL, at least approximately 5×10^10 genome particles / mL, at least approximately 1×10^11 genome particles / mL, at least approximately 5×10^11 genome particles / mL, at least approximately 1×10^12 genome particles / mL, at least approximately 5×10^12 genome particles / mL, at least approximately 6×10^12 genome particles / mL, and at least approximately 7×10^12 genome particles / mL. A titer of at least approximately 8 × 10^12 genome particles / mL, at least approximately 9 × 10^12 genome particles / mL, at least approximately 10 × 10^12 genome particles / mL, at least approximately 15 × 10^12 genome particles / mL, at least approximately 20 × 10^12 genome particles / mL, at least approximately 25 × 10^12 genome particles / mL, at least approximately 50 × 10^12 genome particles / mL, or at least approximately 100 × 10^12 genome particles / mL is administered to the subject. When referring to viral titers, the terms “genome particles (gp)”, “genome equivalent”, or “genome copy (gc)” refer to the number of viral particles containing the recombinant AAV DNA genome, regardless of infectivity or function. The number of genomic particles in a particular vector preparation can be measured, for example, in the embodiments described herein, or, for example, in Clark et al. (1999) Human Gene Therapy, 10:1031-1039; and Veldwijk et al. (2002) Molecular Therapy, 6:272-278.

[0202] The carrier of the present invention can be administered in a volume of fluid. In some cases, the carrier can be administered in volumes of about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, 10.0 mL, 11.0 mL, 12.0 mL, 13.0 mL, 14.0 mL, 15.0 mL, 16.0 mL, 17.0 mL, 18.0 mL, 19.0 mL, 20.0 mL, or greater than 20.0 mL. In some cases, the carrier dose can be expressed as the concentration or titer of the carrier administered to the subject. In this case, the carrier dose can be expressed as the number of carrier genomic units per volume (i.e., genomic units / volume).

[0203] In specific embodiments, the carriers considered herein are administered to subjects at titers of at least about 5 × 10^9 infection units / mL, at least about 6 × 10^9 infection units / mL, at least about 7 × 10^9 infection units / mL, at least about 8 × 10^9 infection units / mL, at least about 9 × 10^9 infection units / mL, at least about 10 × 10^9 infection units / mL, at least about 15 × 10^9 infection units / mL, at least about 20 × 10^9 infection units / mL, at least about 25 × 10^9 infection units / mL, at least about 50 × 10^9 infection units / mL, or at least about 100 × 10^9 infection units / mL. The terms “infectious unit (iU),” “infectious particle” or “replication unit” used in relation to viral titers refer to the number of infectious and reproducible recombinant AAV vector particles measured by the center of infection, also known as the center of replication assay, as described, for example, in the Journal of Virology (J. Virol.), 62:1963-1973, by McLaughlin et al. (1988).

[0204] In specific embodiments, the carriers considered herein are administered to subjects at titers of at least about 5 × 10^10 transduction units / mL, at least about 6 × 10^10 transduction units / mL, at least about 7 × 10^10 transduction units / mL, at least about 8 × 10^10 transduction units / mL, at least about 9 × 10^10 transduction units / mL, at least about 10 × 10^10 transduction units / mL, at least about 15 × 10^10 transduction units / mL, at least about 20 × 10^10 transduction units / mL, at least about 25 × 10^10 transduction units / mL, at least about 50 × 10^10 transduction units / mL, or at least about 100 × 10^10 transduction units / mL. The term “transduction unit (tu)” as used in relation to viral titers refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgenic product, as measured in the examples herein or, for example, in the functional assays described in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) Journal of Virology, 70:520-532 (LFU analysis).

[0205] The carrier dosage is generally determined by the route of administration. In one specific example, intraganglionic injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intrathecal injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intracranial injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In another specific example, intraneural injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.01 mL to approximately 10.0 mL. In yet another example, intraspinal injection may include approximately 1 × 10^9 to approximately 1 × 10^13 carrier genomes in a volume of approximately 0.1 mL to approximately 0.01 mL to approximately 10.0 mL. In yet another specific case, cerebellomedullary cistern infusion may include approximately 0.01 mL to approximately 10.0 mL of vector genomes of approximately 1 × 10^9 to approximately 1 × 10^13. In yet another specific case, subcutaneous injection may include approximately 0.01 mL to approximately 10.0 mL of vector genomes of approximately 1 × 10^9 to approximately 1 × 10^13.

[0206] In one embodiment, the ligand is first administered to the subject after the vector has been administered to the subject.

[0207] In one embodiment, the ligand is administered to the subject at the same time as the vector is administered to the subject.

[0208] In one embodiment, the ligand is administered to the subject after the vector is administered to the subject.

[0209] In one embodiment, the ligand is administered to the subject before the vector is administered to the subject.

[0210] In one embodiment, the ligand is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 hours, days, weeks, months, or years after vector administration. In some cases, a therapeutically effective amount of the ligand may be administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, or more than 30 days after vector delivery. In one specific instance, a therapeutically effective amount of the ligand is administered to the subject at least one week after vector delivery.

[0211] The therapeutically effective amount or dose of the ligand of the present invention can be expressed as mg or μg of the ligand per kilogram of subject body weight. In some cases, the therapeutically effective amount of the ligand may be about 0.001 μg / kg, about 0.005 μg / kg, about 0.01 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 20 μg / kg, about 30 μg / kg, about 40 μg / kg, about 5 μg / kg, or about 5 μg / kg. 0 μg / kg, approximately 60 μg / kg, approximately 70 μg / kg, approximately 80 μg / kg, approximately 90 μg / kg, approximately 100 μg / kg, approximately 120 μg / kg, approximately 140 μg / kg, approximately 160 μg / kg, approximately 180 μg / kg, approximately 200 μg / kg, approximately 220 μg / kg, approximately 240 μg / kg, approximately 260 μg / kg, approximately 280 μg / kg, approximately 300 μg / kg, approximately 320 μg / kg, approximately 340 μg / kg, approximately 360 μg / kg, approximately 380 μg / kg, approximately 4 00μg / kg, approximately 420μg / kg, approximately 440μg / kg, approximately 460μg / kg, approximately 480μg / kg, approximately 500μg / kg, approximately 520μg / kg, approximately 540μg / kg, approximately 560μg / kg, approximately 580μg / kg, approximately 600μg / kg, approximately 620μg / kg, approximately 640μg / kg, approximately 660μg / kg, approximately 680μg / kg, approximately 700μg / kg, approximately 720μg / kg, approximately 740μg / kg, approximately 760μg / kg, approximately 780μg / kg, approximately 800 μg / kg, approximately 820 μg / kg, approximately 840 μg / kg, approximately 860 μg / kg, approximately 880 μg / kg, approximately 900 μg / kg, approximately 920 μg / kg, approximately 940 μg / kg, approximately 960 μg / kg, approximately 980 μg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, or greater than 10 mg / kg.

[0212] In some embodiments, the dose of the ligand administered to the subject is at least about 0.001 μg / kg, at least about 0.005 μg / kg, at least about 0.01 μg / kg, at least about 0.05 μg / kg, at least about 0.1 μg / kg, at least about 0.5 μg / kg, 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, or at least about 20 or higher mg / kg.

[0213] In some embodiments, the dose of the ligand administered to the subject is at least about 0.001 μg / kg to at least about 20 mg / kg, at least about 0.01 μg / kg to at least about 20 mg / kg, at least about 0.1 μg / kg to at least about 20 mg / kg, at least about 1 μg / kg to at least about 20 mg / kg, at least about 0.01 mg / kg to at least about 20 mg / kg, at least about 0.1 mg / kg to at least about 20 mg / kg, or at least about 1 mg / kg to at least about 20 mg / kg, or any range thereof.

[0214] In some respects, the therapeutically effective amount of ligand can be expressed as a molar concentration (i.e., M or mol / L). In some cases, the therapeutically effective amount of ligand can be approximately 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM. nM, 800nM, 900nM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM , 70mM, 80mM, 90mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or greater.

[0215] A therapeutically effective dose of the ligand may be administered once or more daily. In some cases, a therapeutically effective dose of the ligand is administered as needed (e.g., when epilepsy needs to be treated). The ligand may be administered continuously (e.g., daily without interruption for the duration of the treatment regimen). In some cases, the treatment regimen may be less than one week, one week, two weeks, three weeks, one month, or more than one month. In some cases, a therapeutically effective dose of the ligand may be administered for one day, for at least two consecutive days, for at least three consecutive days, for at least four consecutive days, for at least five consecutive days, for at least six consecutive days, for at least seven consecutive days, for at least eight consecutive days, for at least nine consecutive days, for at least ten consecutive days, or for more than ten consecutive days. In certain cases, a therapeutically effective dose of the ligand may be administered for three consecutive days. In some cases, therapeutically effective doses of ligand may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, twenty times, thirty times, thirty times, forty times, or more than forty times per week. In other cases, therapeutically effective doses of ligand may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times per day. In some cases, the therapeutically effective dose of ligand is administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine hours, at least every ten hours, at least every eleven hours, at least every twelve hours, at least every thirteen hours, at least every fourteen hours, at least every fifteen hours, at least every sixteen hours, at least every seventeen hours, at least every eighteen hours, at least every nineteen hours, at least every twenty hours, at least every twenty-one hours, at least every twenty-two hours, at least every twenty-three hours, or at least daily. The dose of ligand may be administered to the subject continuously, or once, twice, three times, four times, or five times daily; once, twice, three times, four times, five times, or six times weekly; once, twice, three times, four times, five times, or six times monthly; once, twice, three times, four times, five times, or six times monthly; or at intervals of even longer. Treatment duration can last from one day, 1, 2 or 3 weeks, 1, 2, 3, 4, 5, 7, 8, 9, 10 or 11 months, 1, 2, 3, 4, 5 or more years or longer.

[0216] In some embodiments, the exogenous ligand is administered to the subject prior to a seizure.

[0217] In some embodiments, an exogenous ligand is administered to the subject during a seizure.

[0218] In some embodiments, an exogenous ligand is administered to the subject after a seizure.

[0219] In some embodiments, the drug is administered 30 minutes before or within 24 hours after a seizure in the subject.

[0220] In some embodiments, the exogenous ligand is automatically administered via (i) a device connected to an automated seizure detection mechanism, or (ii) in response to a seizure predicted by electroencephalogram (EEG) analysis.

[0221] In some cases, the methods and compositions of the present invention are used to treat epilepsy. The compositions described herein can be used to prevent or control epileptic seizures. Epileptic seizures can be classified as tonic-clonic, tonic-clonic, clonic, myoclonic, absent, or asthenic seizures.

[0222] In some embodiments, a method includes introducing a vector containing a modified GPCR into neuronal cells and controlling cell activity by providing a ligand that activates the modified GPCR, thereby alleviating epilepsy in a subject. The method provides significant therapeutic effects on epilepsy without off-target effects, such as systemic central nervous system depression. In some cases, the compositions and methods described herein can prevent or reduce the number of epileptic seizures experienced by a subject by about 5%, about 10%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100%.

[0223] Production methods

[0224] On the other hand, this application provides a method for producing a modified GPCR, comprising modifying a wild-type GPCR at at least one position: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered according to the correspondence with the amino acid sequence of SEQ ID NO:1. The modification includes the following substitutions: a. 113C or 113N; and / or, b. 203G. Further, the modification includes the following substitutions: a. Y113C or Y113N; and / or, b. A203G. In some embodiments, the method comprises modifying the wild-type GPCR at at least one position: a serine / thionine-specific protein kinase A (PKA) phosphorylation site, for example, position 399. Further, the modification includes the following substitution: T399. Further, the modification includes the following substitution: T399A. In some embodiments, the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR.

[0225] Example

[0226] Example 1: Functional Verification of the Mutant hM4Di Receptor

[0227] Principle: Gi / o-coupled GPCRs inhibit the activity of adenylate cyclase by interacting with Gi or Go proteins. Adenylate cyclase is a key enzyme that catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP). cAMP is an important intracellular signaling molecule involved in regulating various physiological processes.

[0228] When Gi / o coupled receptors are activated by their ligands, such as hM4Di activated by CNO, the α subunit of the Gi / o protein inhibits the activity of adenylate cyclase, thereby reducing cAMP production. Therefore, a decrease in intracellular cAMP levels can be considered an indicator of Gi / o coupled receptor activation.

[0229] The experimental steps are as follows:

[0230] 1) Transfection:

[0231] pAAV-EF1α-hM4Di(WT) (as shown in SEQ ID NO:8, capable of expressing hM4Di) and pAAV-EF1α-hM4Di T399A(399) (as shown in SEQ ID NO:9, capable of expressing hM4Di containing the T399A mutation) were transiently transfected into HEK-293T cells. Six hours after transfection, the culture medium was replaced with serum-free medium to reduce the interference of serum factors on the experimental results.

[0232] Experimental grouping and treatment:

[0233] 2) After 24 hours, the cells were divided into three groups for treatment:

[0234] Control group: 0.05 mM IBMX (3-isobutyl-1-methylxanthine) was added to inhibit phosphodiesterase activity and prevent cAMP degradation.

[0235] Isoprenaline group (iso): 100 nM isoprenaline and 0.05 mM IBMX were added, and the treatment lasted for 5 minutes. Isoprenaline is a β-adrenergic receptor agonist that can increase cAMP levels via Gs protein-coupled receptors.

[0236] The isoproterenol + CNO group (iso+CNO): Treatment was first with 100 nM isoproterenol and 0.05 mM IBMX for 5 minutes, followed by treatment with 10 μM CNO (clozapine-N-oxide) for 15 minutes. CNO was used to activate the hM4Di receptor.

[0237] 3) Collect cell samples from each group using a cAMP ELISA kit and determine the cAMP content.

[0238] The experimental results are shown in Figure 1. Isoproterenol effectively increased cAMP levels. The cAMP values ​​of the WT group and the 399 group treated with isoproterenol + CNO (iso + CNO) were lower than those of the iso group, indicating that cAMP levels decreased significantly after CNO activated the receptor.

[0239] In this experiment, isoproterenol increased cAMP levels via Gs protein-coupled receptors, while CNO activation of the hM4Di receptor inhibited adenylate cyclase via the Gi / o protein pathway, leading to a decrease in cAMP levels. The T399A mutant hM4Di receptor, upon CNO activation, caused a decrease in cAMP, with a response intensity similar to that of the WT hM4Di receptor, further validating the functional characteristics of the mutant hM4Di receptor as a Gi / o-coupled GPCR.

[0240] Example 2 - The T399A mutation enhances the receptor's sensitivity to CNO.

[0241] Activation of GPCRs initiates a series of intracellular signaling cascades, including the activation of the MAPK / ERK pathway, which plays a crucial role in cell proliferation, differentiation, and survival. Elevated ERK phosphorylation levels reflect the receptor's activation state and desensitization time. Measuring p-ERK levels allows for the assessment of receptor sensitivity and response intensity to ligands such as CNO.

[0242] The experimental steps are as follows:

[0243] 1) pAAV-EF1α-hM4Di(WT) and pAAV-EF1α-hM4Di T399A(399) were transiently transfected into HEK-293T cells.

[0244] 2) 24 hours after transfection, treat with CNO at 0, 1E-9 to 1E-5M for 5 minutes respectively.

[0245] 3) Collect samples and perform Western blotting analysis to detect p-ERK signals.

[0246] The experimental results are shown in Figure 2. In the sensitivity test, CNO concentrations of 1, 5, 10, 20, 40, and 80 μM acted on wild-type (WT) and 399 receptors in a concentration-dependent manner, with 399 showing a significantly higher response intensity than the wild-type. Then, the CNO concentration was reduced, and the results are shown in Figure 3. It shows that 0.1 μM CNO could significantly activate hM4Di T399A, but the activation effect on the wild-type was weak. CNO in the range of 1E-9 to 1E-5 M showed significantly higher activation intensity of hM4Di T399A than the wild-type, indicating that hM4Di T399A has higher sensitivity to CNO and a stronger response intensity than the wild-type.

[0247] Example 3 - The T399A mutation prolongs receptor desensitization time and reduces receptor desensitization.

[0248] Activation of GPCRs initiates a series of intracellular signaling cascades, including the activation of the MAPK / ERK pathway, which plays a crucial role in cell proliferation, differentiation, and survival. Elevated ERK phosphorylation levels reflect the receptor's activation state and desensitization time. Measuring p-ERK levels allows for the assessment of receptor sensitivity and response intensity to ligands such as CNO.

[0249] The experimental steps are as follows:

[0250] 1) pAAV-EF1α-hM4Di(WT) and pAAV-EF1α-hM4Di T399A(399) were transiently transfected into HEK-293T cells.

[0251] 2) 24 hours after transfection, treat with 10 μM CNO for 0 minutes, 5 minutes, 30 minutes and 60 minutes respectively.

[0252] 3) Collect samples and perform Western blotting analysis to detect p-ERK signals.

[0253] 4) Data analysis: The p-ERK / tubulin ratio was compared using the image analysis software Quantify to compare the changes in p-ERK levels in different treatment groups.

[0254] As shown in Figure 4, the WB analysis showed that the p-ERK signals of 399 and WT were similar at 5 min, but at 30 and 60 min, the p-ERK phosphorylation signal of the 399 group was significantly stronger than that of WT, that is, the effective time of 399 was longer and the desensitization time was prolonged.

[0255] Example 4 - Electrophysiological Study of Brain Slice Patch-Clamp Technique

[0256] By expressing mutant and wild-type hM4Di receptors in the mouse cortex and activating the receptors using CNO, the sensitivity of mutants to CNO and their inhibitory effect on neuronal activity could be evaluated. Specifically, once activated, the hM4Di receptor functions through a Gi / o protein-mediated signaling pathway. In addition to reducing intracellular cAMP levels, it also activates potassium channels (such as GIRK channels). The opening of potassium channels leads to hyperpolarization of the cell membrane, placing neurons in a less excitable state and thus reducing neuronal excitability. Activation of the hM4Di receptor leads to an inhibitory response in neurons through a synergistic effect of multiple mechanisms.

[0257] Experimental steps:

[0258] 1) Selected C57BL / 6J mice of uniform age, sex, and weight, and divided them into two groups. Two different AAV viruses, AAV9:CamKII-hM4Di-T2A-mCherry (as shown in SEQ ID NO:6) and AAV9:CamKII-hM4Di399-T2A-mCherry (as shown in SEQ ID NO:7), were injected into the mouse cortex. A 10-day waiting period was allowed to ensure sufficient receptor expression in neurons.

[0259] 2) Brain slice preparation and patch-clamp recording:

[0260] The brain was sliced ​​into 200 μm thick coronal sections using a vibratory slicer. Neurons expressing mCherry were located under a microscope to ensure that the recorded neurons were transfected with the hM4Di receptor. Recording electrodes were brought into contact with the neurons and a high-resistance seal (>1 GΩ) was formed, followed by gentle suction to create a whole-cell configuration. Electrophysiological signals were low-pass filtered at 3 kHz and digitized at 10 kHz (using a DigiData 1440, Molecular Devices).

[0261] 3) CNO processing and data logging:

[0262] Throughout the recording process, the GABAA receptor blocker picotoxin (50 μM) was consistently applied to inhibit inhibitory synaptic transmission. A current-clamp mode was used, maintaining neurons at -50 mV. After baseline recording, 10 μM CNO was circulated. Action potential firing in neurons under CNO treatment was recorded, and the intensity and duration of the inhibitory effect were monitored.

[0263] 4) Data Analysis:

[0264] Physiological data were analyzed using Clampfit 10 software (Molecular Devices). The firing of neuronal action potentials was recorded under a 10 μM CNO circulation.

[0265] The experimental results are shown in Figure 5. The inhibitory strength of the mutant hM4Di T399A was significantly higher than that of hM4Di(WT) when activated by 10 μM CNO. The inhibition time of the mutant hM4Di T399A was significantly longer than that of hM4Di(WT).

[0266] These results indicate that, upon CNO activation, the mutant hM4Di 399 receptor, under the same conditions, more effectively inhibits neuronal activity than the wild-type receptor, exhibiting stronger inhibitory intensity and longer duration. This further validates the enhanced properties of the mutant hM4Di receptor in terms of CNO sensitivity and signal transduction persistence.

[0267] Example 5 - Verification of Off-Target Effects

[0268] Acetylcholine (ACh) is an endogenous neurotransmitter that can activate various cholinergic receptors, including the M4 muscarinic acetylcholine receptor (CHRM4). To verify whether the mutant hM4Di 399 receptor and its wild-type (WT) receptor are activated by ACh and to assess its off-target effects, changes in p-ERK (phosphorylated extracellular signal-regulated kinase) levels after ACh treatment were analyzed. An increase in p-ERK levels indicates receptor activation. Therefore, if p-ERK levels do not increase after ACh treatment, it indicates that the mutant hM4Di 399 and WT receptors are not activated by ACh.

[0269] Experimental steps:

[0270] (1) Cell culture and transfection:

[0271] HEK-293T cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2.

[0272] Plasmids expressing CHRM4, hM4Di WT, and hM4Di 399 were transfected into HEK-293T cells, respectively.

[0273] (2) ACh treatment:

[0274] 24 hours after transfection, cells in each group were treated with 10 μM ACh for 5 and 30 minutes respectively.

[0275] An untreated group was set up as a control group and marked as 0 minutes.

[0276] (3) Western Blot analysis:

[0277] Cells were collected and lysed to extract total protein. SDS-PAGE electrophoresis was performed, followed by membrane transfer. Western blotting was performed using an anti-p-ERK antibody to detect p-ERK levels.

[0278] (4) Data Analysis:

[0279] The p-ERK level changes in different treatment groups were compared using the p-ERK / tubulin ratio, which was analyzed using the image analysis software Quantify.

[0280] The experimental results, as shown in Figure 6, indicate that the p-ERK level in the CHRM4 transfection group significantly increased after ACh treatment, suggesting that the CHRM4 receptor was activated by ACh. The p-ERK levels in the hM4Di WT and hM4Di 399 transfection groups did not change significantly after ACh treatment, indicating that neither receptor was activated by ACh.

[0281] This verifies that the mutant hM4Di 399 receptor is not activated by ACh, exhibits good specificity, and does not produce off-target effects. This finding further supports the safety and efficacy of the mutant hM4Di 399 receptor in clinical applications.

[0282] Example 6 - Epilepsy Efficacy Verification

[0283] Epilepsy is characterized by recurrent seizures due to excessive neuronal activity. Chemogenetics offers a novel approach to inhibiting this excessive activity in specific neuronal circuits. By introducing inhibitory dreadds into neurons within the seizure-causing region, selectively reducing neuronal firing rates upon ligand administration, it is possible to potentially prevent seizures.

[0284] 4-Aminopyridine (4AP) significantly prolongs the duration of action potentials primarily by blocking voltage-gated potassium channels in neurons. This leads to prolonged presynaptic neuronal depolarization, thereby increasing calcium ion influx and the release of excitatory neurotransmitters such as glutamate. Excess glutamate causes strong and sustained depolarization of postsynaptic neurons. Simultaneously, sustained excitation eventually overwhelms or depletes the brain's inhibitory circuits (particularly the GABAergic system). This disruption of the excitation-inhibition (E / I) balance results in synchronized bursts of abnormal discharges from numerous neurons, triggering epileptic seizures. Therefore, 4AP is widely used in laboratory studies of the pathological mechanisms of epilepsy and in drug testing. By expressing hM4Di at epileptic foci and activating this receptor using CNO, neuronal excitability can be inhibited, thereby reducing epileptic seizures.

[0285] Experimental steps

[0286] 1) AAV injection and expression:

[0287] C57BL / 6J mice of uniform age, sex, and weight were selected and divided into three groups. Three different AAV viruses, namely AAV9:CaMKIIα-mCherry (control), AAV9:CaMKIIα-hM4Di-T2A-mCherry (WT), or AAV9:CaMKIIα-hM4Di 399-T2A-mCherry (399), were injected into the motor cortex of the mice.

[0288] Experiments began 14 days after AAV injection to ensure full expression of the receptor in neurons.

[0289] 2) Acute epilepsy modeling and drug intervention:

[0290] Animals injected with the three viruses were intraperitoneally injected with 1 mg / kg CNO to activate neurons expressing the hM4Di receptor.

[0291] Thirty minutes later, remove the cap of the in situ drug delivery cannula from the mouse motor cortex, insert the delivery tube, and inject 500 nl of 2 mM 4AP at a rate of 0.5 μL / min. Leave the needle in place for 3 minutes after injection to ensure adequate drug diffusion. Note that the surgical site of the cannula is consistent with the viral injection site; precise positioning ensures that only the desired neuronal population is affected, minimizing off-target effects.

[0292] 3) Electroencephalogram (EEG) recording:

[0293] Electroencephalogram (EEG) electrodes were inserted to record brain activity for 4 hours, and the overall seizure situation was analyzed.

[0294] Experimental results:

[0295] The number of seizures, highest seizure severity, and latency to generalization in mice under 4AP-induced epilepsy were recorded and compared among different groups (control, WT, 399). The CaMKIIα-mCherry group showed successful modeling and exhibited typical seizure characteristics.

[0296] The number and highest seizure severity of mice in group 399 were significantly lower than those in group WT during the 4-hour recording period, and the seizure delay was significantly longer in group 399, as shown in Figure 8. This indicates that the mutant hM4Di 399 receptor is more effective in suppressing epilepsy.

[0297] Example 7 - Epilepsy Efficacy Verification

[0298] By expressing hM4Di in epileptic lesions and activating the receptor using olanzapine or clozapine, neuronal excitability can be inhibited, thereby reducing epileptic seizures.

[0299] Experimental steps

[0300] 1) AAV injection and expression:

[0301] C57BL / 6J mice of uniform age, sex, and weight were selected and injected with AAV9:CaMKIIα-hM4Di 399-T2A-mCherry(399) into the motor cortex of the mice.

[0302] Experiments began 14 days after AAV injection to ensure full expression of the receptor in neurons.

[0303] 2) Acute epilepsy modeling and drug intervention:

[0304] Animals injected with the virus were randomly divided into three groups, and injected intraperitoneally with saline, 1 mg / kg olanzapine, or 0.1 mg / kg clozapine, respectively, to activate neurons expressing the hM4Di receptor.

[0305] Thirty minutes later, remove the cap of the in situ drug delivery cannula from the mouse motor cortex, insert the delivery tube, and inject 500 nl of 2 mM 4AP at a rate of 0.5 μL / min. Leave the needle in place for 3 minutes after injection to ensure adequate drug diffusion. Note that the surgical site of the cannula is consistent with the viral injection site; precise positioning ensures that only the desired neuronal population is affected, minimizing off-target effects.

[0306] 3) Electroencephalogram (EEG) recording:

[0307] Electroencephalogram (EEG) electrodes were inserted to record brain activity for 4 hours, and the overall seizure situation was analyzed.

[0308] Experimental results:

[0309] The number of seizures, highest seizure severity, and latency to generalization in mice under 4AP-induced epilepsy were recorded and compared among different groups (saline, clozapine, and olanzapine). The saline group showed successful modeling and exhibited typical seizure characteristics.

[0310] Mice in the clozapine and olanzapine groups had significantly fewer and higher seizure counts and higher seizure severity during the 4-hour recording period compared to the saline group, and significantly longer seizure delay time, as shown in Figure 9. This indicates that the mutant hM4Di 399 receptor is effective in inhibiting epilepsy, and that common hM4Di ligands such as clozapine and olanzapine can activate the mutant hM4Di 399 receptor.

[0311] Example 8 - Study on the effect of activating hM4Di 399 on movement

[0312] The effects of hM4Di 399 activation on motor behavior in mice were verified using the open field test. This is a commonly used experimental method for assessing exploratory behavior, anxiety levels, and general motor abilities in mice.

[0313] Experimental steps

[0314] 1) AAV injection and expression

[0315] C57BL / 6J mice of uniform age, sex, and weight were selected and divided into two groups. Two different AAV viruses, AAV9:CaMKIIα-mCherry (control) and AAV9:CaMKIIα-hM4Di 399-T2A-mCherry (399), were injected into the motor cortex of the mice.

[0316] The experiment began 8 days after AAV injection to ensure full expression of the receptor in neurons.

[0317] 2) Open Field Device Setup

[0318] Prepare a standard open enclosure measuring 50cm × 50cm × 40cm (length × width × height). The interior floor is evenly divided into several grids to facilitate subsequent data analysis. The four walls of the enclosure are opaque, and a camera is mounted on the top to record mouse behavior.

[0319] 3) CNO treatment and testing

[0320] Thirty minutes before the start of the experiment, 1 mg / kg CNO was injected intraperitoneally to activate neurons expressing the hM4Di receptor.

[0321] Place each mouse individually in the center of the open enclosure and immediately start the recording equipment. Allow the mice to explore freely for 10 minutes, recording the entire process. Thoroughly clean the open enclosure after each test to prevent residual odors from affecting the behavior of subsequent mice.

[0322] 3) Behavioral indicator recording:

[0323] Record the following metrics using video analytics software:

[0324] ①Total movement distance: The total distance the mouse moves in 10 minutes.

[0325] ② Central area stay time: The total time that mice spend in the central area of ​​the open field.

[0326] ③ Time spent in the edge area: The total time that mice spend in the edge area of ​​the open field.

[0327] 4) Experimental results:

[0328] As shown in Figure 10, the mice in group 399 showed no significant differences from WT mice in terms of total movement distance, time spent in the central region, time spent in the peripheral region, and average speed, demonstrating that CNO activation of hM4Di T399A has no negative impact on the normal movement behavior of mice.

Claims

A modified human G protein-coupled receptor (GPCR), wherein the modified GPCR has the following properties (i) reduced reactivity to an endogenous ligand compared to a wild-type GPCR; and, (ii) enhanced reactivity to at least one exogenous ligand compared to a wild-type GPCR. The receptor of claim 1, wherein the GPCR is Gi / o-coupled. The receptor of any one of claims 1-2, wherein the wild-type GPCR is muscarinic acetylcholine receptor M4. The receptor according to any one of claims 1 to 3, wherein The modified G protein-coupled receptor (GPCR) comprises an A5.46G and / or Y3.33C mutation compared to a wild-type GPCR. The receptor according to any one of claims 1 to 4, wherein The wild-type GPCR comprises a sequence having at least 75% homology to SEQ ID NO:

1. The receptor according to any one of claims 1 to 5, wherein The modified GPCR comprises at least one modification compared to a wild-type GPCR at the following positions: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The receptor according to claim 6, wherein The modified GPCR comprises the following substitutions compared to a wild-type GPCR: a. 113C or 113N; and / or, b. 203G. The receptor according to claim 7, wherein The modified GPCR comprises the following substitutions compared to a wild-type GPCR: a. Y113C or Y113N; and / or, b. A203G. The receptor according to any one of claims 1 to 8, wherein The modified G protein-coupled receptor (GPCR) comprises at least one modification at a protein kinase A (PKA) phosphorylation site compared to a wild-type GPCR. The receptor according to any one of claims 1 to 9, wherein The modified GPCR comprises at least one modification compared to a wild-type GPCR at the following position: 399, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The receptor according to claim 10, wherein The modified GPCR comprises the following substitution compared to a wild-type GPCR: T399. The receptor according to claim 11, wherein The modified GPCR comprises the following substitution compared to a wild-type GPCR: T399A. The receptor according to any one of claims 1 to 12, wherein The modified GPCR comprises a sequence having at least 75% homology to SEQ ID NOs: 2-3. The receptor according to any one of claims 1 to 13, wherein The exogenous ligand is a ligand capable of binding to a modified GPCR having an amino acid sequence as set forth in SEQ ID NO: 2 or 3. The receptor according to claim 14, wherein The exogenous ligand is selected from clozapine, clozapine oxide (CNO), olanzapine, dechloroclozapine (DCZ), or a derivative thereof. A polynucleotide sequence comprising a polynucleotide encoding a nucleic acid sequence of the modified GPCR of any one of claims 1-15. The polynucleotide sequence of claim 15, wherein the polynucleotide sequence comprises a sequence having at least 75% homology to SEQ ID NOs: 4-5. An expression vector comprising the polynucleotide of any one of claims 16-17. The expression vector of claim 18, wherein the vector is a plasmid, a transposon, a cosmid, a bacterial artificial chromosome, or a viral vector. The vector of claim 19, wherein the vector is an adeno-associated virus (AAV), a herpes virus vector, a retrovirus vector, a vaccinia virus vector, an adenovirus vector, or a lentivirus vector. The vector of any one of claims 18-20, wherein the AAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, Anc80, or a variant thereof. The vector of any one of claims 18-21, wherein the nucleic acid encoding the modified GPCR is operably linked to a promoter. The vector of claim 22, wherein the promoter is one of a constitutive promoter, an inducible promoter, or a tissue-specific promoter. The vector of any one of claims 22-23, wherein the constitutive promoter comprises one or more of a cytomegalovirus CMV immediate early promoter, a viral simian virus 40 SV40, a Moloney murine leukemia virus MoMLV LTR promoter, a Rous sarcoma virus RSV LTR, a herpes simplex virus HSV (thymidine kinase) promoter, H5, P7.5, and P11 promoters from the vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 EGR1, ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase GAPDH, eukaryotic translation initiation factor 4A1 EIF4A1, heat shock 70kDa protein 5 HSPA5, heat shock protein 90kDa beta member 1 HSP90B1, heat shock protein 70kDa HSP70, beta-kinesin beta-KIN, human ROSA 26 promoter, ubiquitin C promoter UBC, phosphoglycerate kinase-1 PGK promoter, cytomegalovirus enhancer / chicken beta-actin CAG promoter, or a beta-actin promoter. The vector of any one of claims 22-24, wherein the promoter is an EF1a promoter. The vector of claim 23, wherein the inducible promoter comprises one of a transcription factor responsive promoter, a tetracycline responsive promoter, an ecdysone responsive promoter, a cumate responsive promoter, a glucocorticoid responsive promoter and an estrogen responsive promoter, a PPAR-gamma promoter, or a RU-486 responsive promoter. The vector of claim 23, wherein the tissue-specific promoter comprises a neuron-specific promoter, a glial cell-specific promoter, a heart-specific promoter, a muscle-specific promoter, a lung-specific promoter, a liver-specific promoter, a kidney-specific promoter, a pancreas-specific promoter, a fat-specific promoter, an endothelial-specific promoter, a retina-specific promoter, a prostate-specific promoter, a skin-specific promoter, a macrophage-specific promoter. The vector of claim 27, wherein the promoter is a neuron-specific promoter comprising: A human synapsin-1 (SYN-1) promoter, a calcium-calmodulin-dependent protein kinase II alpha (CaMKIIa) promoter, a tubulin alpha 1 promoter, a neuron-specific enolase (NSE) promoter, a platelet-derived growth factor beta chain promoter (PDGFB), a TRPV1 promoter, a Nav1.7 promoter, a Nav1.8 promoter, a Nav1.9 promoter, an Advillin promoter, a Drosophila single-minded 1 (SIM1) promoter, an oxytocin (OXT) promoter, an agouti-related protein (AgRP) promoter, a protein kinase C-delta (PKC-delta) promoter, a ghrelin promoter, a glutamic acid decarboxylase (GAD1 / 2) promoter, a choline acetyltransferase (ChAT) promoter, a distal-less homeobox (Dlx) promoter. The vector of claim 27, wherein the promoter is CaMKIIa. The vector of claim 27, wherein the glial cell-specific promoter includes, but is not limited to, a glial fibrillary acidic protein (GFAP) promoter, a Gfabc1D promoter, a myelin basic protein (MBP) promoter, an oligodendrocyte myelin glycoprotein (MOG) promoter, a CD11b promoter, an Iba1 promoter, a Foxj1 promoter. A cell comprising the expression vector of claims 18-30. A pharmaceutical composition comprising the modified GPCR of any one of claims 1-15, or the polynucleotide sequence of any one of claims 16-17, or the expression vector of any one of claims 18-30, or the cell of claim 31, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 32, further comprising the exogenous ligand of claim 15. A method of increasing the responsiveness of a wild-type GPCR to an exogenous ligand, comprising at least one modification to the wild-type GPCR at positions 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The method of claim 34, wherein, The modification comprises the following substitution: a. 113C or 113N; and / or, b. 203G. The method of any one of claims 34-35, wherein The modification comprises the following substitution: a. Y113C or Y113N; and / or, b. A203G. The method of any one of claims 34-36, comprising at least one modification to the wild-type GPCR at position 399, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The method of claim 37, wherein, The modification comprises the following substitution: T399. The method of claim 38, wherein, The modification comprises the following substitution: T399A. The method of any one of claims 34-39, wherein the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR. A method of increasing the potency of an exogenous ligand, comprising, providing the modified GPCR of any one of claims 1-15, or the polynucleotide sequence of claims 16-17, or the expression vector of any one of claims 18-30, or the cell of claim 31, or the pharmaceutical composition of any one of claims 32-33. A modified GPCR obtained by the method of any one of claims 34-40. A method of modulating GPCR activity in a subject, comprising (i) expressing the modified GPCR of any one of claims 1-14 in a target cell of the subject; and (ii) administering an exogenous ligand to the subject. The method of claim 43, wherein the subject is a human. The method of claim 44, wherein the subject is a non-human mammal, a bird, a fish, a reptile, or an amphibian. The method of any one of claims 43-45, wherein the target cell is a neuronal cell, a glial cell, a muscle cell, or an endocrine cell. The method of any one of claims 43-46, wherein the neuronal cell is from the central nervous system (CNS) or the peripheral nervous system (PNS). The method of any one of claims 43-47, wherein the target cell is an excitatory neuronal cell. The method of any one of claims 43-48, wherein the neuronal cell is located at an epileptic lesion or epileptogenic lesion. The method of any one of claims 43-49, further comprising: administering to the subject an effective amount of the polynucleotide of claims 16-17 or the expression vector of any one of claims 18-30. The method of claim 50, wherein the polynucleotide or expression vector is administered by injection. The method of any one of claims 50-51, wherein the polynucleotide or expression vector is in contact with a target cell of the subject. The modified GPCR of claims 1-15 or claim 42, or the polynucleotide sequence of any one of claims 16-17, or the expression vector of any one of claims 18-30, or the cell of claim 31, or the pharmaceutical composition of claims 32-33, or the method of claims 34-40, or the method of claims 43-52, for use in increasing the responsiveness of a GPCR to an exogenous ligand. The modified GPCR of claims 1-15 or claim 42, or the polynucleotide sequence of claims 16-17, or the expression vector of claims 18-30, or the cell of claim 31, or the pharmaceutical composition of claims 32-33, or the method of any one of claims 34-40, or the method of any one of claims 43-52, for use in treating epilepsy in a subject. Use of a modified GPCR according to any one of claims 1-15 or claim 42, or a polynucleotide sequence according to any one of claims 16-17, or an expression vector according to any one of claims 18-30, or a cell according to claim 31, or a pharmaceutical composition according to any one of claims 32-33, for the manufacture of a medicament for the treatment of epilepsy. A method of producing a modified GPCR, comprising at least one modification to a wild-type GPCR at the following positions: 113 and / or 203, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The method of claim 56, wherein, The modification comprises the following substitution: a. 113C or 113N; and / or, b. 203G. The method of any one of claims 56-57, wherein, The modification comprises the following substitution: a. Y113C or Y113N; and / or, b. A203G. A method according to any one of claims 56-58, comprising at least one modification to a wild-type GPCR at the following position: 399, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1. The method of claim 59, wherein, The modification comprises the following substitution: T399. The method of claim 60, wherein, The modification comprises the following substitution: T399A. A method according to any one of claims 56-61, wherein the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR.

Images