M4di variant and use thereof in treatment of pain

Abstract

The present application relates to an M4Di variant and the use thereof in the treatment of pain. 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 pain.

Description

M4Di variants and their use in treating pain TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a mutant of hM4Di receptor. BACKGROUND

[0002] Currently, chemical genetics is a technology that uses engineered receptors and specific ligands to precisely control cellular activities. 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, and high doses of CNO can cause non-specific effects; the defects of shorter effective time and rapid desensitization limit the duration of therapeutic effect and reduce the practicability and effect of long-term treatment. SUMMARY

[0003] The present application provides a mutation of hM4Di receptor at position 399. By solving the key defects of limited ligand sensitivity and rapid desensitization, this innovation provides a more effective and practical solution for long-term chemical genetic therapy, which enhances the sensitivity to ligands (such as CNO) allows the use of lower doses and reduces non-specific effects, and the prolonged desensitization time ensures the sustained therapeutic effect. This development provides new possibilities for the application of chemical genetics in the treatment of pain. Through molecular biology and protein engineering means, the original hM4Di receptor is optimized, which enhances the sensitivity to ligands allows the use of lower doses and reduces non-specific effects, and the prolonged desensitization time ensures the sustained therapeutic effect. Combined with the excitatory neuron-specific promoter, it enhances the targeting and inhibition efficiency of specific neuron subgroups, while reducing the risk of side effects.

[0004] In one aspect, the present application provides a modified human G protein-coupled receptor (GPCR), wherein the modified GPCR has the following properties

[0005] (i) reduced reactivity to an endogenous ligand compared to a wild-type GPCR; and,

[0006] (ii) enhanced reactivity to at least one exogenous ligand compared to the wild-type GPCR.

[0007] In some embodiments, wherein the GPCR is Gi / o coupled.

[0008] In some embodiments, wherein the wild-type GPCR is muscarinic acetylcholine receptor M4.

[0009] In some embodiments, wherein the modified G protein-coupled receptor (GPCR) comprises A5.46G and / or Y3.33C mutations compared to the wild-type GPCR.

[0010] In some embodiments, wherein the wild-type GPCR comprises a sequence having at least 75% homology to SEQ ID NO: 1.

[0011] In some embodiments, wherein the modified GPCR comprises at least one modification at position 113 and / or 203 as compared to a wild-type GPCR, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO: 1.

[0012] In some embodiments, the modified GPCR comprises the following substitutions as compared to a wild-type GPCR:

[0013] a. 113C or 113N; and / or,

[0014] b. 203G.

[0015] In some embodiments, wherein the modified GPCR comprises the following substitutions as compared to a wild-type GPCR:

[0016] a. Y113C or Y113N; and / or,

[0017] b. A203G.

[0018] In some embodiments, the modified G-protein coupled receptor (GPCR) comprises at least one modification at a protein kinase A (PKA) phosphorylation site as compared to a wild-type GPCR.

[0019] In some embodiments, the modified GPCR comprises at least one modification at position 399 as compared to a wild-type GPCR, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO: 1.

[0020] In some embodiments, the modified GPCR comprises the following substitution: T399 as compared to a wild-type GPCR.

[0021] In some embodiments, the modified GPCR comprises the following substitution: T399A as compared to a wild-type GPCR.

[0022] In some embodiments, wherein the modified GPCR comprises a sequence having at least 75% homology to SEQ ID NOs: 2-3.

[0023] In some embodiments, the exogenous ligand is a ligand that is capable of binding to a modified GPCR having an amino acid sequence as set forth in SEQ ID NO: 2 or 3.

[0024] In some embodiments, the exogenous ligand is selected from clozapine, clozapine N-oxide (CNO), olanzapine, dechloroclozapine (DCZ), or a derivative thereof.

[0025] In another aspect, the present application provides a polynucleotide sequence comprising a polynucleotide encoding a nucleic acid sequence of the modified GPCR.

[0026] In some embodiments, wherein the polynucleotide sequence comprises a sequence having at least 75% homology to SEQ ID NO: 4-5.

[0027] In another aspect, the present application provides an expression vector comprising the polynucleotide.

[0028] In some embodiments, wherein the vector is a plasmid, a transposon, a cosmid, a bacterial artificial chromosome, or a viral vector.

[0029] In some embodiments, 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.

[0030] In some embodiments, the AAV vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10, Anc80, or a variant thereof.

[0031] In some embodiments, the nucleic acid encoding the modified GPCR is operably linked to a promoter.

[0032] In some embodiments, the promoter is one of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0033] In some embodiments, the constitutive promoter comprises one or several of cytomegalovirus CMV immediate early promoter, viral simian virus 40 SV40, 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 the vaccinia virus, 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 70 kDa protein 5 HSPA5, heat shock protein 90 kDa beta member 1 HSP90B1, heat shock protein 70 kDa 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 beta-actin promoter.

[0034] In some embodiments, the promoter is an EF1a promoter.

[0035] In some embodiments, 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.

[0036] In some embodiments, 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 cell-specific promoter, a retina-specific promoter, a prostate-specific promoter, a skin-specific promoter, a macrophage-specific promoter.

[0037] In some embodiments, the promoter is a neuron-specific promoter including: 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 glutamate decarboxylase (GAD1 / 2) promoter, a choline acetyltransferase (ChAT) promoter, a distal-less homeobox (Dlx) promoter.

[0038] In some embodiments, the promoter is CaMKIIa.

[0039] In some embodiments, 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.

[0040] In another aspect, the present application provides a cell comprising the expression vector.

[0041] In another aspect, the present 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.

[0042] In some embodiments, the pharmaceutical composition further comprises an exogenous ligand.

[0043] In another aspect, the present application provides 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 by correspondence with the amino acid sequence of SEQ ID NO: 1.

[0044] In some embodiments, the modification comprises the following substitutions:

[0045] a. 113C or 113N; and / or,

[0046] b. 203G.

[0047] In some embodiments, the modification comprises a substitution of:

[0048] a. Y113C or Y113N; and / or,

[0049] b. A203G.

[0050] In some embodiments, the method comprises at least one modification to a wild-type GPCR at position 399, wherein the amino acid positions of the modified GPCR are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1.

[0051] In some embodiments, the modification comprises a substitution of: T399.

[0052] In some embodiments, the modification comprises a substitution of: T399A.

[0053] In some embodiments, the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR.

[0054] In another aspect, the present application provides a method of increasing the potency of an exogenous ligand, comprising the modified GPCR, or the polynucleotide sequence, or the expression vector, or the cell, or the pharmaceutical composition.

[0055] In another aspect, the present application provides a modified GPCR obtained by the method.

[0056] In another aspect, the present application provides a method of modulating GPCR activity in a subject, comprising

[0057] (i) expressing the modified GPCR in a target cell of the subject; and

[0058] (ii) administering an exogenous ligand to the subject.

[0059] In some embodiments, wherein the subject is a human.

[0060] In some embodiments, wherein the subject is a non-human mammal, a bird, a fish, a reptile, or an amphibian.

[0061] In some embodiments, wherein the target cell is an excitable cell.

[0062] In some embodiments, wherein the target cell is a neuronal cell, a glial cell, a muscle cell, or an endocrine cell.

[0063] In some embodiments, wherein the neuronal cell is from the central nervous system (CNS) or the peripheral nervous system (PNS).

[0064] In some embodiments, wherein the neuronal cell is a brain neuronal cell.

[0065] In some embodiments, wherein the neuronal cell is a cerebral cortex neuronal cell.

[0066] In some embodiments, wherein the neuronal cell is a cingulate cortex neuronal cell.

[0067] In some embodiments, wherein the neuronal cell is a pre- or post-cingulate cortex neuronal cell.

[0068] In some embodiments, further comprising: administering to the subject an effective amount of the polynucleotide or the expression vector.

[0069] In some embodiments, wherein the polynucleotide or expression vector is administered by injection.

[0070] In some embodiments, wherein the polynucleotide or expression vector is in contact with the target cell of the subject.

[0071] In another aspect, the present application provides a use of the modified GPCR, the polynucleotide sequence, the expression vector, the cell, the pharmaceutical composition, or the method for increasing the responsiveness of GPCR to exogenous ligand.

[0072] In another aspect, the present application provides a use of the modified GPCR, the polynucleotide sequence, the expression vector, the cell, the pharmaceutical composition, or the method for modulating pain in a subject.

[0073] In another aspect, the present application provides a use of the modified GPCR, the polynucleotide sequence, the expression vector, the cell, the pharmaceutical composition, for the preparation of a medicament for modulating pain.

[0074] In some embodiments, wherein the pain is intractable pain.

[0075] In some embodiments, wherein the pain comprises cancer pain, neuropathic pain.

[0076] In some embodiments, wherein the neuropathic pain comprises central pain, trigeminal neuralgia, shingles pain, diabetic neuropathic pain, intractable headache, phantom limb pain.

[0077] In another aspect, the application provides a method of producing a modified GPCR, comprising at least one modification to a wild-type GPCR at position 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.

[0078] In some embodiments, the modification comprises a substitution of:

[0079] a. 113C or 113N; and / or,

[0080] b. 203G.

[0081] In some embodiments, the modification comprises a substitution of:

[0082] a. Y113C or Y113N; and / or,

[0083] b. A203G.

[0084] In some embodiments, the modification comprises at least one modification to a 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.

[0085] In some embodiments, the modification comprises a substitution of T399.

[0086] In some embodiments, the modification comprises a substitution of T399A.

[0087] In some embodiments, the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR.

[0088] Other aspects and advantages of the application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating the principles of the application. In the detailed description, only exemplary embodiments of the application are shown and described, whereas modifications and variations will be apparent to those skilled in the art from this detailed description. Accordingly, it is to be understood that this application can be practiced otherwise than specifically described, without departing from the spirit and scope of the application involved. BRIEF DESCRIPTION OF DRAWINGS

[0089] The specific features of the application involved are shown in the appended claims. The features and advantages of the application involved can be better understood from the exemplary embodiments described in detail below and from the accompanying drawings. The drawings are briefly described as follows:

[0090] Figure 1 shows the functional validation of the hM4Di mutants provided by the present application as Gi / o-coupled GPCRs.

[0091] Figure 2 and Figure 3 show the sensitivity comparison of the hM4Di mutants provided by the present application compared to hM4Di.

[0092] Figure 4 shows the desensitization time comparison of the hM4Di mutants provided by the present application compared to hM4Di.

[0093] Figure 5 shows the sensitivity of the hM4Di mutants provided by the present application to CNO and the effect of inhibiting neuronal activity.

[0094] Figure 6 shows the off-target validation of the mutants provided by the present application.

[0095] Figure 7 shows the modulatory effect of the hM4Di mutants provided by the present application on the central post-stroke pain (CPSP) model in mice.

[0096] Figure 8 shows the modulatory effect of the hM4Di mutants provided by the present application on the CPSP model in mice.

[0097] Figure 9a and Figure 9b show the modulatory effect of different doses of clozapine, clozapine oxide (CNO) on the CPSP model in mice.

[0098] Figure 10a and Figure 10b show the time course of the therapeutic effect of clozapine or clozapine oxide (CNO) on the CPSP model.

[0099] Figure 11 shows the modulatory effect of the hM4Di mutants provided by the present application on the bone cancer pain (CIBP) model in mice.

[0100] Figure 12 shows the modulatory effect of the hM4Di mutants provided by the present application on the NTG-induced migraine model in mice. DETAILED DESCRIPTION

[0101] The advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present specification.

[0102] Terminology

[0103] The present application discloses a novel therapeutic system which uses a vector to express a modified receptor, the mutated receptor is not sensitive to endogenous neurotransmitters, but is sensitive to exogenous ligands.

[0104] As noted above, receptors that are modified to be activated only by artificial agonists have been known in the art for many years, sometimes referred to as "designer receptors exclusively activated by designer drugs" (DREADDs) or "receptors activated solely by synthetic ligand" (RASSLs). Those skilled in the art can provide such receptors using known methods and apply them to the present application in accordance with the present disclosure. Unless the context requires otherwise, "modified receptor" or similar terms, DREADD and RASSL are used interchangeably herein.

[0105] For example, WO 97 / 35478 describes the preparation of RASSLs. The description of the preparation and properties of RASSLs in that application is hereby incorporated by reference. WO 97 / 35478 describes how to make RASSLs from G protein-coupled receptors, including Gi / o-coupled muscarinic acetylcholine receptors. The description of the construction of RASSLs in WO 97 / 35478 is incorporated herein. In addition, certain definitions relating to RASSLs in WO 97 / 35478 are used herein to be consistent with their art-recognized meanings. The RASSL described therein is a modified G protein-coupled receptor that has reduced binding affinity for a selected natural (i.e., endogenous) ligand of the GPCR (relative to the binding of the wild-type G protein-coupled receptor to the selected ligand), but has normal, near-normal, or preferably enhanced binding affinity for an exogenous (typically synthetic) small molecule. Thus, in the presence of the natural ligand, RASSL-mediated activation of the RASSL-expressing cell is not apparent in vivo, but when exposed to the exogenously introduced small molecule, the activation is apparent. In other words, the exogenous ligand activates the RASSL better than the natural ligand (i.e., the binding of the small molecule ligand activates the RASSL to a greater or greater extent than the binding of a similar concentration of the selected natural ligand).

[0106] In the present 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 a signal mediated by G proteins, resulting in a G protein-coupled cellular response. GPCRs are a large family of evolutionarily related proteins (see WO 97 / 35478). GPCRs interact with a complex of heterotrimeric guanine nucleotide-binding proteins (G proteins), thereby modulating various intracellular signaling pathways, including ion channels. GPCRs can couple with G proteins to G protein-coupled inwardly rectifying potassium channels (GIRKs), altering neuronal excitability and, thus, neurotransmission.

[0107] The preferred GPCR to activate GIRKS is the muscarinic acetylcholine receptor M4, also known as human muscarinic acetylcholine receptor subtype M4. This receptor is a protein that in humans is encoded by the CHRM4 gene. The human muscarinic acetylcholine receptor subtype M4 (hM4) can bind to acetylcholine (Ach), which in turn couples to a G protein-coupled receptor of the Gi / o class, participating in the Gi signaling pathway.

[0108] When two conserved sites on hM4, A5.46G and Y3.33C, are mutated, hM4 no longer binds to acetylcholine, but instead binds efficiently to an exogenous ligand (e.g., CNO). We call the mutated receptor hM4Di. This engineered GPCR includes the following mutations: Y113C / A203G. Preferably, the hM4Di sequence is set forth in SEQ ID NO: 2.

[0109] The G protein-coupled cellular responses relevant to the present application are those that alter cellular excitability. A preferred response is an inhibitory response, in which ligand activation of the receptor triggers a series of intracellular events, such as inhibition of adenylyl cyclase (AC) and, thus, reduction of intracellular cyclic adenosine monophosphate (cAMP) production; activation of potassium ion channels (e.g., GIRK channels), leading to hyperpolarization of the cell and inhibition of action potential firing, among others, which collectively result in inhibition of the cell.

[0110] In the present application, the term "exogenous" or "exogenous" is used in the present disclosure to refer to any molecule, including nucleic acids, proteins or peptides, small molecule compounds, etc., that originates from outside the organism.

[0111] In contrast, the term "endogenous" refers to any molecule that originates from inside the organism (i.e., is naturally produced by the organism).

[0112] The term "endogenous ligand" refers to a molecule inside the organism that can activate the GPCR signaling pathway, causing different excitatory changes in the cell. For example, acetylcholine (Ach) is the endogenous ligand for hM4. Specifically, acetylcholine (Ach) can bind to the human muscarinic acetylcholine receptor subtype M4 (hM4), then couple with Gi / o class G protein-coupled receptors to play a role, involving the Gi / o class signaling pathway.

[0113] The term "exogenous ligand" refers to a molecule outside the organism that can activate the modified GPCR signaling pathway, causing different excitatory changes in the cell. The exogenous ligand is non-naturally occurring or synthetic. For example, clozapine or clozapine oxide is the exogenous ligand for hM4Di. Specifically, hM4Di can bind to the exogenous ligand (e.g., CNO), then couple with Gq class G protein-coupled receptors, and then couple with Gi / o class G protein-coupled receptors to play a role, involving the Gi / o class signaling pathway.

[0114] The "reactivity" described herein refers to the potency of the endogenous ligand or the exogenous ligand to the receptor. For example, the mutant hM4Di receptor can cause a decrease in cyclic adenosine monophosphate (cAMP) after CNO activation, and the degree of cAMP decrease represents the potency of the ligand to the receptor. For another example, the change in the level of ERK phosphorylation caused by the activation of hM4Di receptor by CNO can reflect the activation state and desensitization time of the receptor, and thus the change in the level of ERK phosphorylation represents the potency of the ligand to the receptor. For another example, the activation of Gi / o-coupled receptors can inhibit the excitability of neurons by reducing the level of cAMP and activating potassium channels, and thus reducing the firing of action potentials, and thus the firing of action potentials can reflect the potency of the ligand to the receptor.

[0115] The modified human G protein-coupled receptor (GPCR) is designed to be activated by an exogenous ligand. In this case, the modified human G protein-coupled receptor (GPCR) can not react or substantially less react to an endogenous ligand.

[0116] In the present application, the term "modified" or "modification" in the context of a protein refers to a fragment in which at least one amino acid residue in the reference molecule is replaced, deleted or added. Similarly, in the context of a nucleic acid, the term refers to a fragment in which at least one nucleic acid residue in the reference molecule is replaced, deleted or added.

[0117] In the present application, the term "excitable cell" refers to a cell that can generate an action potential after stimulation, such as a neuron of the central nervous system or the peripheral nervous system, a muscle cell (including striated muscle and smooth muscle), or an endocrine cell.

[0118] In the present application, the term "homology" can generally be equated with sequence "identity". A homologous sequence can include an amino acid sequence that can be 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 a subject sequence. Typically, a homolog will contain the same active sites, etc. as the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues that have similar chemical properties / functionality) or homology can be expressed in terms of sequence identity. In the present application, reference to a sequence having a percentage identity to any of the SEQ ID NOs of the amino acid sequences or nucleotide sequences means a sequence having that percentage identity over the entire length of the referenced SEQ ID NO. To determine sequence identity, a sequence alignment can be performed, which can be done in various ways understood by those skilled in the art, e.g., using the BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared.

[0119] In the present application, the term "wild type" generally refers to naturally occurring or naturally derived.

[0120] In the present application, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymer, either single-stranded or double-stranded. Unless specifically limited, the term encompasses nucleic acids containing analogues of natural nucleotides, which have similar binding properties to the reference nucleic acid (e.g., show sequence information) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, sequences of nucleic acids can include conservatively modified variants thereof, e.g., degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.

[0121] In the present application, the term "expression" generally refers to the transcription and / or translation of a particular nucleotide sequence.

[0122] In the present application, the term "pharmaceutically acceptable" generally refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, appropriate for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0123] In the present application, the term "pharmaceutically acceptable carrier" generally refers to any of the conventional carriers used for that purpose and is limited only by physical-chemical considerations, such as solubility and lack of reactivity with the active binding agent, and by the route of administration. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well-known in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is one that is chemically inert to the active ingredients of the pharmaceutical composition and one that does not have undesirable side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that can be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like; their use is well-known in the art.

[0124] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and preferably are inert at the site of administration and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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 dextrins; 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).

[0125] In the present application, the term "effective amount" or "effective dose" generally refers to an amount that is sufficient to achieve, or at least partially achieve, the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

[0126] In the present application, the term "host cell" or "cell" generally refers to an individual cell, a cell line, or a cell culture that can or has been maintained in a vector comprising an isolated nucleic acid molecule described herein, or that is capable of expressing an isolated nucleic acid molecule described herein. The host cell can include progeny of the original host cell. The progeny can not necessarily be completely identical (in morphology or in genome) to the original parent cell due to natural and intentional mutagenesis or deliberate mutation, but are still capable of expressing the isolated nucleic acid molecule described herein. The host cell can be obtained by transfecting a cell in vitro with a vector described herein. The host cell can be a prokaryotic cell (e.g., E. coli), or a eukaryotic cell (e.g., a yeast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NSO cell, or a neuronal cell). For example, the host cell can be an E. coli cell. For example, the host cell can be a yeast cell. For example, the host cell can be a mammalian cell. For example, the mammalian cell can be an N2A cell.

[0127] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for the transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell comprising the vector.

[0128] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for the transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell comprising the vector.

[0129] In the present application, the term "viral vector" is widely used to refer to a nucleic acid molecule (e.g., a transfer plasmid) or a viral particle that mediates the transfer of a nucleic acid, which includes a nucleic acid element derived from a virus that generally facilitates the transfer or integration of a nucleic acid molecule into a cell genome. The viral particle generally includes various viral components, and sometimes also includes host cell components other than nucleic acids. The viral vector can refer to a virus or a viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself.

[0130] 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.

[0131] 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 clearly 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.

[0132] 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.

[0133] 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.

[0134] 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).

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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 the disease state, eliminating lesions, or improving prognosis. In this application, "treatment" includes any beneficial or desired effect associated with pain relief and may even include minimal pain relief. Treatment may optionally include reducing or alleviating pain, or delaying the progression of pain. "Treatment" does not necessarily mean the complete eradication or cure of a disease or condition or its associated symptoms.

[0141] 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.

[0142] In this application, the term "prevention" generally refers to the preventive application of a combination to a healthy subject to prevent the occurrence of a disease or condition. It may also include the preventive application 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 likelihood 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. In this application, "prevention" refers to a method for preventing, suppressing, or reducing the likelihood of pain occurring or recurring. It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of pain symptoms. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of pain before its onset or recurrence.

[0143] In this application, “managing” or “controlling” or “adjusting” pain means using the compositions or methods covered herein to improve an individual’s quality of life by providing analgesia to a subject suffering from pain.

[0144] In this application, "pain" refers to an uncomfortable and / or unpleasant sensation in the body of the subject. Pain can range from mild to occasional to severe and constant. Pain can be classified as acute or chronic. Pain can be nociceptive pain (i.e., pain caused by tissue damage), neuropathic pain, or psychogenic pain. In some cases, pain is caused by or related to a disease (e.g., cancer, arthritis, diabetes). In other cases, pain is caused by injury (e.g., sports injury, trauma). Non-limiting examples of pain suitable for treatment with the compositions and methods described herein include: neuropathic pain, including peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, back pain, cancer-related neuropathy, HIV / AIDS-related neuropathy, phantom limb pain, carpal tunnel syndrome, central post-stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, pain associated with multiple sclerosis, pain associated with spinal cord injury, pain associated with Parkinson's disease, epilepsy, osteoarthritis pain, rheumatoid arthritis pain, visceral pain, and pain associated with vitamin deficiency; and nociceptive pain, including pain associated with central nervous system trauma, strains / sprains, and burns; myocardial infarction, acute pancreatitis, postoperative pain, post-traumatic pain, renal colic, cancer-related pain, fibromyalgia-related pain, carpal tunnel syndrome-related pain, and back pain.

[0145] 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.

[0146] Invention Details

[0147] Modified human GPCR

[0148] On the one hand, this application provides a modified human G protein-coupled receptor (GPCR), wherein the modified GPCR has the following characteristics

[0149] (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.

[0150] 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) can be selected to treat pain.

[0151] Examples of wild-type human GPCRs may include hM4, the sequence of which is shown in SEQ ID NO: 1.

[0152] 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.

[0153] 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.

[0154] In some embodiments, the modified GPCR is unresponsive or substantially unresponsive to endogenous ligands, and is therefore primarily activated by exogenous ligands.

[0155] 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.

[0156] 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.

[0157] Compared to the modified GPCR (e.g., hM4Di), it further includes at least one modification at the serine / thioine-specific protein kinase A (PKA) phosphorylation site.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] In some embodiments, the exogenous ligands mentioned above include: CNO, clozapine, piperapine, olanzapine, desclozapine, Compound 21, JHU37152, JHU37160, etc.

[0162] 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.

[0163] 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, FSHR93, F2RL3, GPR103L, BAI1, ADORA2B, LH CGR, P2RY1, GRCA, BAI2, ADORA3, TSHR, P2RY2, PGR1, BAI3, P2RY12, GPR54, P2RY4, HGPCR11, CD97, GPR105, LTB4R, P2RY6, SALPR, EMR1, GPR86, LTB4R2, P 2RY11, MAS1, EMR2, GPR87, MRGX1, LGR7, GPR90, EMR3, ADRA1A, MRGX2, 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. ,

[0164] 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%.

[0165] 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.

[0166] expression carrier

[0167] On the other hand, this application provides a vector for expressing modified GPCRs.

[0168] 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.

[0169] 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).

[0170] 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).

[0171] 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, and Anc80.

[0172] In some embodiments, AAV includes poultry AAV, cattle AAV, dog AAV, horse AAV, and sheep AAV.

[0173] The adeno-associated virus vector (AAV vector) may also include a recombinant adeno-associated virus vector (rAAV vector).

[0174] 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.

[0175] In some embodiments, the vector includes a promoter to which a nucleic acid encoding a modified GPCR is operatively linked.

[0176] The promoter can be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0177] 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.

[0178] 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.

[0179] In some embodiments, tissue specificity may include, but is not limited to: neuron-specific promoters, glial cell-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). In some embodiments, the tissue-specific promoter is a neuron-specific promoter or a glial cell-specific promoter. Neuron-specific promoters include 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 no distal homeobox (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.

[0180] In some embodiments, the promoter is CaMKIIα.

[0181] 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.

[0182] cell

[0183] 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.

[0184] Compositions and formulations

[0185] 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.

[0186] In some embodiments, the pharmaceutical composition may further include the exogenous ligand.

[0187] 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.

[0188] 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, intraneural, intraganglionic, and intraventricular administration.

[0189] 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.

[0190] Regulation methods and indications

[0191] On the other hand, this application provides a method for improving the reactivity of wild-type human GPCRs to exogenous ligands, 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 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 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 the wild-type GPCR. Through the above method, the reactivity of wild-type human GPCRs to endogenous ligands (e.g., Ach) is reduced, while the reactivity to exogenous ligands (e.g., CNO) is increased.

[0192] On the other hand, this application describes a method for regulating the GPCR activity of a subject, including...

[0193] (i) expressing an effective dose of the aforementioned modified GPCR in the target cells of the subject; and

[0194] (ii) Administer an effective dose of exogenous ligand to the subject.

[0195] In some implementations, the subject is a human. In other implementations, the subject is a non-human mammal, bird, fish, reptile, or amphibian.

[0196] In some implementations, the target cell is an excitable cell, such as a neuron, muscle cell, or endocrine cell.

[0197] In some embodiments, the neurons are derived from the central nervous system (CNS) or the peripheral nervous system (PNS). For example, the neurons are brain neurons, further, cerebral cortex neurons, even further, cingulate cortex neurons, and even further, anterior cingulate cortex neurons or posterior cingulate cortex neurons.

[0198] 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.

[0199] 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.

[0200] In some embodiments, application may involve a liquid formulation that can be injected into a carrier.

[0201] 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.

[0202] In some embodiments, the carrier can be administered to the subject via intracranial delivery (i.e., direct entry into the brain). In non-limiting examples of intracranial delivery, the carrier of the present invention can be delivered to the cortex of the brain (e.g., the anterior or posterior cingulate cortex) 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 indication to be treated, for example, injection into the sciatic nerve to treat chronic pain. In yet another specific case, the carrier can be administered to the subject via subcutaneous injection, for example, into sensory nerve endings to treat chronic pain.

[0203] 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, 7×1 013, 8×1013, 9×1013, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6×1014, 7×1014, 8×1014, 9×1014, 1×10^15, 2×10^15, 3×1015, 4×10^15, 5×10^15, 6×1015, 7×1015, 8×10^15, 9×1015, 1×10^16, 2×10^16, 3×10164×10^16, 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, 7×10^18 Vector genome units of 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.

[0204] 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.

[0205] 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).

[0206] 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).

[0207] 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).

[0208] 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.

[0209] In one embodiment, the ligand is administered to the subject at the same time as the vector is administered to the subject.

[0210] In one embodiment, the ligand is administered to the subject after the vector is administered to the subject.

[0211] In one embodiment, the ligand is administered to the subject before the vector is administered to the subject.

[0212] 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 administration of the carrier. 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 carrier delivery. In one specific instance, a therapeutically effective amount of the ligand is administered to the subject at least one week after carrier delivery.

[0213] 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.

[0214] 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, or at least about 10 or higher mg / kg.

[0215] 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.

[0216] 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.

[0217] 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 pain relief is required). 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.

[0218] This application further provides compositions and methods for controlling, managing, preventing, or treating pain in a subject. In some embodiments, the compositions and methods considered herein are effective in reducing pain.

[0219] In some implementations, pain includes, but is not limited to, acute pain, chronic pain, neuropathic pain, nociceptive pain, anomalous pain, inflammatory pain, hyperalgesia, neuropathy, neuralgia, diabetic neuropathy, human immunodeficiency virus-associated neuropathy, nerve damage, rheumatoid arthritis pain, osteoarthritis pain, burns, back pain, eye pain, visceral pain, cancer pain (e.g., bone cancer pain), toothache, headache, migraine, carpal tunnel syndrome, fibromyalgia, neuritis, sciatica, pelvic hypersensitivity, pelvic pain, postherpetic neuralgia, postoperative pain, post-stroke pain, and menstrual pain.

[0220] Pain can be classified as treatment-resistant pain. Treatment-resistant pain is defined as pain that does not improve or is tolerated after at least 3 months of standard medical treatment (including but not limited to medication, physical therapy, cognitive therapy, nerve blocks, and other non-invasive or minimally invasive treatments). Characteristics of treatment-resistant pain include: poor response to standard analgesics; potential need for highly individualized treatment plans; involvement of complex physiological and psychological factors; and the need for comprehensive pain management strategies, including both pharmacological and non-pharmacological treatments.

[0221] in,

[0222] 1. Drug therapy refers to patients who have received at least two first-line drugs for 3 months but whose pain relief is still unsatisfactory and / or whose adverse reactions are intolerable.

[0223] 2. Physical therapy aims to relieve pain, restore function, and promote patient recovery through non-pharmacological means. Below are some common physical therapy methods.

[0224] Heat therapy: can raise the pain threshold, relieve muscle spasms, increase blood circulation, and accelerate the removal of pain-causing substances.

[0225] Superficial hyperthermia includes the use of hot water bottles, hot baths, hot compresses, and infrared therapy. Deep hyperthermia involves technologies such as shortwave diathermy, microwaves, and ultrasound to achieve deep heating of the treatment area.

[0226] Cold therapy: Used in the early stages of acute injuries to relieve pain, reduce swelling and inflammation. Common applications include applying ice packs or cold compresses to the injured area, especially in cases of knee swelling.

[0227] Electrotherapy includes electrostatic field therapy, low-frequency electrotherapy (such as TENS), medium-frequency electrotherapy, and high-frequency electrotherapy. TENS (Transcutaneous Electrical Nerve Stimulation) is commonly used to relieve various acute and chronic pain.

[0228] Magnetotherapy: Utilizing the biological effects of magnetic fields, it can help relieve certain types of pain.

[0229] Ultrasound therapy: It promotes blood circulation and relieves muscle tension and pain through the thermal effect generated by high-frequency sound waves.

[0230] Phototherapy, such as infrared therapy and laser therapy, can promote tissue repair and reduce pain.

[0231] Radiofrequency ablation: an interventional treatment that relieves pain by heating local nerve tissue, and is suitable for pain caused by cervical spondylosis, lumbar disc herniation, etc.

[0232] Physiotherapy includes manual therapy, traction, and exercise therapy to help improve joint mobility and reduce pain.

[0233] Acupuncture: By stimulating specific acupoints, it regulates the body's Qi and blood to achieve the purpose of relieving pain.

[0234] Extracorporeal shock wave therapy: used to relieve musculoskeletal pain, such as tennis elbow and myofascial pain syndrome.

[0235] Cold and heat alternation therapy: In specific situations, cold therapy and heat therapy are combined to achieve the best therapeutic effect.

[0236] 3. Cognitive Therapy: This is a psychotherapeutic approach that aims to reduce pain perception and improve patients' functional well-being and quality of life by identifying and changing an individual's negative thought patterns, beliefs, and behavioral responses to pain. This approach posits that an individual's perception of pain, emotional responses, and behavioral coping mechanisms play a significant role in the pain experience, and not merely...

[0237] It is the physiological stimulus itself. Some core components and application strategies of cognitive therapy include:

[0238] Cognitive restructuring: Helping patients identify and challenge negative thoughts that exacerbate pain, such as catastrophic thinking (believing that pain is unbearable or will be permanent), and replace them with more realistic and positive ways of thinking.

[0239] Emotional regulation: Teach patients emotional management skills, such as relaxation training, mindfulness meditation and breathing exercises, to reduce anxiety and depression caused by pain, which often amplify the pain experience.

[0240] Behavioral activation: Encourage patients to gradually resume daily activities, even if it's just small steps, to break the pain-inactivity barrier.

[0241] - This exacerbates the vicious cycle of pain while simultaneously boosting self-efficacy.

[0242] Coping skills training: Provide tools and strategies to help patients cope effectively with pain, including time management and distraction techniques, to reduce the impact of pain on daily life.

[0243] Education and support: Enhance patients’ understanding of the nature of pain, including how physiological and psychological factors interact and the diversity of approaches to pain management, while providing a supportive environment so that patients know they are not alone.

[0244] Goal setting and problem-solving: Working with patients to set realistic short-term and long-term goals and teaching problem-solving skills to overcome obstacles encountered in achieving these goals.

[0245] 4. Nerve block therapy: This involves temporarily blocking the transmission of pain signals to the brain by injecting local anesthetic drugs into specific nerve sites or using physical methods. This treatment aims to directly act on the pain nerves, interrupting the pain transmission pathway, thereby achieving an analgesic effect.

[0246] Pain can be classified as acute or chronic. "Acute pain" refers to pain that begins suddenly and is usually of high quality. Acute pain may be mild and last for a period of time, or it may be severe and last for weeks or months. In most cases, acute pain does not last longer than three months and disappears once the underlying cause of the pain has been treated or healed. However, unrelieved acute pain can lead to chronic pain. "Chronic pain" refers to persistent or recurring pain that lasts longer than the normal course of an acute illness or injury, or lasts for more than three to six months, and has an adverse effect on an individual's health. Chronic pain can be nociceptive pain or neuropathic pain.

[0247] Clinical pain occurs when a patient experiences discomfort and hypersensitivity in their symptoms. Individuals can experience a variety of pain symptoms. These include: 1) spontaneous pain, which may be dull, burning, or tingling; 2) exaggerated pain responses to noxious stimuli (hyperalgesia); and 3) pain induced by normally harmless stimuli (hyperalgesia – Meyer et al., 1994, Textbook of Pain, 13–44). Although patients with various forms of acute and chronic pain may have similar symptoms, their underlying mechanisms may differ, thus requiring different treatment strategies. Pain can also be classified into many different subtypes based on different pathophysiologies, including nociceptive pain, inflammatory pain, and neuropathic pain.

[0248] In some embodiments, the compositions and methods considered herein are effective in reducing nociceptive pain.

[0249] In some embodiments, the compositions and methods considered herein are effective in reducing inflammatory pain.

[0250] In some embodiments, the compositions and methods considered herein are effective in reducing neuropathic pain.

[0251] Nociceptive pain is caused by tissue damage or intense stimulation that can lead to injury. Moderate to severe acute nociceptive pain includes central nervous system trauma, strains / sprains, burns, myocardial infarction and acute pancreatitis, postoperative pain (pain after any type of surgery), post-traumatic pain, renal colic, cancer pain, and back pain. Cancer pain can be chronic pain, such as tumor-related pain (e.g., bone pain, headache, facial pain, or visceral pain) or pain associated with cancer treatment (e.g., post-chemotherapy syndrome, chronic post-operative pain syndrome, or post-radiotherapy syndrome). Cancer pain can also occur in response to chemotherapy, immunotherapy, hormone therapy, or radiation therapy. Back pain may be caused by a herniated or ruptured disc or abnormalities in the lumbar facet joints, sacroiliac joints, paraspinal muscles, or posterior longitudinal ligament. Back pain may subside spontaneously, but in some patients who experience it for more than 12 weeks, it can become a chronic condition, especially debilitating.

[0252] Neuropathic pain can be defined as pain caused or induced by a primary lesion or dysfunction in the nervous system. Causes of neuropathic pain include, for example, peripheral neuropathy, diabetic neuropathy, postherpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, post-central stroke pain, pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy, and vitamin deficiencies.

[0253] Neuropathic pain can be associated with pain disorders, which are conditions, symptoms, or ailments that are related to or caused by pain. Illustrative examples of pain disorders include arthritis, atypical pain, typical trigeminal neuralgia, somatic symptom disorder, pseudoaneurysmia, hyperalgesia, neuralgia, neuritis, neurogenic pain, analgesia, anesthetic hyperalgesia, visceral diseases, chronic pain disorders, migraines / headaches, chronic fatigue syndrome, complex regional pain syndrome, neurotrophic disorders, plantar fasciitis, or cancer-related pain.

[0254] Inflammation is a complex series of biochemical and cellular events that are activated in response to tissue damage or the presence of foreign substances, leading to swelling and pain. Arthritis pain is a common type of inflammatory pain.

[0255] Other types of pain suitable for treatment with the nucleotides, carriers, pharmaceutical compositions, and methods of this application include, but are not limited to, pain caused by musculoskeletal disorders, including myalgia, fibromyalgia, spondylitis, seronegative (non-rheumatoid) arthropathy, non-arthritic rheumatoid arthritis, muscular dystrophy, glycogenolysis, polymyositis, and myositis; cardiac and vascular pain, including pain caused by angina, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, sclerosis, and skeletal muscle ischemia; headaches, such as migraines (including migraine with aura and migraine without aura), cluster headaches, tension headaches, mixed headaches, and headaches associated with vascular diseases; and orofacial pain, including toothache, earache, burning mouth syndrome, and temporomandibular myofascial pain.

[0256] The ability of the compositions and methods considered in this article to reduce the amount of pain experienced by subjects can be determined using a variety of pain scales. Patient self-reports can be used to assess whether pain has been relieved; see, for example, Katz and Melzack (1999) Surg. Clin. North Am. 79:231. Alternatively, observational pain scales can be used. The LANSS Pain Scale can be used to assess whether pain has been relieved; see, for example, Bennett (2001) Pain 92:147. Visual analogue pain scales can be used; see, for example, Schmader (2002) Clin. J. Pain 18:350. The Likert pain scale can be used; for example, where 0 is no pain, 5 is moderate pain, and 10 is the most likely pain. Self-reported pain scales for children include, for example, the Faces Pain Scale; the Wong-Baker Faces Pain Rating Scale; and the Colored Analog Scale. Self-reported pain scales for adults include, for example, the Visual Analog Scale; the Verbal Numerical Rating Scale; the Verbal Descriptor Scale; and the Brief Pain Inventory. Pain measurement scales include, for example, the Alder Hey Triage Pain Score (Stewart et al. (2004) Arch. Dis. Child. 89:625); the Behavioral Pain Scale (Payen et al. (2001) Critical Care Medicine 29:2258); the Brief Pain Inventory (Cleeland and Ryan (1994) Ann. Acad. Med. Singapore 23:129); and the Checklist of Nonverbal Pain Indicators (Feldt (2000) Pain Management Nursing).)1:13); Critical-Care Pain Observation Tool (Gelinas et al. (2006) Am.J.Crit.Care 15:420); Comfort Scale (Ambuel et al. (1992) J.Pediatric Psychol. 17:95); Dallas Pain Questionnaire (Ozguler et al. (2002) Spine 27:1783); Dolorimeter Pain Index (Hardy et al. (1952) Pain Sensations and Reactions Baltimore: The Williams & Wilkins Co.); Revised Faces Pain Scale Scale-Revised (Hicks et al. (2001) Pain 93:173); Face, Legs, Activity, Cry Consolability Scale; McGill Pain Questionnaire (Melzack (1975) Pain 1:277); Descriptor Differential Scale (Gracely and Kwilosz (1988) Pain 35:279); Numeric 11-point Box (Jensen et al. (1989) Clin. J. Pain 5:153); Numeric Rating Scale (Hartrick et al. (2003) Pain Practice (Pract.) 3:310); Wong-Baker Facial Pain Rating Scale; and Visual Analog Scale (Huskisson (1982) Journal of Rheumatology 9:768).

[0257] 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 pain in a subject. The method provides significant analgesia without off-target effects, such as systemic central nervous system depression. In some embodiments, the method provides a reduction of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher in a subject with neuropathic pain compared to an untreated subject.

[0258] Production methods

[0259] 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 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.

[0260] Example

[0261] Example 1: Functional Verification of the Mutant hM4Di Receptor

[0262] 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.

[0263] 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.

[0264] The experimental steps are as follows:

[0265] 1) Transfection:

[0266] 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.

[0267] Experimental grouping and treatment:

[0268] 2) After 24 hours, the cells were divided into three groups for treatment:

[0269] Control group: 0.05 mM IBMX (3-isobutyl-1-methylxanthine) was added to inhibit phosphodiesterase activity and prevent cAMP degradation.

[0270] 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.

[0271] 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.

[0272] 3) Collect cell samples from each group using a cAMP ELISA kit and determine the cAMP content.

[0273] The experimental results are shown in Figure 1. Isorepinephrine effectively increased cAMP levels. The cAMP values ​​of the WT group and the 399 group treated with isorepinephrine + CNO (Iso + CNO) were lower than those of the iso group, indicating that cAMP levels decreased significantly after CNO activated the receptor.

[0274] 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 protein pathway, leading to a decrease in cAMP levels. The 399 mutant hM4Di receptor (399) caused a decrease in cAMP upon CNO activation, 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.

[0275] Example 2 - The T399A mutation enhances the receptor's sensitivity to CNO.

[0276] 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.

[0277] The experimental steps are as follows:

[0278] 1) pAAV-EF1α-hM4Di(WT) and pAAV-EF1α-hM4Di T399A(399) were transiently transfected into HEK-293T cells.

[0279] 2) 24 hours after transfection, treat with CNO at 0, 1E-9 to 1E-5M for 5 minutes respectively.

[0280] 3) Collect samples and perform Western blotting analysis to detect p-ERK signals.

[0281] 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 hM4Di (WT) and the hM4Di399 mutant receptor (399) in a concentration-dependent manner, with 399 showing a significantly higher response intensity than WT. 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 WT was weak. The activation intensity of hM4Di T399A by CNO in the range of 1E-9 to 1E-5 M was significantly higher than that of WT, indicating that hM4Di T399A has a higher sensitivity to CNO and a stronger response intensity than WT.

[0282] Example 3 - T399A mutation prolongs receptor desensitization time and reduces receptor desensitization

[0283] 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.

[0284] The experimental steps are as follows:

[0285] 1) Transfect pAAV-EF1α-hM4Di(WT) and pAAV-EF1α-hM4Di T399A(399) transiently to

[0286] HEK-293T cells.

[0287] 2) 24 hours after transfection, treat with 10 μM CNO for 0 minutes, 5 minutes, 30 minutes and 60 minutes respectively.

[0288] 3) Collect samples and perform Western blotting analysis to detect p-ERK signals.

[0289] 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.

[0290] 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.

[0291] Example 4 - Electrophysiological Study of Brain Slice Patch-Clamp Technique

[0292] By expressing the hM4Di 399 mutant (399) and the hM4Di receptor (WT) in the mouse cortex and activating the receptor using CNO, the sensitivity of the mutant to CNO and its 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, making neurons less excitable and thus reducing neuronal excitability. Activation of the hM4Di receptor works synergistically through multiple mechanisms to lead to an inhibitory response in neurons.

[0293] Experimental steps:

[0294] 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.

[0295] 2) Brain slice preparation and patch-clamp recording:

[0296] 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).

[0297] 3) CNO processing and data logging:

[0298] 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.

[0299] 4) Data Analysis:

[0300] Physiological data were analyzed using Clampfit 10 software (Molecular Devices). The firing of neuronal action potentials was recorded under a 10 μM CNO circulation.

[0301] 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).

[0302] These results indicate that, under the same conditions, the mutant hM4Di T399A receptor, upon CNO activation, 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 T399A receptor in terms of CNO sensitivity and signal transduction persistence.

[0303] Example 5 - Verification of Off-Target Effects

[0304] Acetylcholine (ACh) is an endogenous neurotransmitter that can activate various cholinergic receptors, including the M4 type muscarinic acetylcholine receptor (CHRM4). To verify whether the mutant hM4Di T399A receptor (399) and hM4Di (WT) are activated by ACh and to assess their 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.

[0305] Experimental steps:

[0306] (1) Cell culture and transfection:

[0307] HEK-293T cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2.

[0308] Plasmids expressing CHRM4, hM4Di WT, and hM4Di 399 were transfected into HEK-293T cells, respectively.

[0309] (2) ACh treatment:

[0310] Twenty-four hours after transfection, cells in each group were treated with 10 μMACh for 5 and 30 minutes, respectively.

[0311] An untreated group was set up as a control group and marked as 0 minutes.

[0312] (3) Western Blot analysis:

[0313] 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.

[0314] (4) Data Analysis:

[0315] 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.

[0316] 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.

[0317] 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.

[0318] Example 6 - Establishing a mouse model of central pain (CPSP) after stroke

[0319] Experimental materials: 8-week-old wild-type male C57 mice; type IV collagenase (Sigma, Cat:C4-BIOC); 1mL syringe (Mishawa, 0.4×13mm RWLB); nanoliter micro-operation pump; single-arm digital stereotaxic instrument; animal skull drill.

[0320] Experimental steps

[0321] 1. Preparation before the experiment

[0322] 1) Prepare the necessary materials and equipment, including mice, surgical instruments, syringes, etc.

[0323] 2) Keep the laboratory environment clean and disinfect workbenches and necessary instruments.

[0324] 3) Dissolve type IV collagenase in double-distilled water and adjust the working solution concentration to 1 UI / μL for later use.

[0325] 4) Preparation of anesthetic: Dissolve 12.5g of 2,2,2-Tribromoethanol in 25mL of 2-methyl-2-butanol at 35°C. After dissolving, add 1000mL of physiological saline. Dispense into 50mL tubes for use. Store in the dark at 4°C.

[0326] 2. Experimental Procedure

[0327] 1) Anesthetize the mice: Completely anesthetize the mice using an appropriate anesthetic (500-600uL). After confirming that the mice are unresponsive by pinching their paws, proceed to the next step.

[0328] 2) Assembly of the infusion pump and medication: Inject vegetable oil into the glass electrode (be careful not to introduce air bubbles), attach it to the needle of the infusion pump (tighten it to prevent leakage or detachment), and draw 2000 nL of the pre-prepared type IV collagenase solution into the glass electrode.

[0329] 3) Fixing the mouse: Make an appropriate incision in the skin of the mouse's brain to make it easy to see the Bregma and Lambda points of the skull. Then place the mouse on the operating table and fix it with a stereotactic injection table to ensure that it will not move during the operation. Initially level the mouse to ensure that its head is as horizontal as possible.

[0330] 4) Determine the injection point: Use a mouse brain stereotaxic instrument to determine the coordinates of the injection point according to the brain region to be injected (VPN coordinates: AP-1, ML 1.65, DV-3.4).

[0331] 5) Skull drilling at the injection point: Use an appropriate skull drill to grind the skull surface at the designated injection point to ensure that the injection needle can be successfully inserted into the designated injection site.

[0332] 6) Injection: Slowly inject type IV collagenase solution into the VPN site using a syringe. The injection rate should be controlled at 50 nL / min, and the total injection volume should be 50 nL.

[0333] 7) Waiting before lifting the needle: After the injection is completed, wait for a period of time before slowly lifting the injection needle to ensure that type IV collagenase is fully diffused into the VPN brain region.

[0334] 8) Mice recovery: After injection, remove the mice from the restraints and seal the incision with 3M tissue glue. Then, transfer the mice to the recovery cage and provide appropriate temperature and nutrients during the recovery period.

[0335] 9) Monitoring and recording: Monitor the behavior and health status of mice after surgery and record relevant data for subsequent analysis.

[0336] 3. The experimental results are shown in Figure 7:

[0337] 1) Behavioral assessment showed that mice injected with collagenase had a significantly reduced mechanical pain threshold on the plantar surface of the contralateral hind paw 7 days after injection.

[0338] 2) Ten days after injection, the mechanical pain threshold of the contralateral hind paw plantar surface of mice injected with collagenase continued to decrease.

[0339] Example 7: Therapeutic effects of hM4Di and mutant hM4Di T399A receptors in a mouse CPSP model

[0340] 1. Experimental materials: 8-week-old wild-type male C57 mice; type IV collagenase (Sigma, Cat:C4-BIOC); 1mL syringe (Mishawa, 0.4×13mm RWLB); nanoliter micro-operation pump; single-arm digital stereotaxic instrument; animal skull drill.

[0341] 2. Experimental Procedure: After successful CPSP induction, AAV9:CaMKII-hM4Di(WT) or AAV9:CaMKII-hM4Di 399(399) virus was injected (the virus injection coordinates were in the ACC (anterior cingulate cortex) brain region, with coordinates AP: +0.38, ML: +0.25, DV: -1.12, and the injection dose was 400 nL). The threshold evaluation model of mechanical pain on the contralateral and ipsilateral hind paw plantar surfaces of mice was used, and the classic Von Frey test was employed.

[0342] 1) C57BL / 6J mice were grouped and placed according to the experimental design. They were placed on the mechanical pain test rack 30 minutes before the experiment to ensure stability.

[0343] 2) Apply pressure slowly to the paw area using Vonfrey fibers, wait for the fibers to bend at about 30 degrees for 2-3 seconds, and then record whether the mouse shows obvious avoidance or reaction.

[0344] 3) The up-down method was used to determine the pain threshold of the mice. The test was initiated with a Von Frey force of 0.16g, applied vertically to the palmar surface of the left hind paw for 2 to 3 seconds. This was repeated three times, observing the mouse's behavioral response, such as lifting or licking the hind paw. Two such responses were recorded as a positive response (denoted as X). If the mouse did not show a significant response (denoted as O), a slightly stronger filament (0.4g) was used for stimulation until a significant response was observed. This process was repeated until all filaments were tested. Results were recorded in a table.

[0345] 4) Allow the mice adequate rest time between each test to avoid fatigue or affecting the test results.

[0346] 5) Conduct multiple tests according to the experimental design and record the results of each test.

[0347] 6) 50% PWT calculation: Use the following formula: 50% PWT = 101 / 2Xf + kd3, where Xf is the value of the final Von Frey wire used (in logarithmic units), k is the value measured according to the positive / negative reaction pattern, and d = 0.224 (the average interval between Von Frey wires, in logarithmic units).

[0348] 3. The experimental results are shown in Figure 8:

[0349] On day 7 of CPSP modeling, WT or 399AAV virus was injected into the ipsilateral ACC brain region. On day 15 of modeling, mice were intraperitoneally injected with CNO (3 mg / kg). One hour later, Von Frey assay was performed. The results showed that without CNO treatment, the contralateral paw was abnormally sensitive to mechanical pain stimuli, while after CNO treatment, the threshold of the contralateral hind paw was significantly increased. Moreover, the analgesic effect of 399AAV virus was better than that of WT. All experimental groups of mice showed good efficacy after drug administration, and the analgesic phenotype exhibited by the disease model was significantly improved.

[0350] Example 8 - Exploring the dosage of clozapine and clozapine oxide (CNO) in a mouse CPSP model.

[0351] 1. Experimental materials: CPSP mice injected with AAV9:CaMKII-hM4Di(WT) or AAV9:CaMKII-hM4Di 399(399) AAV virus; 1 mL syringe (Mishawa, 0.4×13 mm RWLB), clozapine-N-Oxide (Tocris, Cat: 34233-69-7), clozapine (Clozapine, Hellobio, Cat: HB6129).

[0352] 2. Experimental procedure: CPSP mice injected with WT or 399AAV virus were given 1 mg / kg or 3 mg / kg Clozapine-N-Oxide via intraperitoneal injection or 1 mg / kg or 2 mg / kg Clozapine via oral gavage. The threshold of mechanical pain was evaluated by the classic Vonfrey test 1 hour after administration.

[0353] 3. Experimental Results

[0354] 1) As shown in Figure 9a: CPSP mice injected with WT or 399AAV virus were treated with CNO via intraperitoneal injection (ip) at doses of 1 mg / kg and 3 mg / kg, respectively. Four hours after treatment with the 3 mg / kg dose, the mechanical pain test results showed a significant increase in the contralateral hind paw threshold. We subsequently selected 3 mg / kg of CNO as the reference dose for subsequent treatments.

[0355] 2) As shown in Figure 9b: CPSP mice injected with WT or 399AAV virus were treated with clozapine orally (po) at doses of 1 and 2 mg / kg. Four hours after treatment with the 2 mg / kg dose, the mechanical pain test showed a significant increase in the contralateral hind paw threshold. We subsequently selected 2 mg / kg of clozapine as the reference dose for subsequent oral treatment.

[0356] Example 9 - Curves showing the therapeutic effects of clozapine or clozapine oxide (CNO) over time in a CPSP model.

[0357] By using rAAV to express hM4Di in pain-excitatory neurons in the ACC brain region, CNO binds to hM4Di and activates the Gi / o protein-coupled inward potassium channel GIRK, causing cell membrane hyperpolarization. This inhibits the firing of action potentials in neurons in the ACC that are involved in pain signal transmission, thereby blocking the descending transmission of pain signals to downstream brain regions and inhibiting the generation of pain.

[0358] By activating hM4Di receptors expressed in neurons, the excitation of pain-related neurons in the anterior cingulate cortex is inhibited, thus treating intractable pain, including but not limited to cancer pain, central pain, trigeminal neuralgia, postherpetic neuralgia, diabetic neuropathy, intractable headache, and phantom limb pain.

[0359] 1. Experimental materials: Refer to Example 8.

[0360] 2. Experimental procedure: CPSP mice injected with WT or 399AAV virus were administered 3 mg / kg of CNO via intraperitoneal injection or 2 mg / kg of clozapine via oral gavage. The threshold of mechanical pain was evaluated using the classic Vonfrey test 0.5, 1, 2, 4, 6, 8 and 24 hours after administration.

[0361] 3. Experimental Results:

[0362] 1) As shown in Figure 10a, CPSP mice injected with WT or 399AAV virus were administered 3 mg / kg of CNO via intraperitoneal injection. The mechanical pain threshold of the contralateral foot was evaluated using the classic Vonfrey test at 0.5, 1, 2, 4, 6, and 8 hours after administration. The results showed that oral administration of CNO for 0.5 hours significantly inhibited mechanical pain, with the optimal therapeutic effect reaching its peak at approximately 4-6 hours. The therapeutic effect gradually disappeared after this point due to metabolism.

[0363] 2) As shown in Figure 10b, CPSP mice injected with WT or 399AAV virus were orally administered clozapine at a dose of 2 mg / kg. The mechanical pain threshold of the contralateral foot was evaluated using the classic Vonfrey test at 0.5, 1, 2, 4, 6, and 8 hours after administration. The results showed that oral administration of clozapine for 0.5 hours significantly inhibited mechanical pain, with the optimal therapeutic effect observed around 4 hours. The therapeutic effect gradually disappeared as the body metabolized thereafter.

[0364] The results showed that the 399 group mice exhibited a significantly higher mechanical pain threshold in the von Frey test than the WT group, indicating that the 399 mutation is more effective in alleviating pain induced by CPSP.

[0365] Example 10 - Therapeutic effect of mutant hM4Di T399A receptor in mouse model of cancer bone pain (CIBP)

[0366] 1. Experimental materials: 8-week-old wild-type female C57 mice; 4T1 tumor cell line (mouse breast cancer cells) for modeling; Hamilton microsyringe; 1mL syringe (Mishawa, 0.4×13mm RWLB); nanoliter micro-operation pump; single-arm digital stereotaxic instrument; animal skull drill.

[0367] 2. Experimental procedure: After successful CIBP induction, either control virus or 399AAV virus was injected. The virus injection coordinates were in the ACC brain region (AP: +0.38, ML: +0.25, DV: -1.12, injection dose: 400 nL). The threshold evaluation model of mechanical pain on the contralateral and ipsilateral hind paw plantar surfaces of mice was used, employing the classic Von Frey test.

[0368] 3. The results are shown in Figure 11:

[0369] 1) The results showed that after injecting mouse breast cancer cells into the left femoral cavity of mice, the mice exhibited obvious mechanical abnormal pain on the model side on day 7.

[0370] 2) On day 12 after CIBP modeling, the right ACC was injected with either the control virus or 399AAV virus. On day 24 after modeling, mice were intraperitoneally injected with CNO. Von Frey assay was performed 30 minutes later. The results showed that the mechanical abnormal pain induced by CIBP surgery was not altered by administration of control AAV to the ACC, but administration of 399AAV virus significantly increased the hind paw threshold on the modeling side in CIBP mice. All experimental groups of mice exhibited good drug efficacy after administration, and the analgesic phenotype of the disease model was significantly improved. The duration of efficacy could last up to 40 days after modeling, or even longer.

[0371] Example 11 - The role of mutant hM4Di receptor in a mouse NTG-induced migraine model

[0372] Nitroglycerin (NTG)-induced migraine: This refers to migraine symptoms experienced by patients when using nitroglycerin (a commonly used vasodilator) to treat cardiovascular disease. This embodiment aims to evaluate the therapeutic effect of chemogenetic techniques on NTG-induced migraine.

[0373] 1. Experimental materials: 8-week-old wild-type male / female C57 mice; nitroglycerin (0.5 mg / tablet); 1 mL syringe (Mishawa, 0.4 × 13 mm RWLB).

[0374] 2. Experimental Procedure: Nitroglycerin (0.5 mg / tablet) was dissolved in DMSO solution by sonication, and the working solution concentration was adjusted to 2.5 mg / mL for later use. According to the experimental design, 100 μL / mouse of diluted NTG solution was administered intraperitoneally to the bilateral ACC brain regions of female / male mice in the experimental group that had been injected with 399AAV virus. The threshold of mechanical pain was evaluated using the classic Von Frey test 1 hour after drug injection.

[0375] 3. The experimental results are shown in Figure 12:

[0376] 4. Before modeling, the pain tolerance of both paws of male and female mice was at a normal level; after receiving intraperitoneal injection of NTG for 0.5 h, the mechanical pain sensitization of both paws of male and female mice was significantly enhanced; after receiving CNO treatment for 1 h, the mechanical pain tolerance of both paws of male and female mice was significantly improved.

Claims

1. 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.

2. The receptor of claim 1, wherein the GPCR is Gi / o coupled.

3. The receptor of any one of claims 1-2, wherein the wild-type GPCR is muscarinic acetylcholine receptor M4.

4. 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.

5. 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.

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

1.

7. The acceptor of claim 6, wherein, The modified GPCR comprises the following substitutions compared to a wild-type GPCR: a. 113C or 113N; and / or, b. 203G.

8. The acceptor of claim 7, wherein, The modified GPCR comprises the following substitutions compared to a wild-type GPCR: a. Y113C or Y113N; and / or, b. A203G.

9. 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.

10. The receptor according to any one of claims 1 to 9, wherein, The modified GPCR comprises at least one modification at position 399 compared to a wild-type GPCR, wherein the amino acid positions of the modified GPCR are numbered in correspondence with the amino acid sequence of SEQ ID NO:

1.

11. The acceptor of claim 10, wherein, The modified GPCR comprises the following substitution compared to a wild-type GPCR: T399.

12. The acceptor of claim 11, wherein, The modified GPCR comprises the following substitution compared to a wild-type GPCR: T399A.

13. 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.

14. The receptor according to any one of claims 1-13, 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.

15. The acceptor of claim 14, wherein, The exogenous ligand is selected from clozapine, clozapine oxide (CNO), olanzapine, dechloroclozapine (DCZ), or a derivative thereof.

16. A polynucleotide sequence comprising a polynucleotide encoding a nucleic acid sequence of the modified GPCR of any one of claims 1-15.

17. The polynucleotide sequence of claim 15, wherein the polynucleotide sequence comprises a sequence having at least 75% homology to SEQ ID NOs: 4-5.

18. An expression vector comprising the polynucleotide of any one of claims 16-17.

19. The expression vector of claim 18, wherein the vector is a plasmid, a transposon, a cosmid, a bacterial artificial chromosome, or a viral vector.

20. 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.

21. 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.

22. The vector of any one of claims 18-21, wherein the nucleic acid encoding the modified GPCR is operably linked to a promoter.

23. The vector of claim 22, wherein the promoter is one of a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

24. 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.

25. The vector of any one of claims 22-24, wherein the promoter is an EF1a promoter.

26. 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.

27. 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 cell-specific promoter, a retina-specific promoter, a prostate-specific promoter, a skin-specific promoter, a macrophage-specific promoter.

28. 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.

29. The vector of claim 27, wherein the promoter is CaMKIIa.

30. The vector of claim 27, wherein a 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.

31. A cell comprising the expression vector of any one of claims 18-30.

32. 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.

33. The pharmaceutical composition of claim 32, further comprising the exogenous ligand of claim 15.

34. 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 position 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.

35. The method of claim 34, wherein, the modification comprises the following substitution: a. 113C or 113N; and / or, b. 203G.

36. The method of any one of claims 34-35, wherein, the modification comprises the following substitution: a. Y113C or Y113N; and / or, b. A203G.

37. 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.

38. The method of claim 37, wherein, the modification comprises the following substitution: T399.

39. The method of claim 38, wherein, the modification comprises the following substitution: T399A.

40. The method of any one of claims 34-39, wherein the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding the wild-type GPCR.

41. A method of increasing the efficacy of an exogenous ligand, comprising, providing 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, or the pharmaceutical composition of any one of claims 32-33.

42. A modified GPCR obtained by the method of any one of claims 34-40.

43. 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.

44. The method of claim 43, wherein the subject is a human.

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

46. The method of any one of claims 43-45, wherein the target cell is an excitable cell.

47. The method of any one of claims 43-46, wherein the target cell is a neuronal cell, a glial cell, a muscle cell, or an endocrine cell.

48. The method of any one of claims 43-47, wherein the neuronal cell is from the central nervous system (CNS) or the peripheral nervous system (PNS).

49. The method of any one of claims 43-48, wherein the neuronal cell is a brain neuronal cell.

50. The method of claim 49, wherein the neuronal cell is a cerebral cortical neuronal cell.

51. The method of claim 50, wherein the neuronal cell is an insular cortical neuronal cell.

52. The method of any one of claims 50-51, wherein the neuronal cell is a preinsular cortical neuronal cell or a postinsular cortical neuronal cell.

53. The method of any one of claims 43-52, further comprising: administering to the subject an effective amount of the polynucleotide of claims 16-17 or the expression vector of claims 18-30.

54. The method of claim 53, wherein the polynucleotide or expression vector is administered by injection.

55. The method of any one of claims 53-54, wherein the polynucleotide or expression vector is in contact with the target cell of the subject.

56. 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 any one of claims 32-33, or the method of any one of claims 34-40, or the method of claims 43-55, for use in increasing the responsiveness of a GPCR to an exogenous ligand.

57. 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 any one of claims 32-33, or the method of any one of claims 34-40, or the method of any one of claims 43-55, for use in modulating pain in a subject.

58. Use of 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 any one of claims 31, or the pharmaceutical composition of any one of claims 32-33, for the manufacture of a medicament for modulating pain.

59. The use of any one of claims 57-58, wherein the pain is intractable pain.

60. The use of claim 59, wherein the pain comprises cancer pain, neuropathic pain.

61. The use of claim 59, wherein the neuropathic pain comprises central pain, trigeminal neuralgia, shingles pain, diabetic neuropathic pain, intractable headache, phantom limb pain.

62. A method of producing a modified GPCR, comprising making at least one modification to a wild-type GPCR at position 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.

63. The method of claim 62, wherein, the modification comprises the following substitution: a. 113C or 113N; and / or, b. 203G.

64. The method of any one of claims 62-63, wherein, the modification comprises the following substitution: a. Y113C or Y113N; and / or, b. A203G.

65. The method of any one of claims 62-64, comprising making at least one modification to a 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.

66. The method of claim 65, wherein, the modification comprises the following substitution: T399.

67. The method of claim 66, wherein, the modification comprises the following substitution: T399A.

68. The method of any one of claims 62-67, wherein the modification is performed on a polynucleotide comprising a nucleic acid sequence encoding a wild-type GPCR.

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