Targeted chemogenetic agents and uses thereof

Abstract

This invention is directed to compositions comprising polynucleotide constructs comprising a portion encoding a chemogenetically modified G protein-coupled protein receptor (GPCR); and a portion encoding an amino acid sequence comprising a c-terminal tail of another protein, thereby targeting the construct toward a particular localization in a neuron. Also provided for are methods of using the same.

Description

[0001] Attorney Docket No. 5470.992.WO

[0002] TARGETED CHEMOGENETIC AGENTS AND USES THEREOF RELATED APPLICATION INFORMATION

[0003] This application claims the benefit of U.S. Provisional Application Serial No.

[0004] 63 / 913,567, filed November 7, 2025 and U.S. Provisional Application Serial No. 63 / 762,343, filed February 24, 2025, the disclosure of which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING A Sequence Listing in XML format, entitled 5470-992WO_ST26.xml, 51,610 bytes in size, generated on February 23, 2026, and filed herewith, is hereby incorporated by reference into the specification for its disclosures.

[0006] STATEMENT OF GOVERNMENT SUPPORT

[0007] This invention was made with government support under Grant Number DK116195 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] FIELD OF THE INVENTION

[0009] This invention is directed to compositions comprising polynucleotide constructs comprising a portion encoding a chemogenetically modified G protein-coupled protein receptor (GPCR); and a portion encoding an amino acid sequence comprising a c-terminal tail of another protein, thereby targeting the construct toward a particular localization in a neuron. Also provided for are methods of using the same.

[0010] BACKGROUND OF THE INVENTION

[0011] Chemogenetics provide a robust platform for on-demand modulation of GPCR signaling in vitro and in vivo. However, the most commonly used chemogenetic platform, known as DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), was invented 20 years ago, and few improvements to the original technology have been reported.

[0012] The present invention overcomes previous shortcomings in the art by providing improved engineered chemogenetic tools with enhanced and selective targeting for specified neuronal subdomains.Attorney Docket No. 5470.992.WO

[0013] SUMMARY OF THE INVENTION

[0014] One aspect of the present invention provide a polynucleotide construct comprising a portion encoding a chemogenetically modified G protein-coupled protein receptor (GPCR); and a portion encoding an amino acid sequence comprising a c-terminal tail of any one of Cholinergic Nicotinic Alpha 4 Receptor ("Chma4"), G-protein-coupled Receptor for Serotonin 5-hydroxytryptamine IB Serotonin Receptor ("HTR1B"), Neurexin la ("NRX1A"), a G-protein-coupled Receptor for 5-HT2A serotonin receptor (“2ACT”) and / or Sodium Channel Protein Type 2a ("Scn2a") proteins.

[0015] In some embodiments, the construct localizes intracellularly to the somatodendritic domain of a neuron (i.e., to a somatodendritic portion of a neuron, e.g., the cell body and / or dendrites).

[0016] In some embodiments, the construct localizes intracellularly to the axonal domain of a neuron (i.e., to an axonal portion of a neuron, e.g., the axons).

[0017] A further aspect of the invention provide a polypeptide construct encoded by the polynucleotide construct of the present invention.

[0018] Also provided are nucleic acid molecules encoding a polypeptide construct of the present invention, vectors comprising a polynucleotide construct of the present invention, vectors encoding a polypeptide construct of the present invention, and compositions and isolated cells comprising the same.

[0019] Another aspect of the invention provides a method of treating a subject in need thereof, comprising administering an effective amount of a polynucleotide construct, polypeptide, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention.

[0020] Another aspect of the invention provides a method of expressing a construct in a cell, comprising contacting a cell with an amount of a polynucleotide construct, polypeptide, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention.

[0021] Also provided for are uses of a polynucleotide construct, polypeptide, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention, for therapeutic or research purposes in vitro, ex vivo, or in vivo.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows panels with schematic diagrams describing the strategy for somatodendritic targeted DREADDS. Panel A shows a cartoon of the designed somatodendritically targeted excitatory DREADD hM3Dq-mCitrene-2ACT. Panel B showsAttorney Docket No. 5470.992.WO

[0023] the AF3 predicted structure. Panel C shows typical results in transduced cortical neurons (DIV18). Panel D shows typical results for hM3Dq-mCitrene and hM3Dq-mCitrene-2ACT.

[0024] FIG. 2 shows fluorescent microscopy images indicating localization of one of the tested constructs. The top four panels show the localization of GFP transduced in mouse cortex using an hM3Dq-mCitrene-2ACT (two top, left panels) an hM3Dq-mCitrene vector (two top, right panels) in HTR2A-Cre mice. The bottom four panels show the improved targeting of hM3Dq-mCitrene-2ACT (two bottom, left panels) compared to hM3Dq-mCitrene (two bottom, right panels) to the apical dendrites of 5-HT2A expressing neurons.

[0025] FIG. 3 shows fluorescent microscopy images indicating localization of the tested constructs (Panel A) and data plots of calcium influx measurements (Panel B). Panel A shows the expression of the various constructs in DIV18 mouse cortical neurons transduced with AAV8-expressing constructs. Panel B shows the raw calcium flux measurements in transfected HEK-T cells showing that all tested constructs were functional with greater responses than the control vector.

[0026] FIGS. 4A-4G shows schematic maps of construct designs for hM3Dq-CHRNA (FIG.

[0027] 4A), hM3Dq-mCitrene-CHRNA (FIG. 4B), hM3Dq-lBCT (FIG. 4C), hM3Dq-NRXlA (FIG. 4D), hM3Dq-mCitrene-NRXlA (FIG. 4E), hM3Dq-Scn2A (FIG. 4F), and hM3Dq-mCitrene-Scn2a (FIG. 4G).

[0028] FIGS. 5A-5B show photos of fluorescent imaging to identify neuronal targeting of DREADD constructs indicated by name on the lefthand side of each of FIG. 5A and FIG. 5B.

[0029] Arrows indicate the location of axon(s). hM3Dq shows non-specific cellular targeting; hM3Dq-2ACT shows somatodendritic targeting; hM3Dq-lBCT shows somatodendritic and axonal targeting. White arrows = axon (MAP2 negative process).

[0030] FIG. 6 shows data plots indicating that hM3Dq constructs with axonal targeting sequences are functional via calcium influx measurements. HEK cells were transfected with different plasmids containing possible axonal targeting motifs (e.g., Chrna4, Htlb, Nrxnla, Scn2al). After 48 hours, cells were treated with 10 pM CNO and cellular calcium influx was measured.

[0031] FIG. 7 shows a schematic diagram of exemplar additional generic embodiments of axonally targeted DREADD S.

[0032] FIG. 8 shows detailed construct maps of each of Signal-HA-hM3Dq-linker-mScarlet-linker-2A-CT-Stop (Panel A) and Signal-HA-hM4Di-linker-mScarlet-linker-2A-CT-Stop (Panel B) example constructs.Attorney Docket No. 5470.992.WO

[0033] FIG. 9 shows a schematic diagram of exemplar additional generic embodiments of dendritically targeted DREADDS.

[0034] FIG. 10 shows images of fluorescent staining demonstrating selective somatodendritic targeting of Sig-HAhM3D(Gq)-mCitrene-2ACT to the somatodendritic compartment of cortical pyramidal neurons.

[0035] FIG. 11 shows a schematic diagram of the GRK2 / 3 dependent phosphorylation sites in the C-terminus of 5-HT2A receptor.

[0036] FIG. 12 shows a schematic diagram of HTR2A and HTR1B and their C-terminal tails.

[0037] FIG. 13 shows a schematic diagram of the M3 and M4 DREADD constructs.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0041] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0042] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature orAttorney Docket No. 5470.992.WO

[0043] combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0044] As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0045] The term "and / or" when used in describing two or more items or conditions refers to situations where all named items or conditions are present or applicable, or to situations wherein only one (or less than all) of the items or conditions is present or applicable.

[0046] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0047] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

[0048] As used herein, the term "comprising", which is synonymous with "including", "containing", and "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term of art that means that the named elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0049] As used herein, the phrase "consisting of excludes any element, step, or ingredient not specified in the claim. When the phrase "consists of appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0050] As used herein, the phrase "consisting essentially of limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0051] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids areAttorney Docket No. 5470.992.WO

[0052] represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §§1.831 - 1.835 and established usage.

[0053] As used herein, the term "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single-stranded. The nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0054] The terms "nucleic acid segment," "nucleotide sequence," "nucleic acid molecule," or more generally "segment" will be understood by those in the art as a functional term that includes both genomic DNA sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, small regulatory RNAs, operon sequences and smaller engineered nucleotide sequences that express or may be adapted to express, proteins, polypeptides or peptides. Nucleic acids of the present disclosure may also be synthesized, either completely or in part, by methods known in the art.

[0055] "Amino acid sequence" and terms such as "peptide", "polypeptide", and "protein" are used interchangeably herein, and are not meant to limit the amino acid sequence to the complete, native amino acid sequence ( / .< ., a sequence containing only those amino acids found in the protein as it occurs in nature) associated with the recited protein molecule.

[0056] The term "sequence identity," as used herein, has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art.

[0057] A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

[0058] In a similar manner, percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.

[0059] The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that in one embodiment, theAttorney Docket No. 5470.992.WO

[0060] percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of nucleotides in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

[0061] As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.

[0062] A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.

[0063] As used herein, the term "chimera," and / or "chimeric" refer to an amino acid sequence (e.g., polypeptide, e.g., a viral genome) generated non-naturally by deliberate human design comprising, among other components, an amino acid sequence of a protein of interest and / or a modified variant and / or active fragment thereof (a "backbone"), wherein the protein of interest comprises modifications (e.g., substitutions such as singular residues and / or contiguous regions of amino acid residues) from different wild type reference sequences (chimera). The generated chimera may optionally be linked to other amino acid segments (fusion protein). The different components of the designed chimera may provide differing and / or combinatorial function. Structural and functional components of the designed chimera may be incorporated from differing and / or a plurality of source material.

[0064] A "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.

[0065] As used herein with respect to nucleic acids, the term "operably linked" refers to a functional linkage between two or more nucleic acids. For example, a promoter sequence may be described as being "operably linked" to a heterologous nucleic acid sequence because the promoter sequence initiates and / or mediates transcription of the heterologous nucleic acid sequence. In some embodiments, the operably linked nucleic acid sequences are contiguous and / or are in the same reading frame.

[0066] A "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.

[0067] As used herein, an "isolated" polynucleotide (e.g., an "isolated nucleic acid" or an "isolated nucleotide sequence") means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, theAttorney Docket No. 5470.992.WO

[0068] cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. Optionally, but not necessarily, the "isolated" polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0069] An "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. Optionally, but not necessarily, the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0070] Furthermore, an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier.

[0071] The term "endogenous" refers to a component naturally found in an environment, i.e., a gene, nucleic acid, miRNA, protein, cell, or other natural component expressed in the subject, as distinguished from an introduced component, i.e., an "exogenous" component.

[0072] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0073] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein and refer to a nucleic acid molecule and / or nucleotide sequence that is not naturally occurring in the virus. Generally, the heterologous nucleic acid may comprise an open reading frame that encodes a protein, protein fragment, peptide or nontranslated RNA of interest (e.g., for delivery to a cell or subject). In some embodiments, a heterologous nucleic acid of the invention may comprise and / or encode a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and / or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR, or any combination thereof).Attorney Docket No. 5470.992.WO

[0074] As used herein with respect to nucleic acids, the term "fragment" refers to a nucleic acid that is reduced in length relative to a reference nucleic acid and that comprises, consists essentially of and / or consists of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference nucleic acid. Such a nucleic acid fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, the nucleic acid fragment comprises, consists essentially of or consists of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive nucleotides. In some embodiments, the nucleic acid fragment comprises, consists essentially of or consists of less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive nucleotides.

[0075] As used herein with respect to polypeptides, the term "fragment" refers to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of and / or consists of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference polypeptide. Such a polypeptide fragment may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, the polypeptide fragment comprises, consists essentially of or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive amino acids. In some embodiments, the polypeptide fragment comprises, consists essentially of or consists of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive amino acids.

[0076] As used herein with respect to nucleic acids, the term "functional fragment" or "active fragment" refers to nucleic acid that encodes a functional fragment of a polypeptide.

[0077] As used herein with respect to polypeptides, the term "functional fragment" or "active fragment" refers to polypeptide fragment that retains at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of at least one biological activity of the full-length polypeptide (e.g., the ability to up-or down-regulate gene expression). In some embodiments, the functional fragment actually has a higher level of at least one biological activity of the full-length polypeptide.Attorney Docket No. 5470.992.WO

[0078] As used herein, the term "modified," as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof. Modified sequences may also be referred to as "modified variant(s)."

[0079] A "vector" refers to a compound used as a vehicle to carry foreign genetic material into another cell, where it can be replicated and / or expressed. A cloning vector containing foreign nucleic acid is termed a recombinant vector. Examples of nucleic acid vectors are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes. Recombinant vectors typically contain an origin of replication, a multicloning site, and a selectable marker. The nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Expression vectors (expression constructs or expression cassettes) are for the expression of the exogenous gene in the target cell, and generally have a promoter sequence that drives expression of the exogenous gene. Insertion of a vector into the target cell is referred to transformation or transfection for bacterial and eukaryotic cells, although insertion of a viral vector is often called transduction. The term "vector" may also be used in general to describe items that serve to carry foreign genetic material into another cell, such as, but not limited to, a transformed cell or a nanoparticle.

[0080] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts may be referred to as "transcription products" and encoded polypeptides may be referred to as "translation products." Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression product itself, e.g., the resulting nucleic acid or protein, may also be said to be "expressed." An expression product can be characterized as intracellular, extracellular, or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell. A substance is "secreted" by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

[0081] The term "enhance" or "increase" refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold, and / or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,Attorney Docket No. 5470.992.WO

[0082] 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more, or any value or range therein.

[0083] The term "inhibit" or "reduce" or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0084] A "subject" of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a mammal, a reptile, a bird, an amphibian, or a fish. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, efc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, efc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human. The terms "subject" and "patient" are in some embodiments used interchangeably herein, such as but not limited to in reference to a human subject or patient.

[0085] A "subject in need" of the methods of the invention can be any subject known or suspected to have a disorder and / or an illness to which the methods of the present invention disclosed herein may provide beneficial health effects, or a subject having an increased risk of developing the same.

[0086] As used herein the term "control" refers to a comparative sample and / or other reference source for a control subject.

[0087] A "sample" or "biological sample" of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.

[0088] As used herein, the term "detectable moiety" refers to any moiety which allows for the detection and / or selection of the construct to of which it is a component e.g., in an in vitro assay, e.g., in a scan performed in vivo. Non-limiting examples of detectable moieties include, but are not limited to, molecular reporter tags such as HA-Tag and / or FLAG-tag. In some embodiments, a detectable moiety may be a fluorescence reporter tag such as, but not limitedAttorney Docket No. 5470.992.WO

[0089] to, GFP, eGFP, RFP, YFP, BFP, CFP, mCherry, mScarlet (e.g., mScarlet3), mCitrene, or the like. In some embodiments, the one or more reporter tag may be a hemagglutinin ("HA") tag.

[0090] A detectable moiety may also include a selection moiety, e.g., a selection marker, e.g., an antibiotic resistance sequence. Non-limiting examples of antibiotic resistance sequences include sequences which confer resistance to blasticidin, neomycin, puromycin, tetracycline, and / or any combination thereof.

[0091] As used herein, the term "chemogenetics" or "chemogenetic" refers to the field of use and generation of macromolecules, including but not limited to proteins, engineered to interact with previously unrecognized small molecule chemical actuators (e.g., activators and / or inhibitors). A "chemogenetic protein" refers to a protein engineered to have an operative state (activated) and inoperative state (unactivated) with respect to the activity of a target brain cell. For example, a chemogenetic receptor in an operative state may be configured to react with other molecules in a cell to provide activation or inhibition of existing activity of a target brain cell through biochemical reactions.

[0092] Chemogenetics (Armbruster and Roth 2005 Neuropsychopharmacology 30:S265; Armbruster et al. 2007 PNAS 104:5163-5168) and optogenetics (Boyden et al. 2005 Nat Neurosci 104:1263-1268; Li et al. 2005 PNAS 102:17816-17821) have transformed neuroscience by providing tools to specifically modulate the activity of defined neuronal and non-neuronal cells in vivo and in vitro. The most widely used chemogenetic platform is known as DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) which was invented 20 years ago.

[0093] As used herein, the term "DREADD" refers to Designer Receptors Exclusively Activated by Designer Drugs as in Armbruster et al. 2007 PNAS 104(12): 5163-5168 and the review Roth, B.L., 2016 Neuron 89:683-694, the disclosures of each of which are incorporated herein by reference in their entirety. Additional description of the terms as used herein, such as but not limited to "DREADD", can be found in these references.

[0094] DREADDs are modified versions of natural activating or inhibitory GPCRs, engineered to respond to synthetic molecules rather than endogenous ligands (Armsbruster et al. 2007 PNAS 104(12):5163-5168). The term "GPCR" or "G protein-coupled protein receptor" refers to a class of cell surface receptors which are seven-pass transmembrane proteins that bind a specific ligand. Ligand binding activates the G-proteins to modulate downstream signaling. GPCRs have been shown to be involved in a wide variety of biological processes, including initiating signaling pathways in inflammation and neurotransmission.Attorney Docket No. 5470.992.WO

[0095] DREADDs can be classified as Gi-based DREADDs (Gi-DREADDs), Gq-based DREADDs (Gq-DREADDs), and Gs- and P-Arrestin-DREADDs. Exemplary DREADDs include hM3Dq, hM4Di, GsD, R165L P-Arr DREADD, hM4Dnr n, KORD (K-opioid-derived DREADD) and others identifiable to a person skilled in the art. For example, Gi-DREADDs include hM2Di, hM4Di, and KORD. hM2Di and hM4Di can be activated by chemical actuators such as clozapine-N-oxide (CNO), DREADD agonist 21 (Thompson et al.

[0096] 2018 ACS Pharmacol & Translational Science 1:61-72), and perlapine. Gq-DREADDs include hMlDq, hM3Dq and hM5Dq DREADD. Gq-DREADD can be activated by chemical actuators such as CNO, a pharmacologically inert metabolite of the atypical antipsychotic drug clozapine. Gs-DREADDs are created by swapping the intracellular regions of the turkey erythrocyte P adrenergic receptor for equivalent regions of a rat M3 DREADD to create a rat eGs-DREADD. P-Arrestin-DREADDs are DREADDs signaling exclusively via P-arrestin.

[0097] Only a few modifications from the original DREADD design have been reported (Guettier et al. 2009 PNAS 106:19197-19202; Nakajima and Wess 2012 Mol Pharmacol 82(4):575-582; Stachniak et al. 2014 Neuron 82:797-808; Inoue et al. 2019 Cell 177:1933-1947). Additional DREADDs based on k-opioid (Vardy et al. 2015 Neuron 86:936-946) and the HCAR2-hydroxy carboxylic acid (niacin) (Kang et al. 2024 Cell 187:7433-7449) have also been reported along with modified chemogenetic cargos for sensing extracellular antigens (Kalogriopoulos et al. 2025 Nature 637:230-239). There have also been efforts to improve chemogenetic actuators (Chen et al. 2015 ACS Chem Neurosci 6:476-484) with deschloroclozapine being the preferred ligand for the muscarinic-based DREADDs (Nagai et al. 2020 Nat Neurosci 23 : 1157- 1167).

[0098] The present invention overcomes previous shortcomings in the art by providing a suite of engineered chemogenetic cargos with enhanced and selective targeting for specified neuronal subdomains.

[0099] One aspect of the present invention provides a polynucleotide construct comprising: a portion encoding a chemogenetically modified G protein-coupled protein receptor (GPCR); and a portion encoding an amino acid sequence comprising a c-terminal tail of any one of Cholinergic Nicotinic Alpha 4 Receptor ("Chma4"), G-protein-coupled Receptor for Serotonin 5-hydroxytryptamine IB Serotonin Receptor ("HTR1B"), Neurexin la("NRXlA"), a G-protein-coupled Receptor for 5-HT2A serotonin receptor (“2ACT”) and / or Sodium Channel Protein Type 2a ("Scn2a") proteins.

[0100] In some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5 to about 20 (e.g., about 5, 6, 7, 8, 9, 10,Attorney Docket No. 5470.992.WO

[0101] 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or any value or range therein) amino acids of the c-terminal tail of Chrna4, HTR1B, NRX1A, 2ACT and / or Scn2a. For example, in some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the c-terminal tail of Chrna4, or about 5 to about 20, about 10 to about 15, about 5 to about 15, about 5 to about 10, about 10 to about 20, amino acids of the c-terminal tail of Chrna4. In some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the c-terminal tail of HTR1B, or about 5 to about 20, about 10 to about 15, about 5 to about 15, about 5 to about 10, about 10 to about 20, amino acids of the c-terminal tail of HTR1B. In some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the c-terminal tail of NRX1A, or about 5 to about 20, about 10 to about 15, about 5 to about 15, about 5 to about 10, about 10 to about 20, amino acids of the c-terminal tail of NRX1 A. In some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the c-terminal tail of 2ACT, or about 5 to about 20, about 10 to about 15, about 5 to about 15, about 5 to about 10, about 10 to about 20, amino acids of the c-terminal tail of 2ACT. In some embodiments, the amino acid sequence comprising the c-terminal tail may comprise, consist essentially of, or consist of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the c-terminal tail of Scn2a, or about 5 to about 20, about 10 to about 15, about 5 to about 15, about 5 to about 10, about 10 to about 20, amino acids of the c-terminal tail of Scn2a.

[0102] In some embodiments, the chemogenetically modified GPCR may comprise an Allelespecific GPCR, a Receptor Activated Solely by Synthetic Ligand (RASSL), or a Designer Receptor Exclusive Activated by Designer Drugs (DREADD).

[0103] In some embodiments, the DREADD may comprise hMIDq, hM2Di, hM3Dq, hM4Di, hM5Dq, KORD and / or HCAD.

[0104] In some embodiments, the construct may have binding capacity for a ligand.

[0105] The ligand may be any ligand which functions as an actuator of the construct. In some embodiments, the ligand may be a clozapine-n-oxide (CNO) molecule, dechloroclozapine, olanzapine, FCH-2296413, Compound 21, perlapine molecule, salvinorin B molecule, and / or any derivative thereof.

[0106] In some embodiments, the construct may encode a fusion protein.Attorney Docket No. 5470.992.WO

[0107] In some embodiments, the construct may further comprise one or more linker (e.g., a flexible linker).

[0108] The one or more linker may be any linker which retains the function of the construct as a whole. In some embodiments, the one or more linker may be positioned within the portion encoding the amino acid sequence comprising a c-terminal tail (e.g., wherein the amino acid sequence comprising a c-terminal tail comprises two separated portions of amino acid sequence). In some embodiments, the one or more linker may be positioned upstream of (i.e., 5' to) the portion encoding the amino acid sequence comprising a c-terminal tail.

[0109] In some embodiments, the construct may further comprise one or more promoter and / or enhancer (e.g., CMV enhancer / promoter).

[0110] In some embodiments, the construct may further comprise one or more detectable moiety, e.g., a detection and / or selection moiety. A detection and / or selection moiety of the present invention may be any moiety which allows for the detection and / or selection of the construct e.g., in an in vitro assay, e.g., in a scan performed in vivo. Non-limiting examples of detection / selection moi eties include, but are not limited to, molecular reporter tags such as HA-Tag and / or FLAG-tag. For example, in some embodiments, the construct may further comprise and / or encode one or more reporter tag. In some embodiments, one or more reporter tag may be a fluorescence tag (e.g., fluorescent reporter, e.g., mCherry, mScarlet (e.g., mScarlet3), mCitrene, or the like). In some embodiments, the one or more reporter tag may be a hemagglutinin ("HA") tag.

[0111] In some embodiments, the one or more reporter tag may be positioned within the portion encoding the amino acid sequence comprising a c-terminal tail (e.g., wherein the amino acid sequence comprising a c-terminal tail comprises two separated portions of amino acid sequence).

[0112] In some embodiments, the one or more reporter tag may be positioned upstream of (i.e., 5' to) the portion encoding the amino acid sequence comprising a c-terminal tail.

[0113] In some embodiments, the construct may further comprise and / or encode a signal sequence (e.g., a nucleic acid signal sequence, e.g., encoding a signal peptide). In some embodiments, the signal sequence may be a cleavable signal sequence upstream of (5' to) one or more reporter tag.

[0114] In some embodiments, the construct may further comprise a polyA tail (e.g., bGH poly A, SV40 polyA).Attorney Docket No. 5470.992.WO

[0115] In some embodiments, the construct may localize intracellularly to the somatodendritic domain of a neuron (i.e., to a somatodendritic portion of a neuron, e.g., the cell body and / or dendrites).

[0116] In some embodiments, the construct may localize intracellularly to the axonal domain of a neuron (i.e., to an axonal portion of a neuron, e.g., the axons).

[0117] In some embodiments, the portion encoding the amino acid sequence of a c-terminal tail may encode a c-terminal tail of human Chma4, human HTR1B, human NRX1A, human 2ACT and / or human Scn2a protein.

[0118] In some embodiments, the portion encoding the amino acid sequence comprising a c-terminal tail may encode a serine (S) to alanine (A) substitution at one or more amino acid positions in the c-terminal tail (e.g., thereby eliminating phosphorylation at the one or more positions). In some embodiments, the one or more positions subject to a serine (S) to alanine (A) substitution in the c-terminal tail may comprise those one or more positions comprising a serine as shown in bold and underline in dendritically-targeted example constructs (3) and / or (4) below.

[0119] M3 construct amino acid sequence:

[0120] MYPYDVPDYAMTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAG NFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYF LLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCVASNASVMNLLVISF DRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGEC FIQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENF VHPTGS SRSC S S YELQQQ SMKRSNRRK YGRCHF WFTTKS WKP S SEQMDQDHS S SD S WNNNDAAASLENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTKLPSSDNLQVPEEE LGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKST ATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNI MVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDK KKRRKQQYQQRQSVIFHKRAPEQAL* (SEQ ID NO:1)

[0121] M4 construct amino acid sequence:

[0122] MYPYDVPDYAMANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSLVT VVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKGYWPLGAV VCDLWLALDCVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTKMAGLMIAAAWVL SFVLWAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAGFYLPVVIMTVLYIH ISLASRSRVHKHRPEGPKEKKAKTLAFLKSPLMKQSVKKPPPGEAAREELRNGKLEEAttorney Docket No. 5470.992.WO

[0123] APPPALPPPPRPVADKDTSNESSSGSATQNTKERPATELSTTEATTPAMPAPPLQPRA LNPASRWSKIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARKFASIARNQVRKKR QMAARERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDTVWSIGYWLCYVNS TINPACYALCNATFKKTFRHLLLCQYRNIGTAR* (SEQ ID NO:2)

[0124] Construct "5"; axonallv targeted M3 amino acid sequence:

[0125] MKTIIALSYIFCLVFAYPYDVPDYAMTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTV THFGS YNVSRAAGNF S SPDGTTDDPLGGHTVWQ VVFIAFLTGILALVTIIGNIL VIVSF KVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCV ASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFW QYFVGKRTVPPGECFIQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELA GLQASGTEAETENFVHPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKP S SEQMDQDHS S SDSWNNND AAASLENS AS SDEEDIGSETRAIYSIVLKLPGHSTILNS TKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDT AKTSDVNSSVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLS AILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFR TTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQALGGSGGGSGMDSTEAVIKE FMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLRVTKGGPLPFSWDILSPQFM YGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAVSVAQDTSLEDGTLIYKV KLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMALRLKDGGRYLADF KTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGGSGGSGGGS GRFKCTS* (SEQ ID NO:3)

[0126] Construct "6"; axonallv targeted M4 amino acid sequence:

[0127] MKTIIALSYIFCLVFAYPYDVPDYAMANFTPVNGSSGNQSVRLVTSSSHNRYETVEM VFIATVTGSLSLVTVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYT VYIIKGYWPLGAVVCDLWLALDCVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTK MAGLMIAAAWVLSFVLWAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAG FYLPVVIMTVLYIHISLASRSRVHKHRPEGPKEKKAKTLAFLKSPLMKQSVKKPPPGE AAREELRNGKLEEAPPPALPPPPRPVADKDTSNESSSGSATQNTKERPATELSTTEAT TPAMPAPPLQPRALNPASRWSKIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARK FASIARNQVRKKRQMAARERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDT VWSIGYWLCYVNSTINPACYALCNATFKKTFRHLLLCQYRNIGTARGGSGGGSGMD STEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLRVTKGGPLPFSWAttorney Docket No. 5470.992.WO

[0128] DILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAVSVAQDTSLE DGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMALRLKD GGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGG SGGSGGGSGRFKCTS* (SEQ ID NO:4)

[0129] Construct "3"; dendriticallv targeted M3 amino acid sequence:

[0130] MKTIIALSYIFCLVFAYPYDVPDYAMTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTV THFGS YNVSRAAGNF S SPDGTTDDPLGGHTVWQ VVFIAFLTGILALVTIIGNIL VIVSF KVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCV ASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFW QYFVGKRTVPPGECFIQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELA GLQASGTEAETENFVHPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKP S SEQMDQDHS S SDSWNNND AAASLENS AS SDEEDIGSETRAIYSIVLKLPGHSTILNS TKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDT AKTSDVNSSVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLS AILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFR TTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQALQYKENKKPLQLILVNTIPA LAYKSSQLQMGQKKNSKQDAKTTDNDCSMVALGKQHGGSGGGSGMDSTEAVIKE FMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLRVTKGGPLPFSWDILSPQFM YGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAVSVAQDTSLEDGTLIYKV KLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMALRLKDGGRYLADF KTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGGSGGSGGGS GSEEASKDNSDGVNEKVSCV* (SEQ ID NO:5)

[0131] Construct "4"; dendriticallv targeted M4 amino acid sequence:

[0132] MKTIIALSYIFCLVFAYPYDVPDYAMANFTPVNGSSGNQSVRLVTSSSHNRYETVEM VFIATVTGSLSLVTVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYT VYIIKGYWPLGAVVCDLWLALDCVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTK MAGLMIAAAWVLSFVLWAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAG FYLPVVIMTVLYIHISLASRSRVHKHRPEGPKEKKAKTLAFLKSPLMKQSVKKPPPGE AAREELRNGKLEEAPPPALPPPPRPVADKDTSNESSSGSATQNTKERPATELSTTEAT TPAMPAPPLQPRALNPASRWSKIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARK FASIARNQVRKKRQMAARERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDT VWSIGYWLCYVNSTINPACYALCNATFKKTFRHLLLCQYRNIGTARQYKENKKPLQAttorney Docket No. 5470.992.WO

[0133] LILVNTIPALAYKSSQLQMGQKKNSKQDAKTTDNDCSMVALGKQHGGSGGGSGMD STEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLRVTKGGPLPFSW DILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAVSVAQDTSLE DGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMALRLKD GGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGG SGGSGGGSGSEEASKDNSDGVNEKVSCV* (SEQ ID NO:6)

[0134] In some embodiments, a polynucleotide construct of the present invention may comprise, consist essentially of, or consist of a nucleotide sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:7.

[0135] (DNA sequence of SOMATODENDRITIC ALLY TARGETED EXCITATORY DREADD)

[0136] In some embodiments, a polynucleotide construct of the present invention may comprise, consist essentially of, or consist of a nucleotide sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to:

[0137] SEQ ID NO:8

[0138] (DNA SEQUENCE OF SOMATODENDRITIC ALLY TARGETED INHIBITORY CHEMOGENETIC CARGO)

[0139] Another aspect of the present invention provides a polypeptide construct encoded by a polynucleotide construct of the present invention.

[0140] For example, in some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:9 (hMD3Dq-CHRNA).

[0141] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 10 (hM3Dq-mCitrene-CHRNA).Attorney Docket No. 5470.992.WO

[0142] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 11 (hM3Dq-1BCT).

[0143] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 12 (hM3Dq-mCitrene-lBCT).

[0144] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 13 (hM3Dq-NRX1A).

[0145] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 14 (hM3Dq-mCitrene-NRX 1 A) .

[0146] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 15 (hM3Dq-Scn2a).

[0147] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,Attorney Docket No. 5470.992.WO

[0148] 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 16 (hM3Dq-mCitrene- S cn2 A) .

[0149] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:5 (Construct 3, with serines).

[0150] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:17 (construct 3, with alanines).

[0151] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:6 (construct 4, with serines).

[0152] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO: 18 (construct 4, with alanines).

[0153] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:3 (construct 5).

[0154] In some embodiments, a polypeptide construct of the present invention may comprise, consist essentially of, or consist of an amino acid sequence with at least 70% (70, 71, 72, 73,Attorney Docket No. 5470.992.WO

[0155] 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to: SEQ ID NO:4 (construct 6).

[0156] It would be understood that the modifications described above provide multiple examples of how the amino acid sequences described herein can be obtained and that, due to the degeneracy of the amino acid codons, numerous other modifications can be made to a nucleotide sequence encoding a polypeptide to obtain the desired amino acid sequence.

[0157] Another aspect of the present invention provides a nucleic acid molecule encoding a polypeptide construct of the present invention.

[0158] It will be understood by those skilled in the art that the nucleic acid(s) of the present invention may be operably associated with appropriate control sequences. For example, the nucleic acid may be operably associated with expression control elements, such as transcription / translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, and the like.

[0159] Those skilled in the art will further appreciate that a variety of promoter / enhancer elements may be used depending on the level and tissue-specific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0160] Promoter / enhancer elements can be native to the target cell or subject to be treated and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0161] Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the nucleic acid sequence(s). Inducible promoters / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and / or smooth muscle), neural tissue specific or preferred (including brain-specific), eye (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter / enhancer elements. In one embodiment, a CNS cell-specific or CNS cell-preferred promoter is used. Examples of neuron-specific or preferred promotersAttorney Docket No. 5470.992.WO

[0162] include, without limitation, neuronal-specific enolase, synapsin, and MeCP2. Examples of astrocyte-specific or preferred promoters include, without limitation, glial fibrillary acidic protein and SlOOp. Examples of ependymal cell-specific or preferred promoters include, without limitation, wdrl6, Foxjl, and LRP2. Examples of microglia-specific or preferred promoters include, without limitation, F4 / 80, CX3CR1, and CDllb. Examples of oligodendrocyte-specific or preferred promoters include, without limitation, myelin basic protein, cyclic nucleotide phosphodiesterase, proteolipid protein, Gtx, and SoxlO. Use of a CNS cell-specific or preferred promoter can increase the specificity achieved by the chimeric AAV vector by further limiting expression of the heterologous nucleic acid to the CNS. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoters / enhancer elements include, but are not limited to, a Tet on / off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.

[0163] In embodiments wherein the nucleic acid sequence(s) is transcribed and then translated in the target cells, specific initiation signals are generally employed for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0164] The nucleic acid can be incorporated into a delivery vector, such as a viral delivery vector. To illustrate, the nucleic acid of the invention can be packaged in an AAV particle, an adenovirus particle, a herpesvirus particle, a baculovirus particle, or any other suitable virus particle. Moreover, the nucleic acid can be operably associated with a promoter element. Promoter elements are described in more detail herein.

[0165] Another aspect of the invention provides a vector comprising a polynucleotide construct of the present invention and / or a nucleic acid molecule of the present invention.

[0166] Another aspect of the invention provides a vector encoding a polypeptide construct of the present invention.

[0167] Further provided is a pharmaceutical formulation comprising a AAV particle, nucleic acid molecule, and / or the vector of the present invention in a pharmaceutically acceptable carrier.

[0168] In some embodiments, the present invention provides a composition comprising a vector of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of delivery, the carrier mayAttorney Docket No. 5470.992.WO

[0169] be either solid or liquid. For inhalation delivery, the carrier will be respirable, and will preferably be in solid or liquid particulate form.

[0170] By "pharmaceutically acceptable" it is meant a material that is not toxic or otherwise undesirable, z.e., the material may be administered to a subject without causing any undesirable biological effects.

[0171] Another aspect of the present invention provides a composition comprising a polynucleotide construct, polypeptide construct, nucleic acid molecule, and / or vector of the present invention, optionally also comprising a pharmaceutically acceptable carrier, diluent and / or adjuvant.

[0172] Another aspect of the present invention provides an isolated (e.g., in vitro, e.g., ex vivo) cell comprising a polynucleotide construct, polypeptide construct, nucleic acid molecule, vector, and / or composition of the present invention.

[0173] In some embodiments, the cell, e.g., the isolated cell, may be a mammalian cell (e.g., a mouse, a rat, a monkey, a non-human primate, a human, a patient).

[0174] Another aspect of the present invention provides a method of treating a subject in need thereof, comprising administering an effective amount of a polynucleotide construct, polypeptide construct, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention.

[0175] Another aspect of the present invention provides a method of expressing a construct in a cell, comprising contacting a cell with an amount of a polynucleotide construct, polypeptide construct, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention.

[0176] Also provided is use of a polynucleotide construct, polypeptide construct, nucleic acid molecule, vector, composition, and / or isolated cell of the present invention, for therapeutic or research purposes in vitro, ex vivo, or in vivo.

[0177] A further aspect of the invention relates to kits for use in the methods of the invention. The kit can comprise the polynucleotide construct, polypeptide construct, nucleic acid molecule encoding the polynucleotide construct, vector, and / or composition of the invention in a form suitable for use in a clinical and / or research in vitro laboratory setting. The kit can comprise the polynucleotide construct, polypeptide construct, nucleic acid molecule encoding the polynucleotide construct, vector, and / or composition of the invention in a form suitable for use in administration to a subject or in a form suitable for compounding into a formulation. The kit can further comprise other therapeutic agents, carriers, buffers, containers, devices for administration, and the like. The kit can further comprise labels and / or instructions, forAttorney Docket No. 5470.992.WO

[0178] treatment of a disorder. Such labeling and / or instructions can include, for example, information concerning the amount, frequency and method of administration of the polynucleotide construct, polypeptide construct, nucleic acid molecule encoding the polynucleotide construct, vector, and / or composition of the invention.

[0179] The kit can further comprise other labeling agents, solid supports, carriers, buffers, containers, devices for administration, and the like. The kit can further comprise labels and / or instructions, for detection of a disorder.

[0180] The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.

[0181] EXAMPLES

[0182] Example 1: Generation and Testing of Neuronal Targeting DREADDS with Improved Chemogenetic Cargos.

[0183] Somatodendritic DREADDS'. Initial experiments suggested that the C-terminus of HTR2A (5-HT2A serotonin receptor) may encode a somatodendritic targeting sequence (FIG.

[0184] 12). Studies were then performed by inserting a GFP variant upstream of the targeting sequence (FIG. 1 panel A) to yield hM3Dq-mCitrene-2ACT and FIG. 1 panel B shows the predicted structure of the final chimeric protein expressed in a lipid bilayer. We then expressed such variants in HEK-293 cells to determine functionality.

[0185] As shown in FIG. 1 panel C, hM3Dq-mCitrene-2ACT showed a greater response to a maximum concentration of clozapine-N-oxide (CNO) indicating that this modification may enhance targeting and / or signaling. We also created a modified lentiviral vector to express hM3Dq-mCitrene-2ACT in neurons and found excellent somatodendritic targeting when compared with hM3Dq-mCitrene (FIG. 1 panel D).

[0186] We next created a modified AAV-based targeting system to selectively express hM3Dq-mCitrene-2ACT (FIG. 2, top left panels) or hM3Dq-mCitrene (FIG. 2, top right panels) selectively in genetically defined neurons. As shown in FIG. 2, bottom four panels, excellent somatodendritic targeting of hM3Dq-mCitrene-2ACT (FIG. 2, bottom left panels) compared with hM3Dq-mCitrene (FIG.2, bottom right panels) was obtained after expression in HTR2A-expressing neurons via a HTR2A-Cre driver mouse line. We next performed singleAttorney Docket No. 5470.992.WO

[0187] neuron patch studies on transduced neurons and observed a strong depolarization and enhanced firing following bath exposure of DCZ.

[0188] Axonal targeting DREADDs. We next utilized a similar procedure to create axonally-targeted excitatory and inhibitory DREADDs. For these experiments we explored the following potential targeting sequences from ion channels and GPCRs which are presynaptically localized: CHRNA (SWKDNCRRLIESMHKMANAPRFWP,- SEQ ID NO:35), HTR1B (KQAFHKLIRFKCAG,' SEQ ID NO:36), NRX1A (KKNKDKEYYV, SEQ ID NO:37), SCN2Aa (CLDILFAFT, SEQ ID NO:38). As shown, all tested constructs showed variable degrees of axonal targeting in vitro. When these constructs were transiently expressed in HEK-293 cells larger responses compared with the control (hm3Dq-mCitrene) were observed (FIG. 3; Panel A).

[0189] Example 2: Construct Designs for Somatodendritic Targeting of Chemogenetic Cargos.

[0190] A nonlimiting list of AXON ALLY-TARGETED DREADDs created with and without linkers and fluorescent protein markers, based on the generic M3 and M4 DREADD constructs (FIG.

[0191] 13) include those constructs described below.

[0192] hM3Dq-CHRNA

[0193] PROTEIN SEQUENCE; hM3Dq=lower case; c-terminus of CHRNA=upper case mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilvi vsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrakr ttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalSVVKDNCRRLIESMHKM ANAPRFWP (SEQ ID NO:9)

[0194] hM3Dq-mCitrene-CHRNA mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilvi vsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrakr ttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLMCFAR YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSVV KDNCRRLIESMHKMANAPRFWP (SEQ ID NO: 10)Attorney Docket No. 5470.992.WO

[0195] hM3Dq-lBCT

[0196] PROTEIN SEQUENCE; hM3Dq=lower case; HTR1B c-terminus: upper case Mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilv ivsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrak rttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalKQAFHKLIRFKCAG (SEQ ID NO: 11)

[0197] hM3Dq-mCitrene-lBCT mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilvi vsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrakr ttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLMCFAR YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKQA FHKLIRFKCAG (SEQ ID NO: 12)

[0198] hM3Dq-NRXlA

[0199] PROTEIN SEQUENCE; hM3Dq=lower case; NRX1 A c-terminus=UPPER CASE Mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilv ivsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrak rttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalKKNKDKEYYV (SEQ ID NO: 13)

[0200] hM3Dq-mCitrene-NRXlA mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilvi vsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrakr ttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLMCFAR YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDAttorney Docket No. 5470.992.WO

[0201] FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKKKN KDKEYYV (SEQ ID NO: 14)

[0202] hM3Dq-Scn2a

[0203] PROTEIN SEQUENCE; hM3Dq=lower case; SCN2Aa c-terminus=UPPER CASE Mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilv ivsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrak rttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalCLDILFAFT (SEQ ID NO: 15)

[0204] hM3Dq-mCitrene-Scn2a Mtlhnnsttsplfpnissswihspsdaglppgtvthfgsynvsraagnfsspdgttddplgghtvwqvvfiafltgilalvtiignilv ivsfkvnkqlktvnnyfllslacadliigvismnlfttyiimnrwalgnlacdlwlaidcvasnasvmnllvisfdryfsitrpltyrak rttkragvmiglawvisfvlwapailfwqyfvgkrtvppgecfiqflseptitfgtaiagfympvtimtilywriyketekrtkelagl qasgteaetenfvhptgssrscssyelqqqsmkrsnrrkygrchfwfttkswkpsseqmdqdhsssdswnnndaaaslensass deedigsetraiysivlklpghstilnstklpssdnlqvpeeelgmvdlerkadklqaqksvddggsfpksfsklpiqlesavdtakt sdvnssvgkstatlplsfkeatlakrfalktrsqitkrkrmslvkekkaaqtlsaillafiitwtpynimvlvntfcdscipktfwnlgy wlcyinstvnpvcyalcnktfrttfkmlllcqcdkkkrrkqqyqqrqsvifhkrapeqalMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLMCFAR YPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGID FKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKCLDI LFAFT (SEQ ID NO: 16)

[0205] To verify axonal targeting we employed an approach identical to that of Xia et al. (2003 Neuroscience 122:907-920; the disclosure of which are incorporated herein by reference in their entirety) using cortical neurons in vitro, using hM3Dq-mCitrene as comparison and hM3Dq-mCitrene-2ACT for a non-axonally targeted DREADD. Maps of the constructs described herein are provided in FIGS. 4A-4G.

[0206] FIGS. 5 A and 5B show fluorescent targeting by the tested constructs. To verify functionality we transfected the constructs above using pcDNA3 into HET cells and Ca++ flux following CNO administration was measured, as shown in FIG. 6.

[0207] In addition to the above-described constructs, axonally targeted DREADDs using the generic construct as exemplified in FIG. 7 are generated for mammalian cell expression.

[0208] To achieve efficient somatodendritic and surface expression, the following chemogenetic cargos were created (FIG. 8):

[0209] • Sig-HA-hM3D(Gq)-mScarlet3-2ACT

[0210] • Sig-HA-hM4D(Gi)-mScarlet3-2ACTAttorney Docket No. 5470.992.WO

[0211] These were also created without the mScarlet3 indicator as follows:

[0212] • Sig-HA-hM3D(Gq)-linker-2ACT

[0213] • Sig-HA-hM4D(Gi)-linker-2ACT

[0214] These were also created with additional fluorescent reporters such as:

[0215] • Sig-HA-hM3D(Gq)-mCitrene-2ACT

[0216] • Sig-HA-hM4D(Gi)-mCitrene-2ACT

[0217] Additionally it is contemplated that any fluorescent reporter could be used as follows where XFP=fluorescent reporter protein:

[0218] • Sig-HA-hM3D(Gq)-XFP-2ACT

[0219] • Sig-HA-hM4D(Gi)-XFP-2ACT

[0220] Non-limiting features of these constructs are:

[0221] • A specific cleavable signal sequence upstream of an HA-tag to enhance surface expression

[0222] • A flexible linker before the mScarlet3

[0223] • A flexible linker before the 2ACT sequence

[0224] • An intact C-terminal fragment of the human 5-HT2A sequence to promote somatodendritic targeting

[0225] In addition to the above-described constructs, dendritically targeted DREADDs using the generic construct as exemplified in FIG. 9 are generated for mammalian cell expression.

[0226] As proof of concept, experiments were performed showing successful selective somatodendritic targeting of Sig-HAhM3D(Gq)-mCitrene-2ACT to the somatodendritic compartment of cortical pyramidal neurons (FIG. 10).

[0227] Example 3: Additional Variations Of Constructs Generated.

[0228] Data was generated that indicated that the C-terminus of the 5-HT2A receptor is phosphorylated by GRK2 (FIG. 11).

[0229] S469 is part of the canonical PDZ-binding motif in the 5-HT2A receptor and is essential for targeting to the somatodendritic domain of cortical neurons (Xia et al 2003 J Biological Chemistry 278:21901-21908; Xia et al. 2003 Neuroscience 122:907-920). S453, S457 and S461 provide a putative arrestin recognition site for phosphorylated Ser residues based on homology with the 5-HT2B receptor (Cao et al. 2022 Neuron 110:3154-3167).

[0230] In view thereof, in some embodiments of the invention, for example wherein chronic chemogenetic activation may be used, modified cargoes as below are contemplated:Attorney Docket No. 5470.992.WO

[0231] Sig-HA-hM3D(Gq)-linker-2ACT (S453A, S457A, S461A) wherein the three phosphorylatable Ser residues are replaced by the non-phosporylatable residue Ala.

[0232] Also provided are embodiments of the constructs, for example, wherein the cargos thereof omit a cleavable signal sequence, HA-tag and / or XFP reporter, for example to reduce potential immunogenicity due to foreign non-human protein sequences. These constructs find use for example in long-term chemogenetic therapeutic applications:

[0233] • hM3D(Gq)-linker-2ACT

[0234] • hM4D(Gi)-linker-2ACT

[0235] The foregoing and the examples provided herewith are illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

Attorney Docket No. 5470.992.WOWHAT IS CLAIMED IS:

1. A polynucleotide construct comprising:a portion encoding a chemogenetically modified G protein-coupled protein receptor (GPCR); anda portion encoding an amino acid sequence comprising a c-terminal tail of any one of Cholinergic Nicotinic Alpha 4 Receptor ("Chma4"), G-protein-coupled Receptor for Serotonin 5-hydroxytryptamine IB Serotonin Receptor ("HTR1B"), Neurexin la ("NRX1 A"), a G-protein-coupled Receptor for 5-HT2A serotonin receptor (“2ACT”) and / or Sodium Channel Protein Type 2a ("Scn2a") proteins.

2. The polynucleotide construct of claim 1, wherein the amino acid sequence comprising the c-terminal tail comprises, consists essentially of, or consists of about 5 to about 20 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or any value or range therein) amino acids of the c-terminal tail of Chrna4, HTR1B, NRX1 A, 2ACT and / or Scn2a.

3. The polynucleotide construct of claim 1 or 2, wherein the chemogenetically modified GPCR comprises an Allele-specific GPCR, a Receptor Activated Solely by Synthetic Ligand (RASSL), or a Designer Receptor Exclusive Activated by Designer Drugs (DREADD).

4. The polynucleotide construct of claim 3, wherein the DREADD comprises hMIDq, hM2Di, hM3Dq, hM4Di, hM5Dq, KORD and / or HCAD.

5. The polynucleotide construct of any one of claims 1-3, wherein the construct has binding capacity for a ligand.

6. The polynucleotide construct of claim 5, wherein the ligand is a clozapine-n-oxide (CNO) molecule, dechloroclozapine, olanzapine, FCH-2296413, Compound 21, perlapine molecule, salvinorin B molecule, and / or any derivative thereof.

7. The polynucleotide construct of any one of claims 1-6, wherein the construct encodes a fusion protein.Attorney Docket No. 5470.992.WO8. The polynucleotide construct of any one of claims 1-7, further comprising one or more linker (e.g., a flexible linker).

9. The polynucleotide construct of claim 8, wherein the one or more linker is positioned within the portion encoding the amino acid sequence comprising a c-terminal tail (e.g., wherein the amino acid sequence comprising a c-terminal tail comprises two separated portions of amino acid sequence).

10. The polynucleotide construct of claim 8, wherein the one or more linker is positioned upstream of (i.e., 5' to) the portion encoding the amino acid sequence comprising a c-terminal tail.

11. The polynucleotide construct of any one of claims 1-10, further comprising one or more promoter and / or enhancer (e.g., CMV enhancer / promoter).

12. The polynucleotide construct of any one of claims 1-11, further comprising and / or encoding one or more reporter tag.

13. The polynucleotide construct of any one of claims 1-12, wherein the one or more reporter tag is positioned within the portion encoding the amino acid sequence comprising a c-terminal tail (e.g., wherein the amino acid sequence comprising a c-terminal tail comprises two separated portions of amino acid sequence).

14. The polynucleotide construct of any one of claims 1-12, wherein the one or more reporter tag is positioned upstream of (i.e., 5' to) the portion encoding the amino acid sequence comprising a c-terminal tail.

15. The polynucleotide construct of any one of claims 12-14, wherein the one or more reporter tag is a fluorescence tag (e.g., fluorescent reporter, e.g., mCherry, mScarlet (e.g., mScarlet3), mCitrene, or the like).

16. The polynucleotide construct of any one of claims 12-15, wherein the one or more reporter tag is an hemagglutinin ("HA") tag.Attorney Docket No. 5470.992.WO17. The polynucleotide construct of any one of claims 1-16, further comprising and / or encoding a signal sequence (e.g., a nucleic acid signal sequence, e.g., encoding a signal peptide).

18. The polynucleotide construct of claim 17, wherein the signal sequence is a cleavable signal sequence upstream of (5' to) one or more reporter tag.

19. The polynucleotide construct of any one of claims 1-18, further comprising a polyA tail (e.g., bGH polyA, SV40 polyA).

20. The polynucleotide construct of any one of claims 1-19, wherein the construct localizes intracellularly to the somatodendritic domain of a neuron (i.e., to a somatodendritic portion of a neuron, e.g., the cell body and / or dendrites).

21. The polynucleotide construct of any one of claims 1-19, wherein the construct localizes intracellularly to the axonal domain of a neuron (i.e., to an axonal portion of a neuron, e.g., the axons).

22. The polynucleotide construct of any one of claims 1-21, wherein the portion encoding the amino acid sequence of a c-terminal tail encodes a c-terminal tail of human Chrna4, human HTR1B, human NRX1 A, human 2ACT and / or human Scn2a protein.

23. The polynucleotide construct of any one of the preceding claims, wherein the portion encoding the amino acid sequence comprising a c-terminal tail encodes a serine (S) to alanine (A) substitution at one or more amino acid positions in the c-terminal tail (e.g., thereby eliminating phosphorylation at the one or more positions).

24. The polynucleotide construct of claim 23, wherein the one or more positions subject to a serine (S) to alanine (A) substitution in the c-terminal tail comprises those one or more positions comprising a serine as shown in bold on pages 17-19 in constructs (3) and / or (4).

25. The polynucleotide construct of any one of the preceding claims, comprising, consisting essentially of, or consisting of a nucleotide sequence with at least 70% (70, 71, 72,Attorney Docket No. 5470.992.WO73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:7.

26. The polynucleotide construct of any one of the preceding claims, comprising, consisting essentially of, or consisting of a nucleotide sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:8.

27. A polypeptide construct encoded by the polynucleotide construct of any one of claims 1-26.

28. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:9.

29. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 10.

30. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 11.

31. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 1232. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79,Attorney Docket No. 5470.992.WO80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 13.

33. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 14.

34. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 15.

35. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 16.

36. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:5.

37. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 17.

38. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:6.

39. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79,Attorney Docket No. 5470.992.WO80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO: 18.

40. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:3.

41. The polypeptide construct of claim 27, comprising, consisting essentially of, or consisting of an amino acid sequence with at least 70% (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% or any value or range therein) sequence identity to SEQ ID NO:4.

42. A nucleic acid molecule encoding the polypeptide construct of any one of claims 28-41.

43. A vector comprising the polynucleotide construct of any one of claims 1-26 or the nucleic acid molecule of claim 42.

44. A vector encoding the polypeptide construct of any one of claims 27-41.

45. A composition comprising the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, and / or the vector of claim 43 or 44, optionally also comprising a pharmaceutically acceptable carrier, diluent and / or adjuvant.

46. An isolated (e.g., in vitro, e.g., ex vivo) cell comprising the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, the vector of claim 43 or 44, and / or the composition of claim 45.

47. The cell of claim 46, wherein the cell is a mammalian cell (e.g., a mouse, a rat, a monkey, a non-human primate, a human, a patient).Attorney Docket No. 5470.992.WO48. A method of treating a subject in need thereof, comprising administering an effective amount of the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, the vector of claim 43 or 44, the composition of claim 45, and / or the isolated cell of claim 46 or 47.

49. A method of expressing a construct in a cell, comprising contacting a cell with an amount of the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, the vector of claim 43 or 44, and / or the composition of claim 45.

50. Use of the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, the vector of claim 43 or 44, and / or the composition of claim 45, for therapeutic or research purposes in vitro, ex vivo, or in vivo.

51. A kit comprising the polynucleotide construct of any one of claims 1-26, the polypeptide construct of any one of claims 27-41, the nucleic acid molecule of claim 42, the vector of claim 43 or 44, and / or the composition of claim 45, and optionally instructions for the use thereof.

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