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WO2025102068A1 - Modulation de la soif - Google Patents

Modulation de la soif Download PDF

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Publication number
WO2025102068A1
WO2025102068A1 PCT/US2024/055510 US2024055510W WO2025102068A1 WO 2025102068 A1 WO2025102068 A1 WO 2025102068A1 US 2024055510 W US2024055510 W US 2024055510W WO 2025102068 A1 WO2025102068 A1 WO 2025102068A1
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WIPO (PCT)
Prior art keywords
asprosin
ptprd
mice
peptide
flox
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PCT/US2024/055510
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English (en)
Inventor
Atul Chopra
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Case Western Reserve University
University Hospitals Cleveland Medical Center
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Case Western Reserve University
University Hospitals Cleveland Medical Center
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Publication of WO2025102068A1 publication Critical patent/WO2025102068A1/fr
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

  • BACKGROUND [0004]
  • Psychogenic polydipsia also known as primary polydipsia, manifests in individuals with several mental ailments, most prominently schizophrenia, alongside anxiety, affective disorders, anorexia nervosa, autism, and personality disorders.
  • Primary polydipsia affects 6-20% of all psychiatric inpatients, with 10-20% of schizophrenic patients experiencing polydipsia, and a third of them developing water intoxication and hyponatremia necessitating hospitalization.
  • Embodiments described herein relate to a method of regulating internal water balance and/or modulating thirst in a subject in need thereof, and particularly relates to a method of treating thirst disorders, such as polydipsia or excessive thirst, and adipsia or absence of thirst.
  • thirst disorders such as polydipsia or excessive thirst, and adipsia or absence of thirst.
  • PTPR ⁇ protein tyrosine phosphatase receptor type ⁇
  • Purkinje neuron-specific asprosin receptor (Ptprd) deletion results in reduced water intake and renders null asprosin’s dipsogenic effect.
  • an anti-asprosin mAb effectively treated a mouse model of neuropsychiatric disease that manifests polydipsia, with prolonged efficacy from a single dose.
  • modulating asprosin-PTPR ⁇ -cerebellum signaling and/or activity can be used as a method or therapy for treating thirst disorders, such as polydipsia, adipsia, or hypodipsia in a subject in need thereof, and particularly thirst disorders associated with mental ailments.
  • a method of regulating internal water balance and/or thirst in a subject in need thereof includes administering to the subject a therapeutically effective amount of an agent that modulates asprosin mediated PTPR ⁇ signaling and/or activity.
  • a therapeutically effective amount of an agent that inhibits asprosin mediated PTPR ⁇ signaling and/or activity can be administered to a subject in need thereof to decrease excessive thirst and/or fluid intake.
  • a therapeutically effective amount of an agent that promotes or enhances asprosin mediated PTPR ⁇ signaling and/or activity can be administered to a subject in need thereof to increase thirst and/or fluid intake.
  • a method of treating polydipsia in a subject in need thereof can include administering to the subject a therapeutically effective amount of an agent that inhibits asprosin mediated PTPR ⁇ signaling and/or activity.
  • the agent, which inhibits asprosin mediated PTPR ⁇ signaling and/or activity can include at least one of a small molecule, nucleic acid, peptide, protein, or antibody that inhibits asprosin binding to PTPR ⁇ or PTPR ⁇ signaling or activity.
  • the which inhibits asprosin mediated PTPR ⁇ signaling or activity can include an antibody or antigen binding fragment that specifically binds to asprosin and inhibits asprosin mediated PTPR ⁇ signaling and/or activity.
  • the anti-asprosin antibody or antigen binding fragment thereof can specifically bind to an asprosin peptide having an amino acid sequence of KKKELNQLEDRYDKDYLSGELGDNLKMK (SEQ ID NO: 1).
  • an anti- asprosin antibody or antigen binding fragment thereof that specifically binds to SEQ ID NO: 1 can include a heavy chain variable region and/or light variable region that includes three heavy chain CDRs and/or three light chain CDR of an antibody produced by hybridoma cell line deposited with the American Type Culture Collection under accession number ATCC PTA-123085.
  • the anti-asprosin antibody or antigen binding fragment thereof can be a fully human or humanized antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof can be a monospecific or bispecific antibody or antigen binding fragment thereof.
  • the agent which inhibits asprosin mediated PTPR ⁇ signaling or activity, can be a peptide that includes an amino acid sequence substantially identical to an extracellular portion of the amino acid sequence of PTPR ⁇ that binds to asprosin.
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about
  • the peptide can have a binding affinity KD to asprosin less than about 10 ⁇ M less than about 1 ⁇ M, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 10 nM, less about 1 nM, or less than about 500 pM.
  • KD binding affinity
  • FIGs.2(A-H) illustrate asprosin activates cerebellar Purkinje neurons.
  • A Representative brain sections from mouse (left) and human (right) brains pre-incubated with GFP (top) or free asprosin (bottom) subjected to competitive binding assay with AP-tagged asprosin. Scale bars, 10 ⁇ m.
  • E Representative image of recorded Pcp2 + neurons under brightfield and fluorescence microscopy and representative action potential firing traces of Pcp2 + neurons after puff treatment of control GFP or recombinant asprosin.
  • G Representative resting membrane potential trace of Pcp2 + neurons in response to 30 nM recombinant mammalian asprosin or GFP in the presence of a cocktail of synaptic blockers including 1 ⁇ M tetrodotoxin (TTX), 30 ⁇ M AP-5, 30 ⁇ M CNQX and 50 ⁇ M bicuculline.
  • FIGs.3(A-Q) illustrate Purkinje neuron activation enhances water intake in mice.
  • A Schematic of the experimental strategy using the Cre-dependent AAV expressing hSyn- DIO- hM3Dq-mCherry to activate the Purkinje neurons of the Pcp2-cre mice in lobes V–VI of the cerebellum.
  • B Representative image of recorded Pcp2-cre neurons expressing hM3Dq-mCherry under brightfield and fluorescence microscopy.
  • Figs.4(A-I) illustrate Purkinje neuron-specific Ptprd deletion leads to hypodipsia without affecting food intake.
  • Figs.5(A-O) illustrate Purkinje neuron-specific Ptprd deletion does not affect motor learning or coordination.
  • FIG. 1 Schematic of ErasmusLadder experimental paradigm showing sessions one and two: ‘training’ during which mice are trained to walk across the ErasmusLadder, a horizontal ladder apparatus with pressure-sensitive stepping rungs (blue, default stepping rungs) and lower rungs that capture missteps (gray rungs).
  • mice are challenged with a cerebellum-dependent learning paradigm in which obstacle rungs are presented (red, unconditioned stimulus (US)) during paired trials preceded by a conditioning stimulus (CS) tone separated by an interstimulus interval (ISI) of 250 ms.
  • US unconditioned stimulus
  • CS conditioning stimulus
  • ISI interstimulus interval
  • Figs.6(A-C) illustrate Purkinje neuron-specific Ptprd deletion abolishes water deprivation induced purkinje neuron activation.
  • Figs.7(A-P) illustrate asprosin activates purkinje neurons in vivo, and Purkinje neuron specific-Ptprd deletion renders mice unresponsive to the dipsogenic effects of asprosin.
  • A Representative action potential firing traces of Purkinje neurons from Pcp2-cre (control, WT) and Pcp2-cre; Ptprd flox/flox (KO) mice, treated with recombinant asprosin.
  • D Representative action potential firing traces of Purkinje neurons from Pcp2-cre (control, WT) and Pcp2-cre; Ptprd flox/flox (KO) mice, treated with norepinephrine (NE).
  • G Schematic experimental strategy using the Cre-dependent AAV expressing hSyn-FLEX-GCaMP7f for optical recording of activated Purkinje neurons of the Pcp2-cre (control) and Pcp2-cre; Ptprd flox/flox (KO) mice subjected to i.c.v. or i.v. injection of GFP or recombinant asprosin.
  • FIGs.8(A-G) illustrate AgRP neuron-specific Ptprd deletion does not affect water intake.
  • (B- C) 24h food and water intake of 14-week-old male and 8-week-old female Ptprd f/f and AgRP- cre;Ptprd f/f mice maintained on ad libitum chow diet (n 4/group).
  • (D) Body weight of female Ptprd f/f and AgRP-cre;Ptprd f/f mice on normal chow (week 5), and after 5 weeks of high fat diet (Week 1O; n 9/ group).
  • C Schematic of coronal brain section showing Purkinje neurons recorded in different places.
  • Figs.10(A-L) illustrate chemogenetic activation of purkinje neurons enhances water intake without affecting food intake or body weight.
  • Figs.11(A-J) illustrate specific Ptprd deletion affects water, isotonic and hypertonic saline intake.
  • Figs.13(A-I) illustrate Purkinje neuron-specific Ptprd loss results in increased regularity of Purkinje neuron complex spikes.
  • A Schematic representation of an in vivo awake single-unit recording.
  • (A-C) GCaMP7 fluorescent response of Pcp2cre neurons in response to hypertonic stress (3M NaCl and 2M mannitol) and hypovolemic stress (30% polyethylene glycol; PEG; n 5 wild type mice in each treatment).
  • D-F 2h water intake (D,E) and 48 h water intake (F) of control (Pcp2-cre;Ptprd +/+ ) and knockout (Pcp2-cre;Ptprd flox/flox ) mice injected with 3M NaCl (D), 2M mannitol (E) and 30% PEG (F).
  • Error bars represent mean ⁇ s.e.m. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, and ****p ⁇ 0.0001; by two-tailed unpaired Student’s t-test.
  • Figs.15 illustrates a schematic showing Purkinje Neurons modulate thirst. Asprosin activates the cerebellar Purkinje neurons via the Ptprd receptor, leading to rapid manifestation of water drinking behavior.
  • DETAILED DESCRIPTION [0036] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
  • the term "about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “one or more of a, b, and c” means a, b, c, ab, ac, bc, or abc. The use of “or” herein is the inclusive or.
  • chimeric protein refers to a fusion of a first amino acid sequence encoding a polypeptide with a second amino acid sequence defining a domain (e.g., polypeptide portion) foreign to or heterologous with and not substantially homologous with the domain of the first polypeptide.
  • a chimeric protein may present a foreign domain, which is found (albeit in a different protein) in an organism, which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • expression refers to the process by which nucleic acid is translated into peptides or is transcribed into RNA, which, for example, can be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or is selected, include splicing of the mRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.
  • the term "genetic therapy” and grammatical variants thereof involves the transfer of heterologous DNA to cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought.
  • the DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced.
  • the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product; it may encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product.
  • Genetic therapy may also be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced.
  • the heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
  • the term "gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • heterologous nucleic acid sequence is typically DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
  • a heterologous nucleic acid sequence may also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA.
  • heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, and DNA that encodes other types of proteins, such as antibodies.
  • Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.
  • parenteral and parenterally means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into a target tissue, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • administered systemically means the administration of a compound, drug or other material other than directly into a target tissue, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • polynucleotide sequence means the administration of a compound, drug or other material other than directly into a target tissue, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • polynucleotide sequence means the administration of a compound, drug or other material other than directly into a target tissue, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • polynucleotide sequence means the administration of a compound, drug or other material
  • peptide or “polypeptide” are used interchangeably herein and refer to compounds consisting of from about 2 to about 90 amino acid residues, inclusive, wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond.
  • a peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (see Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989)).
  • a "peptide” can comprise any suitable L-and/or D-amino acid, for example, common a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g., P- alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
  • the amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group.
  • Suitable protecting groups for amino and carboxyl groups and means for adding or removing protecting groups are known in the art. See, e.g., Green & Wuts, PROTECTING GROUPS IN ORGANIC SYNTHESIS (John Wiley & Sons, 1991).
  • the functional groups of a peptide can also be derivatized (e.g., alkylated) using art-known methods.
  • Peptides can be synthesized and assembled into libraries comprising a few too many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be as described herein or using other suitable methods to determine if the library comprises peptides which can sequester asprosin.
  • peptides can then be isolated by suitable means.
  • portion when referring to a polypeptide include any polypeptide that retains at least some biological activity referred to herein (e.g., inhibition of an interaction such as binding).
  • Polypeptides as described herein may include portion, fragment, variant, or derivative molecules without limitation, as long as the polypeptide still serves its function.
  • Polypeptides or portions thereof of the present invention may include proteolytic fragments, deletion fragments and in particular, or fragments that more easily reach the site of action when delivered to an animal.
  • a "portion" or “fragment” polypeptide (including a domain) will be understood to mean a polypeptide of reduced length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residue(s) (e.g., peptide(s)) relative to a reference polypeptide, respectively, and comprising, consisting essentially of and/or consisting of a polypeptide of contiguous residues, respectively, identical or almost identical (e.g., 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% identical) to the reference polypeptide.
  • residue(s) e.g., peptide(s)
  • residue(s) e.g.
  • homologues Different nucleic acids or proteins having homology are referred to herein as "homologues.”
  • the term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • homologue refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • compositions and methods described herein further comprise homologues to the nucleotide sequences of this invention.
  • Orthologous and “orthologs” as used herein refers to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation.
  • a homologue or ortholog of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 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%, 99.5% or 100%) to the nucleotide sequence described herein.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence as compared to a reference polypeptide.
  • the nucleotide sequences can be substantially identical over at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides).
  • a substantially identical nucleotide or protein sequence performs substantially the same function as the nucleotide (or encoded protein sequence) to which it is substantially identical.
  • a polynucleotide and/or recombinant nucleic acid construct described herein can be codon optimized for expression.
  • a polynucleotide, nucleic acid construct, expression cassette, and/or vector described herein may be codon optimized for expression in an organism (e.g., an animal, a plant, a fungus, an archaeon, or a bacterium).
  • the codon optimized nucleic acid constructs, polynucleotides, expression cassettes, and/or vectors of the invention have about 70% to about 99.9% (e.g., 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%, 99.5%.99.9% or 100%) identity or more to the reference nucleic acid constructs, polynucleotides, expression cassettes, and/or vectors but which have not been codon optimized.
  • a polynucleotide or nucleic acid construct described herein may be operatively associated with a variety of promoters and/or other regulatory elements for expression in an organism or cell thereof.
  • a polynucleotide or nucleic acid construct described herein may further comprise one or more promoters, introns, enhancers, and/or terminators operably linked to one or more nucleotide sequences.
  • a promoter may be operably associated with an intron.
  • a promoter associated with an intron maybe referred to as a "promoter region".
  • a polynucleotide sequence (DNA, RNA) is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
  • the term "operatively linked” includes having an appropriate start signal ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.
  • the term "linked,” or “fused” in reference to polypeptides refers to the attachment of one polypeptide to another.
  • a polypeptide may be linked or fused to another polypeptide (at the N-terminus or the C-terminus) directly (e.g., via a peptide bond) or through a linker (e.g., a peptide linker).
  • linker in reference to polypeptides is art-recognized and refers to a chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion polypeptide protein.
  • a linker may be comprised of a single linking molecule (e.g., a single amino acid) or may comprise more than one linking molecule.
  • the linker can be an organic molecule, group, polymer, or chemical moiety such as a bivalent organic moiety.
  • the linker may be an amino acid or it may be a peptide.
  • the linker is a peptide.
  • a "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operably associated with the promoter.
  • the coding sequence controlled or regulated by a promoter may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue- specific promoters for use in the preparation of recombinant nucleic acid molecules, e.g., "synthetic nucleic acid constructs" or "protein-RNA complex.” These various types of promoters are known in the art.
  • the term "therapeutically effective” means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes, symptoms, or sequelae of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination, of the causes, symptoms, or sequelae of a disease or disorder.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Preferred vectors are those capable of one or more of, autonomous replication and expression of nucleic acids to which they are linked.
  • expression vectors Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • wild type or “WT” refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo.
  • nucleic acid refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • the agents, compounds, polypeptides, proteins, etc. used in the methods described herein are considered to be purified and/or isolated prior to their use. Purified materials are typically "substantially pure", meaning that a nucleic acid, polypeptide or fragment thereof, or other molecule has been separated from the components that naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and other organic molecules with which it is associated naturally.
  • a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
  • isolated materials have been removed from their natural location and environment. In the case of an isolated or purified domain or protein fragment, the domain or fragment is substantially free from amino acid sequences that flank the protein in the naturally-occurring sequence.
  • isolated DNA means DNA has been substantially freed of the genes that flank the given DNA in the naturally occurring genome.
  • isolated DNA encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.
  • administering to the subject means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject to relieve, cure or reduce the symptoms associated with a disease, disorder or condition, e.g., a pathological condition. Oral administration is one way of administering the instant compounds to the subject.
  • subject and patient are used interchangeably to refer to a human.
  • the terms “pediatric subject” or “pediatric patient” are used interchangeably to refer to a human less than 18 years of age.
  • an “adult patient” refers to a human 18 years of age or older.
  • An “adolescent patient” or “adolescent subject” is a subject typically about 12 to 18, such as 12 to 17 or 13 to 18, years old.
  • Embodiments described herein relate to a method of regulating internal water balance and/or modulating thirst in a subject in need thereof, and particularly relates to a method of treating thirst disorders, such as polydipsia or excessive thirst, and adipsia or absence of thirst.
  • Figs.1(O, P) and 16-17 we further found healthy wild-type (WT) mice injected with the anti-asprosin monoclonal antibody (mAb) experienced notable reduction in 24-hour water intake, alongside decreased urine output.
  • WT wild-type mice
  • mAb monoclonal antibody
  • Administration of an anti-asprosin mAb effectively treated a mouse model of neuropsychiatric disease that manifests polydipsia, with prolonged efficacy from a single dose.
  • modulating asprosin-PTPR ⁇ -cerebellum signaling and/or activity can be used as a method or therapy for treating thirst disorders, such as polydipsia, adipsia, or hypodipsia in a subject in need thereof, and particularly thirst disorders associated with mental ailments.
  • modulating asprosin mediated PTPR ⁇ signaling and/or activity can include inhibiting or decreasing asprosin mediated PTPR ⁇ signaling and/or activity to decrease excessive thirst or fluid intake and treat polydipsia in a subject in thereof.
  • Asprosin mediated signaling and/or activity of PTPR ⁇ can be decreased or inhibited in several ways including: direct inhibition of asprosin binding to or activation of the PTPR ⁇ (e.g., by using small molecules, antibodies, and/or peptide antagonists); activation of genes and/or proteins that decrease or inhibit one or more of, asprosin mediated activity and/or signaling of the PTPR ⁇ (e.g., by decreasing the expression or activity of the genes and/or proteins); promotion of genes and/or proteins that are downstream mediators of the PTPR ⁇ activity (e.g., by decreasing the expression and/or activity of the mediator genes and/or proteins); introduction of genes and/or proteins that negatively regulate one or more of activity and/or signaling of PTPR ⁇ (e.g., by using recombinant gene expression vectors, recombinant viral vectors or recombinant polypeptides); or gene replacement with, for instance, a hypomorphic mutant of the PTPR ⁇ (e.g.,
  • the agent, which inhibits asprosin mediated PTPR ⁇ signaling or activity can include, for example, at least one of a small molecule, nucleic acid, peptide, protein, or antibody that inhibits asprosin binding to PTPR ⁇ or PTPR ⁇ signaling and/or activity.
  • the agent that inhibits asprosin mediated PTPR ⁇ signaling and/or activity can include an antibody or antigen binding fragment thereof that specifically binds to asprosin or PTPR ⁇ .
  • the antibody or antigen binding fragment thereof can be any immunologic binding agent, such as IgG, IgM, IgA, IgD and IgE.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily in a laboratory setting.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single domain antibodies
  • scFv single chain Fv
  • the techniques for preparing and using various antibody- based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • Antibodies of the disclosure may specifically bind their target.
  • the phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologics.
  • a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample.
  • Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • the agent which inhibits asprosin mediated PTPR ⁇ signaling and/or activity, can be an anti-asprosin antibody, such as an anti-asprosin monoclonal antibody (mAb).
  • mAb anti-asprosin monoclonal antibody
  • Anti-asprosin mAbs may be generated and employed as inhibitors of asprosin for the use in an individual.
  • the anti-asprosin mAbs are used in methods of treating polydipsia.
  • the immunogen for the monoclonal antibodies may be the entire asprosin polypeptide or may be a fragment thereof.
  • An example sequence for generating a monoclonal antibody to asprosin is HuFbn12838:2865 KKKELNQLEDKYDKDYLSGELGDNLKMK (SEQ ID NO:1).
  • the ant-asprosin antibody binds an epitope on the amino acid sequence of SEQ ID NO:1.
  • the epitope may be all of the amino acid sequence of SEQ ID NO:1 or it may be a fragment of SEQ ID NO:1.
  • the epitope is a continuous sequence of amino acids, although in some cases the epitope binds a three- dimensional configuration of amino acid that may or may not be continuous in form. In some cases, the epitope is between 5 and 20, 5 and 15, 5 and 10, 8 and 20, 8 and 15, 8 and 10, 10 and 20, or 10 and 15 amino acids in length.
  • the epitope may comprise, consist of, or consist essentially of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acids of SEQ ID NO: 1, and in some embodiments the amino acids are continuous in SEQ ID NO:1 whereas in other cases they are not continuous in SEQ ID NO:1.
  • the ant-asprosin antibody is an isolated antibody or antigen-binding portion that specifically binds a peptide comprising, consisting essentially of, or consisting of SEQ ID NO:1.
  • an isolated anti-asprosin antibody or antigen- binding portion that specifically binds a peptide of SEQ ID NO: 1 can be produced by the hybridoma cell line deposited with the American Type Culture Collection under accession number ATCC PTA-123085.
  • the anti-asprosin antibody or antigen binding fragment thereof comprises the same CDRs of the heavy and light chain polypeptide sequences as an antibody produced by a hybridoma having deposit accession number ATCC PTA-123085.
  • the anti-asprosin antibody or antigen binding fragment thereof can include a heavy chain variable region and/or light variable region that includes three heavy chain CDRs and/or three light chain CDR of an antibody hybridoma having deposit accession number ATCC PTA-123085.
  • the disclosure also encompasses one or more isolated cells of a hybridoma having deposit accession number ATCC PTA-123085 and also the hybridoma cell line having deposit accession number ATCC PTA-123085.
  • Antibodies produced by any cell lines of the disclosure are encompassed herein. Specific embodiments include isolated and purified monoclonal antibodies produced by the continuous hybridoma cell line having deposit accession number PTA-123085.
  • Monoclonal antibodies may be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the anti-asprosin monoclonal antibodies may be made using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975), or may be made by recombinant DNA methods [Cabilly, et al., U.S. Pat. No.4,816,567].
  • a mouse or other host animal such as a hamster is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)].
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against asprosin.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem.107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104 (Academic Press, 1986). Examples of culture media for this purpose include Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells can serve as a source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci.81, 6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • chimeric or “hybrid” antibodies are prepared that have the binding specificity of an anti-asprosin monoclonal antibody herein.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for asprosin and another antigen-combining site having specificity for a different antigen.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • antibodies against asprosin are humanized.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non- human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332, 323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human Accordingly, such “humanized” antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Human monoclonal antibodies can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J.
  • transgenic animals e.g., mice
  • J H antibody heavy chain joining region
  • antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
  • the antibody to asprosin can be a bispecific antibody.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for asprosin, the other one is for any other antigen, and preferably for another receptor or receptor subunit.
  • bispecific antibodies specifically binding asprosin and an asprosin receptor or two different asprosin receptors are within the scope of the present invention.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions.
  • the first heavy chain constant region (CH1) containing the site necessary for light chain binding in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.
  • This approach is disclosed in co-pending application Ser. No.07/931,811 filed Aug.17, 1992. [0102]
  • For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121, 210 (1986).
  • the agent which inhibits asprosin mediated PTPR ⁇ signaling and/or activity, can include a peptide that is a peptide mimetic of the asprosin ligand binding domain of PTPR ⁇ (i.e., PTPR ⁇ -lbd).
  • the peptide or peptide mimetic of the asprosin ligand binding domain of PTPR ⁇ can have an amino acid sequence substantially identical to an extracellular portion of the amino acid sequence of PTPR ⁇ that binds to asprosin.
  • Peptide mimetics of the PTPR ⁇ -lbd when ectopically introduced into the circulation of a subject in need thereof inhibit asprosin induced or mediated PTPR ⁇ signaling in neural cells resulting in a corresponding decrease in excessive thirst.
  • the peptide mimetic or peptide can bind to and sequester asprosin in the circulation of the subject.
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 300,
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to about 10 to about 1240, about 20 to about 1220, about 30 to about 1210, about 40 to about 1200, about 50 to about 1190, about 60 to about 1180, about 70 to about 1170, about 80 to about 1160, about 90
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 2.
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200 consecutive amino acids
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200 consecutive
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80 consecutive amino acids of SEQ ID NO: 4.
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 consecutive amino acids of SEQ ID NO: 5.
  • the peptide can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 consecutive amino acids of SEQ ID NO: 6.
  • the peptide has a binding affinity K D to asprosin less than about 10 ⁇ M, less than about 1 ⁇ M, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 10 nM, less about 1 nM, or less than about 500 pM.
  • the peptides described herein can be subject to other various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
  • peptides that have an amino acid sequence substantially identical to an extracellular portion of the amino acid sequence of PTPR ⁇ that binds to asprosin can correspond to or be substantially homologous with, rather than be identical to, the sequence of a recited polypeptide where one or more changes are made and it retains the ability to inhibit or reduce one or more of the activity, signaling, and/or function of asprosin mediated polydipsia.
  • the peptide can be in any of a variety of forms of polypeptide derivatives that include amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides and the like derivatives.
  • the peptide can also include substitutions of amino acid residues. It will be appreciated that the conservative substitution can also include the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite binding activity.
  • "Chemical derivative” refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those polypeptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
  • Polypeptides described herein may also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.
  • One or more of peptides of the peptides described herein can also be modified by natural processes, such as posttranslational processing, and/or by chemical modification techniques, which are known in the art.
  • Modifications may occur in the peptide including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
  • Modifications comprise for example, without limitation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, amidation, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, hydroxylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination (for reference
  • Peptides and/or proteins described herein may also include, for example, biologically active mutants, variants, fragments, chimeras, and analogues. Fragments encompass amino acid sequences having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus (C- terminus), or from the interior of the protein. Analogues of the invention involve an insertion or a substitution of one or more amino acids. Variants, mutants, fragments, chimeras and analogues may function as inhibitors of asprosin mediated polydipsia (without being restricted to the present examples).
  • the polypeptides described herein may be prepared by methods known to those skilled in the art.
  • the peptides and/or proteins may be prepared using recombinant DNA.
  • one preparation can include cultivating a host cell (bacterial or eukaryotic) under conditions, which provide for the expression of peptides and/or proteins within the cell.
  • the purification of the polypeptides may be done by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity or other purification technique typically used for protein purification.
  • the purification step can be performed under non-denaturating conditions.
  • the protein may be renatured using techniques known in the art.
  • the peptide is an exogenous peptide that can be recombinantly produced and systemically administered to the subject by, for example, parenteral or intravenous administration.
  • the peptide includes at least one heterologous or foreign moiety, such as a heterologous or foreign polypeptide.
  • the at least one heterologous polypeptide can include, for example, an antibody or antigen binding fragment thereof, a glucagon-like peptide-1 receptor (GLP-1R) agonist, a Fc portion of an immunoglobulin, an albumin peptide, an albumin binding domain (ABD), a signal peptide, or a combination thereof.
  • GLP-1R glucagon-like peptide-1 receptor
  • the moiety is fused to the N-terminus or C- terminus of the peptide.
  • the heterologous moiety is inserted between two amino acids within the peptide.
  • the peptide can further comprise two, three, four, five, six, seven, or eight heterologous sequences. In some embodiments, all the heterologous moieties are identical. In some embodiments, at least one heterologous moiety is different from the other heterologous moieties. In some embodiments, the disclosure can comprise two, three, four, five, six, or more than seven heterologous moieties in tandem.
  • the heterologous moiety increases the half-life (is a "half- life extender") of the peptide.
  • the heterologous moiety is a peptide or a polypeptide with either unstructured or structured characteristics that are associated with the prolongation of in vivo half-life when incorporated in the peptide.
  • Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, the C-terminal peptide (CTP) of the ⁇ subunit of human chorionic gonadotropin, a HAP sequence, an XTEN sequence, a transferrin or a fragment thereof, a PAS polypeptide, polyglycine linkers, polyserine linkers, albumin-binding moieties, or any fragments, derivatives, variants, or combinations of these polypeptides.
  • the heterologous polypeptide includes an immunoglobulin constant region or a portion thereof, transferrin, albumin, or a PAS sequence.
  • a heterologous moiety includes von Willebrand factor or a fragment thereof.
  • a heterologous polypeptide can include an attachment site (e.g., a cysteine amino acid) for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these elements.
  • a heterologous moiety comprises a cysteine amino acid that functions as an attachment site for a non-polypeptide moiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these elements.
  • a first heterologous polypeptide is a half-life extending molecule which is known in the art
  • a second heterologous moiety is a half-life extending molecule which is known in the art.
  • the first heterologous polypeptide e.g., a first Fc polypeptide
  • the second heterologous polypeptide e.g., a second Fc polypeptide
  • the second heterologous polypeptide is a second Fc polypeptide, wherein the second Fc polypeptide is linked to or associated with the first heterologous polypeptide, e.g., the first Fc polypeptide.
  • the second heterologous polypeptide e.g., the second Fc polypeptide
  • the first heterologous moiety e.g., the first Fc polypeptide
  • a linker or associated with the first heterologous moiety by a covalent or non-covalent bond.
  • the heterologous polypeptide comprises, consists essentially of, or consists of at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2500, at least about 3000, or at least about 4000 amino acids.
  • the heterologous polypeptide comprises, consists essentially of, or consists of about 100 to about 200 amino acids, about 200 to about 300 amino acids, about 300 to about 400 amino acids, about 400 to about 500 amino acids, about 500 to about 600 amino acids, about 600 to about 700 amino acids, about 700 to about 800 amino acids, about 800 to about 900 amino acids, or about 900 to about 1000 amino acids.
  • a heterologous polypeptide improves one or more pharmacokinetic properties of the peptide without significantly affecting its biological activity or function.
  • a heterologous polypeptide increases the in vivo and/or in vitro half-life of the peptide.
  • a heterologous polypeptide facilitates visualization or localization of the peptide. Visualization and/or location of the peptide can be in vivo, in vitro, ex vivo, or combinations thereof.
  • a heterologous polypeptide increases stability of the peptide.
  • stability refers to an art-recognized measure of the maintenance of one or more physical properties of the peptide in response to an environmental condition (e.g., an elevated or lowered temperature).
  • the physical property can be the maintenance of the covalent structure of the peptide (e.g., the absence of proteolytic cleavage, unwanted oxidation or deamidation).
  • the physical property can also be the presence of the peptide in a properly folded state (e.g., the absence of soluble or insoluble aggregates .
  • the stability of the peptide is measured by assaying a biophysical property of the peptide, for example thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g., ability to bind to a protein, receptor or ligand), etc., and/or combinations thereof.
  • biochemical function is demonstrated by the binding affinity of the interaction.
  • a measure of protein stability is thermal stability, i.e., resistance to thermal challenge.
  • the heterologous moiety or polypeptide can include an Fc portion of an immunoglobulin.
  • the Fc portion of an immunoglobulin can be linked to the peptide to form a fusion or chimeric polypeptide or protein.
  • Fusion or chimeric polypeptides or proteins that can combine the Fc regions of IgG with one or more domains of another protein, such as various cytokines and soluble receptors, are known. These chimeric proteins can be fusions of human Fc regions and human domains of another protein. These chimeric proteins would then be a "humanized Fc chimera", which would be advantageous as a human therapeutic. (See, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, (1996); U.S. Pat. Nos.5,116,964 and 5,541,087).
  • the fusion polypeptide can be a homodimeric protein linked through cysteine residues in a hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule without the CH1 domains and light chains. Due to the structural homology, such Fc fusion proteins exhibit in vivo pharmacokinetic profile comparable to that of human IgG with a similar isotype.
  • This approach has been applied to several therapeutically important cytokines, such as IL-2 and IFN- ⁇ , and soluble receptors, such as TNF-Rc and IL-5-Rc (See, for example, U.S. Pat. Nos. 5,349,053, 6,224,867 and 7,250,493).
  • the peptide-Fc fusion polypeptide or chimera is a chimeric molecule that includes a human sequence encoded extracellular portion of Ptprd fused to a human Fc fragment.
  • the heterologous polypeptide can include a glucagon-like peptide 1 receptor (GLP-1R) agonist that is linked to the peptide to form a fusion polypeptide or protein.
  • GLP-1R agonist can include a which binds to and activates the GLP-1 receptor like GLP-1 (glucagon-like peptide 1).
  • Physiological actions of GLP-1 and/or of the GLP-1R agonist are described e.g., in Nauck, M. A.
  • Human GLP-1(7-37) possesses the amino acid sequence of SEQ ID NO: 7.
  • Human GLP-1(7-36)amide possesses the amino acid sequence of SEQ ID NO: 8.
  • peptides with GLP-1R agonistic activity are disclosed in US 2006/0003417, US 2019/0085043, and small organic compounds with GLP-1R agonistic activity are disclosed in Chen et al.2007, PNAS, 104, 943-948, No.3 or Knudsen et al., 2007, PNAS, 104, 937-942.
  • the peptide can linked directly to heterologous peptide or indirectly to the heterologous polypeptide with a linker.
  • the linker can include a structural unit that is inserted in between two or more other units (e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide) and couple these two or more other units with each other to create one molecule.
  • the coupling of the two units is preferably by covalent bond(s).
  • the linker as used herein also refers to a structural unit that can be attached to the N- or C-terminus of two or more other units (e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide), wherein said two or more other units are directly coupled together.
  • two or more other units e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide
  • the linker as used herein also refers to combinations of the preceding definitions, i.e., one structural unit is inserted in between the two or more other units (e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide) and one or more further structural units is/are attached to the N- or C-terminus of two or more other units (e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide).
  • one structural unit is inserted in between the two or more other units (e.g., two or more peptides or polypeptides or proteins or a peptide and a protein a polypeptide and a protein, a peptide and a polypeptide) and one or more further structural units is/are attached to the N- or C
  • the linker can include, for example, additional residues that may be added at either terminus of an peptide for the purpose of conveniently linking other the polypeptides, proteins or other such as detectable moieties, labels, solid matrices, or carriers.
  • Amino acid residue linkers are usually at least one residue and can be 2 or more residues, more often about 2 to about 1000 or more amino acids in length, for example, about 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 100 or more amino acids in length.
  • the linker can be a flexible peptide linker that links the peptide to other polypeptides, proteins, and/or molecules, such as detectable moieties, labels, solid matrices, or carriers.
  • a flexible peptide linker can be about 20 or fewer amino acids in length.
  • a peptide linker can contain about 12 or fewer amino acid residues, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
  • Typical amino acid residues used for linking are glycine, serine, tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
  • a peptide linker comprises two or more of the following amino acids: glycine, serine, alanine, and threonine.
  • the peptide linker can include a functional moiety conferring one or more additional functions beyond that of linking the peptide and the heterologous moiety or polypeptide.
  • the linker can be added for improved or independent folding of one or both of the polypeptides forming the fusion polypeptide or protein and/or for avoiding sterical hindrance and/or for introducing further desired functionalities, e.g., entry sites for covalent attachment of additional moieties, tags for protein purification, protease cleavage sites, protein stabilization and/or half-life extension of the protein.
  • Linkers can include between 0, 1 to 1000 amino acids.
  • the linker can also be absent (i.e., 0 amino acids).
  • Typical linker types can e.g., be helical or non-helical, wherein helical linkers are thought to act as rigid spacers separating two domains and non-helical linkers contain proline or are rich in proline, which also leads to structural rigidity and isolation of the linker from the attached domains. This means that both linker types are likely to act as a scaffold to prevent unfavorable interactions between folding domains.
  • the be a moiety conferring increased stability and/or half-life to the fusion polypeptide such as a) an XTENylation, rPEG or PASylation or HESylation sequence or Elastin-like polypeptides (ELPs); b) an entry site for covalent modification of the fusion protein, such as a cysteine or lysine residue; c) a moiety with intra- or extracellular targeting function, such as a protein-binding scaffold (such as an antibody, antigen-binding fragment, or other proteinaceous non-antibody binding scaffold), a nucleic acid (such as an aptamer, PNA, DNA or the like); d) a protease cleavage site such as a Factor Xa cleavage site or a cleavage site for another (preferably extracellular) protease; or e) an albumin binding domain (ABD); or f) an amino acid sequence comprising one or more histidine
  • the peptide or fusion polypeptide may be modified to include a signal peptide that promotes secretion of the expressed peptide or fusion polypeptide from a cell.
  • the characteristics of the signal peptides are well known in the art, and the signal peptides conventionally having 16 to 30 amino acids, but they may include more or less number of amino acid residues.
  • Conventional signal peptides consist of three regions of the basic N-terminal region, a central hydrophobic region, and a more polar C- terminal region.
  • the signal peptide can include, for example, an IL2 or IL15 signal peptide.
  • modulating asprosin mediated PTPR ⁇ signaling and/or activity can include enhancing or increasing asprosin mediated PTPR ⁇ signaling and/or activity to increase thirst and/or fluid intake and treat adipsia or hypodipsia in a subject in thereof.
  • Asprosin mediated signaling and/or activity of PTPR ⁇ can be enhanced, increased and/or promoted in several ways including: direct enhancement of asprosin mediated activity of the PTPR ⁇ (e.g., by using asprosin and/or peptide agonists); activation of genes and/or proteins that enhance, increase, and/or promote one or more of, the activity, signaling, and/or function of asprosin or PTPR ⁇ (e.g., by increasing the expression or activity of the genes and/or proteins); promotion of genes and/or proteins that are downstream mediators of the PTPR ⁇ activity (e.g., by enhancing the expression and/or activity of the mediator genes and/or proteins); introduction of genes and/or proteins that positively regulate one or more of, activity, signaling, and/or function of PTPR ⁇ (e.g., by using recombinant gene expression vectors, recombinant viral vectors or recombinant polypeptides); or gene replacement with, for instance, a hypermorphic mutant
  • a agent that enhances, increases, and/or promotes one or more of the activity and/or signaling of the PTPR ⁇ can include a therapeutic peptide or small molecule that binds to and/or complexes PTPR ⁇ to enhance the activity, signaling, and/or function of PTPR ⁇ .
  • the PTPR ⁇ agonist is a therapeutic polypeptide or protein that when introduced into the circulation of a subject in need thereof can bind and/or complex to the membrane bound PTPR ⁇ to induce PTPR ⁇ activity and/or signaling in neural cells, such as Purkinje neurons.
  • the PTPR ⁇ agonist can include asprosin and/or an analogue thereof.
  • Asprosin is encoded by FBN1 gene and belongs to a post-translationally modified product of fibrillin. Asprosin is released as the C-terminal propeptide (aa2732- 2871) from profibrillin-1 and is cleaved by pro-proteinase furin (Jensen et al., 2014). In some embodiments, asprosin can have an amino acid sequence consisting of SEQ ID NO: 9.
  • the asprosin and/or an analogue thereof can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100,
  • the asprosin and/or an analogue thereof can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to about 10 to about 140, about 20 to about 120, about 30 to about 110, to about 100, about 50 to about 90, about 60 to about 80, consecutive amino acids of SEQ ID NO: 9.
  • the asprosin and/or an analogue thereof can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 9.
  • the asprosin and/or an analogue thereof can have an amino acid sequence at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100
  • the asprosin and/or an analogue thereof has a binding affinity K D to PTPR ⁇ less than about 10 ⁇ M, less than about 1 ⁇ M, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 10 nM, less about 1 nM, or less than about 500 pM.
  • the asprosin and/or an analogue thereof described herein can be subject to other various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
  • asprosin and/or an analogue thereof that have an amino acid sequence substantially identical to asprosin and/or an analogue thereof that binds to PTPR ⁇ can correspond to or be substantially homologous with, rather than be identical to, the sequence of a recited polypeptide where one or more changes are made and it retains the ability to increase or promote one or more of activity, signaling, and/or function of PTPR ⁇ .
  • the asprosin and/or an can be in any of a variety of forms of polypeptide derivatives that include amides, conjugates with proteins, cyclized polypeptides, polymerized polypeptides, analogs, fragments, chemically modified polypeptides and the like derivatives.
  • the peptides or proteins that modulate (e.g., inhibit or promote) asprosin mediated PTPR ⁇ signaling and/or activity can be expressed from a cell, in vivo or ex vivo, using a vector that includes a nucleic acid encoding the peptide or protein.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy.
  • Vectors include, for example, viral vectors (such as adenoviruses (Ad), adeno- associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a target cell.
  • viral vectors such as adenoviruses (Ad), adeno- associated viruses (AAV), and retroviruses
  • liposomes and other lipid-containing complexes such as adenoviruses (Ad), adeno- associated viruses (AAV), and retroviruses
  • liposomes and other lipid-containing complexes such as liposomes and other lipid-containing complexes
  • other macromolecular complexes capable of mediating delivery of a polynucleotide to a target cell.
  • the vector can include an expression cassette.
  • the expression cassette can include a nucleic acid molecule which comprises the peptide or fusion polypeptide coding sequences (e.g., coding sequences for the peptide, heterologous polypeptide, and optional linker), promoter, and may include other regulatory sequences therefor, which cassette may be engineered into a genetic element and/or packaged into the capsid of a viral vector (e.g., a viral particle).
  • a viral vector e.g., a viral particle
  • such an expression cassette for generating a viral vector contains the peptide and heterologous polypeptide sequences described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • the expression cassette typically contains a promoter sequence as part of the expression control sequences.
  • the promoter can include a liver-specific promoter thyroxin binding globulin (TBG).
  • TBG liver-specific promoter thyroxin binding globulin
  • vectors described herein can include a CB7 promoter.
  • CB7 is a chicken ⁇ -actin promoter with cytomegalovirus enhancer elements.
  • other liver-specific promoters may be used.
  • TTR minimal enhancer/promoter alpha-antitrypsin promoter, LSP (845 nt)25 (requires intron-less scAAV).
  • promoters such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO 2011/126808 and WO 2013/04943), or a promoter responsive to physiologic cues may be used in the vectors described herein.
  • an cassette and/or a vector may contain other appropriate control sequences, such as transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA.
  • enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulin promoter/alpha1-microglobulin/bikunin enhancer), amongst others. These control sequences can be operably linked to the peptide sequences.
  • the nucleic acid encoding the therapeutic peptides or protein may be modified to include a signal sequence that promotes secretion of the expressed therapeutic peptide from a cell.
  • the nucleic acid can include cDNA encoding the extracellular domain of PTPR ⁇ and IL2 signal sequence. A number of such modifications are known in the art and can be applied by the skilled practitioner.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide (such as one or more transcriptional regulatory sequences).
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated.
  • a variety of such marker genes have been described, including positive/negative) markers (see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton, S., WO 94/28143, published Dec.8, 1994).
  • Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts.
  • the vector can include an adeno-associated virus (AAV) viral vector.
  • AAV viral vector is an AAV DNase-resistant particle having an AAV protein capsid into which is packaged nucleic acid sequences for delivery to target cells.
  • An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV.
  • AAV serotypes may be selected as sources for capsids of AAV viral vectors (DNase resistant viral particles) including, e.g., AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh74, AAVrh10, AAV5, AAV7, AAVS3, AAVHSC, AAV2.7m8, AAV-LK03, AAV8/Olig001, AAV2i8, AAVhu37, AAV2tYF, AAVh1, AAVhu68, AAVrh.8, AAVrh9, AAV.PHP.B., AAV.PHP.eB, AAV.PHP.S, AAV/BBB, AAV-DJ, AAVr3.45, AAV-sh10, AAV2(Y444F), AAV4, AAV-RPF2, AAV3b, AAVrh64R1, or variants of any of the known or mentioned AAVs or AAVs yet to be discovered.
  • AAV viral vectors DNase resistant viral particles
  • an AAV cap for use in the viral vector can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of the aforementioned AAV Caps or its encoding nucleic acid.
  • the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins.
  • the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomers from two or three different AAVs or recombinant AAVs.
  • an rAAV composition comprises more than one of the aforementioned Caps.
  • the ITRs are the only AAV components required in cis in the same construct as the gene.
  • the coding sequences for the replication (rep) and/or capsid (cap) are removed from the AAV genome and supplied in trans or by a packaging cell line in order to generate the AAV vector.
  • a pseudotyped AAV may contain ITRs from a source which differs from the source of the AAV capsid.
  • a chimeric AAV capsid may be utilized. Still other AAV components may be selected.
  • AAV sequences are described herein and may also be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. [0166] Methods for generating and isolating AAV viral vectors that can be used for delivery to a subject are known in the art. See, e.g., U.S. Pat. No.7,790,449; U.S. Pat.
  • a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap.
  • a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs.
  • AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
  • helper functions i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase
  • helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
  • the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors.
  • baculovirus-based vectors For reviews on these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos.
  • the peptides or proteins described herein may be expressed via viral vectors other than rAAV.
  • viral vectors other viral vectors that can be used herein include herpes simplex virus (HSV)-based vectors.
  • HSV vectors deleted of one or more immediate early genes (IE) are advantageous because they are generally non-cytotoxic, persist in a state similar to latency in the target cell, and afford efficient target cell transduction.
  • Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid.
  • Retroviruses such as C-type retroviruses and lentiviruses, might also be used in the application.
  • retroviral vectors may be based on murine leukemia virus (MLV).
  • MMV murine leukemia virus
  • MLV-based vectors may contain up to 8 kb of heterologous (therapeutic) DNA in place of the viral genes.
  • the heterologous DNA may include a tissue-specific promoter and a nucleic acid encoding the peptide. In methods of delivery to neural cells, it may also encode a ligand to a tissue specific receptor.
  • Additional retroviral vectors that might be used are replication-defective lentivirus-based vectors, including human immunodeficiency (HIV)-based vectors.
  • Lentiviral vectors are advantageous they are capable of infecting both actively dividing and non-dividing cells.
  • Lentiviral vectors for use in the application may be derived from human and non-human (including SIV) lentiviruses.
  • lentiviral vectors include nucleic acid sequences required for vector propagation as well as a tissue-specific promoter operably linked to a peptide encoding nucleic acid.
  • a lentiviral vector can be employed. Lentiviruses have proven capable of transducing different types of CNS neurons (Azzouz et al., (2002) J Neurosci.22: 10302-12) and may be used in some embodiments because of their large cloning capacity. [0172] A lentiviral vector may be packaged into any lentiviral capsid. The substitution of one particle protein with another from a different virus is referred to as “pseudotyping”.
  • the vector capsid may contain viral envelope proteins from other viruses, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV).
  • MMV murine leukemia virus
  • VSV vesicular stomatitis virus
  • Alphavirus-based vectors such as those made from semliki forest virus (SFV) and Sindbis virus (SIN) might also be used in the application. Use of alphaviruses is described in Lundstrom, K., Intervirology 43:247-257, 2000 and Perri et al., Journal of Virology 74:9802-9807, 2000.
  • Recombinant, replication-defective alphavirus vectors are advantageous because they are capable of high-level heterologous (therapeutic) gene expression, and can infect a wide target cell range.
  • Alphavirus replicons may be targeted to specific cell types by displaying on their virion surface a functional heterologous ligand or binding domain that would allow selective binding to target cells expressing a cognate binding partner.
  • Alphavirus replicons may establish latency, and therefore long-term heterologous nucleic acid expression in a target cell. The replicons may also exhibit transient heterologous nucleic acid expression in the target cell.
  • hybrid viral vectors may be used to deliver a nucleic acid encoding a peptide to a target neuron, cell, or tissue. Standard techniques for the construction of hybrid vectors are well-known to those skilled in the art. Such techniques can be found, for example, in Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y.
  • Double-stranded AAV genomes in adenoviral capsids containing a combination of AAV and adenoviral ITRs may be used to transduce cells.
  • an AAV vector may be placed into a “gutless”, “helper-dependent” or “high-capacity” adenoviral vector.
  • Adenovirus/AAV hybrid vectors are discussed in Lieber et al., J. Virol.73:9314-9324, 1999. Retrovirus/adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186, 2000.
  • Retroviral genomes contained within an adenovirus may integrate within the target cell genome and effect stable gene expression.
  • Other nucleotide sequence elements, which facilitate expression of the peptide and cloning of the vector are further contemplated.
  • the presence of enhancers upstream of the promoter or terminators downstream of the coding region can facilitate expression.
  • a tissue-specific promoter can be fused to nucleotides encoding the peptides described herein. By fusing such tissue specific promoter within the adenoviral construct, transgene expression is limited to a particular tissue.
  • tissue specific promoters can be determined, using the recombinant adenoviral system.
  • non-viral methods may also be used to introduce a nucleic acid encoding a peptide into a target cell.
  • a review of non-viral methods of gene delivery is provided in Nishikawa and Huang, Human Gene Ther.12:861- 870, 2001.
  • An example of a non-viral gene delivery method according to the application employs plasmid DNA to introduce a nucleic acid encoding a peptide into a cell. Plasmid- based gene delivery methods are generally known in the art.
  • Synthetic gene transfer molecules can be designed to form multimolecular aggregates with plasmid DNA. These aggregates can be designed to bind to a target cell. Cationic amphiphiles, including lipopolyamines and cationic lipids, may be used to provide receptor-independent nucleic acid transfer into target cells. [0181] In addition, preformed or cationic lipids may be mixed with plasmid DNA to generate cell-transfecting complexes. Methods involving cationic lipid formulations are reviewed in Felgner et al., Ann. N.Y. Acad. Sci.772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev.20:221-266, 1996.
  • DNA may also be coupled to an amphipathic cationic peptide (Fominaya et al., J. Gene Med. 2:455-464, 2000).
  • Methods that involve both viral and non-viral based components may be used according to the application.
  • EBV Epstein Barr virus
  • a method involving a DNA/ligand/polycationic adjunct coupled to an adenovirus is described in Curiel, D. T., Nat. Immun.13:141-164, 1994.
  • the nucleic acid encoding the peptides of fusion polypeptide can be introduced into the target cell by transfecting the target cells using electroporation techniques. Electroporation techniques are well known and can be used to facilitate transfection of cells using plasmid DNA.
  • Vectors that encode the expression of the peptides can be delivered in vivo to the target cell in the form of an injectable preparation containing pharmaceutically acceptable carrier, such as saline, as necessary. Other pharmaceutical carriers, formulations and dosages can also be used in accordance with the present application.
  • the peptide or proteins can be expressed for any suitable length of time within the target cell, including transient expression and stable, long-term expression.
  • the agents used for modulating asprosin mediated PTPR ⁇ signaling and/or activity including, for example, anti-asprosin antibodies, peptides, proteins, or vectors, described herein, may be formulated with one or more pharmaceutically acceptable carrier or excipients to provide a pharmaceutical composition.
  • the agents described herein may be combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and a pH acceptable for administration, such as parenteral administration.
  • one or more pharmaceutically acceptable anti- microbial agents may be added. Meta-cresol and phenol are preferred pharmaceutically acceptable microbial agents.
  • One or more pharmaceutically acceptable salts may be added to adjust the ionic strength or tonicity.
  • excipients may be added to further adjust the isotonicity of the formulation.
  • Glycerin is an example of an isotonicity-adjusting excipient.
  • Pharmaceutically acceptable suitable for administration to a human or other animal does not contain toxic elements or undesirable contaminants and does not interfere with the activity of the active compounds therein.
  • the agents described herein may be formulated as a solution formulation or as a lyophilized powder that can be reconstituted with an appropriate diluent.
  • a lyophilized dosage form is one in which the agent is stable, with or without buffering capacity to maintain the pH of the solution over the intended in-use shelf-life of the reconstituted product.
  • a pharmaceutically-acceptable salt form of the agents described herein can also be provided.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
  • the therapeutic agent e.g., antibody, peptide, fusion polypeptide, or vector expressing the therapeutic peptide or fusion polypeptide
  • Systemic administration can include, for example, parenteral administration, such as intramuscular, intravenous, intraarticular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration.
  • the agent can also be administered orally, transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally.
  • the therapeutic peptide, fusion polypeptide, or vector can be administered to the subject via intravenous administration using an infusion pump to deliver daily, weekly, or doses of the therapeutic agent.
  • Pharmaceutically acceptable of the therapeutic agent can be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps.
  • the therapeutic agent can be formulated in liquid solutions, typically in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the therapeutic agent may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the injection can be, for example, in the form of a bolus injection or continuous infusion (such as using infusion pumps) of the therapeutic agent.
  • a therapeutically effective amount of the agent which inhibits asprosin mediated PTPR ⁇ signaling and/or activity, can be administered to treat polydipsia or excessive thirst in a subject in need thereof.
  • the polydipsia or excessive thirst can be primary polydipsia or secondary polydipsia.
  • the primary polydipsia can be associated with or result from a neurological disease or disorder.
  • the neurological disease or disorder can include, for example, social isolation stress, depression, anxiety, schizophrenia, affective disorders, anorexia nervosa, autism, and personality disorders or a combination of such neurological diseases or disorders.
  • the anxiety disorder can include one or more generalized anxiety disorder, phobia, social anxiety disorder, social phobia, panic disorder, panic attack, post- traumatic stress disorder, separation anxiety disorder, selective mutism, agoraphobia, or an anxiety disorder induced by a substance/medication or due to a medical condition.
  • the subject is experiencing at least one symptom of polydipsia, besides excessive thirst, wherein the at least one symptom includes persistent dry mouth, excessive urination, a headache, nausea, cramps, slow reflexes, slurred speech, low energy, confusion, seizures, or any combination thereof.
  • the subject has been diagnosed with schizophrenia only.
  • the subject is experiencing water intoxication and hyponatremia.
  • a therapeutically effective amount of the agent, which inhibits asprosin mediated PTPR ⁇ signaling or activity is administered prior to the onset of polydipsia or a neurological disease or disorder in the subject.
  • a therapeutically effective amount of the agent, which inhibits asprosin mediated PTPR ⁇ signaling or activity is administered to a subject at risk of developing polydipsia.
  • the subject can be identified as suffering from polydipsia.
  • the subject suffers from polydipsia.
  • the subject is a human or a non-human mammal.
  • the subject is an elderly subject.
  • the fluid intake comprises drinking behavior.
  • fluid intake does not comprise eating behavior.
  • inhibiting fluid intake comprises, consists essentially of, or consists of inhibiting drinking behavior.
  • the method of inhibiting fluid intake is a method of ameliorating, inhibiting, delaying the onset of, reducing the severity of, or preventing polydipsia.
  • the amount, volume, concentration, and/or dosage of the therapeutic agent e.g., antibody, peptide, fusion polypeptide, or vector
  • the amount, volume, concentration, and/or dosage of the therapeutic agent depends on many factors, including the subject’s size, body surface area, age, the particular composition to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Specific variations of the above noted amounts, volumes, concentrations, and/or dosages of the therapeutic agent can readily be determined by one skilled in the art using the experimental methods described below.
  • Doses may be in the range of 0.01 to 10 mg/kg body weight. In an embodiment, the doses may be in the range of 0.05 to 5 mg/kg body weight. In another embodiment, the doses may be in the range of 0.01 to 1 mg/kg body weight. In still another embodiment, the doses may be in the range of 0.05 to 0.5 mg/kg body weight. In still another embodiment, the doses may be in the range of 0.05 to 1 mg/kg body weight. [0199]
  • the therapeutic agent e.g., peptide, fusion polypeptide, or vector can be administered at an interval of one week or greater.
  • the doses may be administered at an interval of 1 week or greater.
  • the doses may be administered at an interval of 2 weeks or greater.
  • the doses may be administered at an interval of 3 weeks or greater.
  • the doses may be administered at an interval of 3 days, 4 days, 5 days, 6 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days, or greater.
  • the doses may be administered at a frequency of once a week, twice a week, once every other week, or twice per month, three times per month, and the like.
  • the method comprises administering the therapeutic agent (e.g., antibody, peptide or fusion polypeptide) at a dose of from about 0.01 mg/kg to about 10 mg/kg, about 0.02 mg/kg to about 10 mg/kg, from about 0.03 mg/kg to about 10 mg/kg, about 0.04 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 10 mg/kg, about 0.06 mg/kg to about 10 mg/kg, from about 0.07 mg/kg to about 10 mg/kg, from about 0.08 mg/kg to about 10 mg/kg, from about 0.09 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 0.15 mg/kg to about 10 mg/kg, from about 0.2 mg/kg to about 10 mg/kg, from about 0.25 mg/kg
  • the upper limit of the above ranges may be about 5 mg/kg.
  • the doses may be administered at an interval of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days, or greater.
  • the doses may be administered at a frequency of once a week, twice a week, once every other week, or twice per month, three times per month, and the like.
  • the therapeutic agent e.g., antibody, peptide or fusion polypeptide
  • the therapeutic agent may be administered at a dose of from about 0.01 mg/kg to about 1 mg/kg, about 0.02 mg/kg to about 1 mg/kg, from about 0.03 mg/kg to about 1 mg/kg, about 0.04 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 1 mg/kg, about 0.06 mg/kg to about 1 mg/kg, from about 0.07 mg/kg to about 1 mg/kg, from about 0.08 mg/kg to about 1 mg/kg, from about 0.09 mg/kg to about 1 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, from about 0.16 mg/kg to about 1 mg/kg, from about 0.2 mg/kg to about 1 mg/kg, from about 0.24 mg/kg to about 1 mg/kg, from about 0.3 mg/kg to about 1 mg/kg, from about 0.35 mg/kg to about 1 mg/kg, from about 0.4 mg/kg to about 1 mg
  • the doses may be administered at an interval of 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 30 days, 40 days, or greater.
  • the doses may be administered at a frequency of once a week, twice a week, once every other week, or twice per month, three times per month, and the like.
  • the method comprises administering the therapeutic agent (e.g., antibody, peptide or fusion polypeptide) at a dose of from about 0.1 mg/kg to about 5 mg/kg, from about 0.2 mg/kg to about 5 mg/kg, from about 0.3 mg/kg to about 5 mg/kg, from about 0.4 mg/kg to about 5 mg/kg, from about 0.5 mg/kg to about 5 mg/kg, from about 0.6 mg/kg to about 5 mg/kg, from about 0.7 mg/kg to about 5 mg/kg, from about 0.8 mg/kg to about 5 mg/kg, from about 0.9 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 1.1 mg/kg to about 5 from about 1.2 mg/kg to about 5 mg/kg, from about 1.3 mg/kg to about 5 mg/kg, from about 1.4 mg/kg to about 5 mg/kg, from about 1.5 mg/kg to about 5 mg/kg, from about 1.6 mg/kg to about 5 mg/kg,
  • the doses may be administered at a frequency of once a week, twice a week, once every two weeks, once per month, twice per month, three times per month, and the like.
  • the therapeutic agent e.g., antibody, peptide or fusion polypeptide
  • the therapeutic agent is administered at a dose of 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, 0.2 mg/kg, 0.21 mg/kg, 0.22 mg/kg, 0.23 mg/kg, 0.24 mg/kg, 0.25 mg/kg, 0.26 mg/kg, 0.27 mg/kg, 0.28 mg/kg, 0.29 mg/kg, or 3 mg/kg at an interval of 1 week or two weeks.
  • the two weeks interval schedule may be replaced with a frequency of every other week.
  • the therapeutic agent e.g., antibody, peptide or fusion polypeptide
  • the therapeutic agent is administered at a dose of 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, 0.2 mg/kg at an interval of 1 week or 10 days.
  • therapeutic agent e.g., antibody, peptide or fusion polypeptide
  • a pharmaceutically acceptable formulation used to administer the therapeutic agent can also be formulated to provide sustained delivery of the active compound to a subject.
  • the formulation may deliver the active compound for at least one, two, three, or four weeks, inclusive, following initial administration to the subject.
  • a subject to be treated in accordance with the method described herein can be treated with the therapeutic agent for at least 30 days (either by repeated administration or by use of a sustained delivery system, or both).
  • Approaches for sustained delivery include use of a polymeric capsule, a minipump to deliver the formulation, a biodegradable implant, or implanted transgenic autologous cells (see U.S.
  • Implantable infusion pump systems e.g., INFUSAID pumps (Towanda, PA)); see Zierski et al., 1988; Kanoff, 1994) and osmotic pumps (sold by Alza Corporation) are available commercially and otherwise known in the art.
  • Another mode of administration is via an implantable, externally programmable infusion pump.
  • Infusion pump systems and reservoir systems are also described in, e.g., U.S. Patents No.5,368,562 and No.4,731,058.
  • Vectors encoding the therapeutic peptides or fusion polypeptides can often be administered less frequently than other types of therapeutics.
  • an effective amount of such a vector can range from about 0.01 mg/kg to about 5 or 10 mg/kg, inclusive; administered daily, weekly, biweekly, monthly or less frequently.
  • the pharmaceutical compositions can be administered to any subject that can experience the beneficial effects of inhibition of asprosin-mediated polydipsia. Foremost among such animals are humans, although the present invention is not intended to be so limited. [0211] The invention is further illustrated by the following example, which is not intended to limit the scope of the claims.
  • Example [0212] We found that cerebellar Purkinje neurons, known to regulate the coordination and learning of complex movements, are activated by a peripherally generated hormone, asprosin; that Purkinje neuron activity is necessary and sufficient for the generation of thirst; and that inhibition of asprosin signaling in neurons, while causing hypodipsia, does not affect Purkinje neuron-mediated motor coordi- nation and learning. Our findings highlight a powerful and clinically relevant neural circuit for the modulation of thirst.
  • mice Two different strains of Ptprd mice were used in this work and were maintained as heterozygous, as previously described.
  • B6;129-Ptprd ⁇ tm1Yiw > mice were purchased from the RIKEN BioResource Center.
  • Mice with Ptprd cKO potential (C57BL/6NA ⁇ tm1Brd>Ptprd ⁇ tm2a(KOMP)Wtsi>/WtsiOrl; MEXY mice) was purchased from Wellcome Trust Sanger Institute and crossed to Flpase + mice to remove the neomycin selection cassette and Lacz reporter, thereby making a conditional allele, as described previously.
  • homozygous conditionally ready floxed mice (Ptprd tm2c(KOMP)Wtsi) were mated with Pcp2-cre mice to create purkinje neuron specific Ptprd knock-out (Pcp2cre;Ptprd flox/flox ).
  • homozygous conditionally ready floxed mice (Ptprd tm2c(KOMP)Wtsi) were mated with AgRP- IRES-Cre (C57BL/6-Agrptm1(cre)Lowl) to create AgRP neuron specific Ptprd knock-out, as previously described 8 .
  • Rosa26-LSL-tdTOMATO mice (Jackson Laboratory, JAX# 007914) were mated with the above described Pcp2cre and Pcp2cre;Ptprd flox/flox mice for the generation of Pcp2cre-Rosa26-LSL-tdTOMATO mice and Pcp2cre;Ptprd flox/flox -Rosa26- LSL-tdTOMATO, respectively.
  • Littermates from in-house mating served as controls in all experiments, except for WT lean mice that were bought from the Jackson Laboratory and used for experimentation after acclimation to the mouse housing facility.
  • mice were housed in microventilator cages on a 12-hour light cycle in an animal facility maintained at 20-25°C and 40-60% humidity. Mice had ad libitum access to water and normal chow (5V5R, Lab Supply), dustless pellet diet (F0173, Bio-Serv) or Teklad High Fat Diet (Envigo; TD.06414), unless otherwise specified. Animal housing, husbandry, and euthanasia were conducted under animal protocols approved by the Case Western Reserve University Institutional Animal Care and Use Committee 0042). The general health of mice was monitored by the CWRU animal resource center. Validation of Ptprd deletion in purkinje neurons [0215] Validation of Ptprd deletion was done as previously described.
  • mice were anesthetized with inhaled isoflurane and perfused with saline followed by 10% Formalin. Brain sections (25 ⁇ m in thickness) were collected and then subjected to immunofluorescence for Ptprd and tdTOMATO. Briefly, one series of the brain sections were blocked (5% normal donkey) for 1 hour.
  • the brain sections were incubated overnight with rabbit anti-Ptprd antibody (1:1000 dilution; # A15713, ABclonal) on a shaker at 4°C overnight. The next day, the brain sections were incubated by the donkey anti-rabbit Alexa Fluor 488 (1:500, A21206, Invitrogen) for 2 hours. Sections were mounted on slides and cover-slipped with DAPI mounting medium. Fluorescence images were taken using the Leica TCS SP5 fluorescence microscope with OptiGrid structured illumination. Purkinje neurons co-expressed by tdTOMATO and Ptprd were counted and averaged in at least four consecutive coronal brain sections from each mouse and this data was treated from one biological sample.
  • NIS-Elements-AR (Nikon) software was used for the analysis of Purkinje neuron morphology parameters, including cell number, cell body (soma) diameter, fiber length, synapse density and dendritic density. Alkaline phosphatase staining [0217] Human asprosin was cloned into pAPtag-5 (AP-TAG Kit B, GenHunter Corporation; Q202). HEK293T cells were grown up in 10 cm dishes and transfected with 15 ug of pAPtag-5_Asprosin according to the manufacturer's protocol (FuGENE® HD, Promega). 16 hours after transfection, the media was replaced with serum-free DMEM media.
  • AP-tagged asprosin was secreted into the media and collected over the course of 4 days. Media was concentrated to ⁇ 500 ⁇ L 15 mL Amicon centrifugal filters. Brains of adult wild-type C57BL/6 mice were dissected and frozen in 0CT and sectioned coronally. Frozen sections were washed with HBS buffer and then rinsed with HBAH buffer (GenHunter). AP-tagged asprosin protein was added to slides and incubated for 90 minutes at room temperature in a moist chamber. Sections were washed with HBAH buffer and then fixed for 15 seconds with acetone-formaldehyde fixative.
  • Metabolic caging, water, and food intake assessment [0218] Metabolic caging experiments were performed at the Cardiovascular Research Institute Mouse Metabolic and Phenotyping Core at CWRU (IACUC# 2019-0029). Mice were housed with a 12-h light/dark cycle (7 am/7 pm) at 22°C with controlled humidity. Metascreen software (V2.3.15.11) controlled system data acquisition.
  • Respirometry (V02, VC02, H20 vapor), activity and ad libitum food and water intake measures were collected individually using a Promethion metabolic cage system (Sable Systems, Las Vegas, NV, USA). Gas analyzers were calibrated before each run. Gas measurements were multiplexed over 8 cages and baselined to a cage-equivalent volume of room air twice per 5-minute cycle while maintaining a 2L/min/cage, negative pressure-derived flow rate. Acquired data was processed using Macro Interpreter (V2.34) running Macro V2.33.3- slice1hr. Energy expenditure was calculated using the Weir equation (Weir, 1949).
  • mice were acclimated to sipper bottles (with water) for 4 days, followed by a 7-day paradigm consisting of sequential ad libitum access to isotonic saline (for 2 days), water 2 days), hyper- tonic saline (for 1 day) and water (for 2 days). Post acclimation, 48-h intake of isotonic and hypertonic saline was recorded. Additionally, the water intake of mice was recorded in response to i.p.
  • hypertonic saline 3 M NaCl
  • isotonic saline 154 mM
  • mannitol 2 M
  • 30% PEG 30% PEG
  • 300 ⁇ l PEG (30% in PBS vehicle) was administered subcutaneously
  • NaCl and mannitol total volume, 150 ⁇ l
  • Water intake was recorded for 2 h post isotonic saline, mannitol and hypertonic saline treatment and for 48 h post PEG treatment.
  • Manual measurement of water intake was done in 8-week-old Pcp2-cre; Ptprd flox/flox and Pcp2-cre; Ptprd +/+ mice in all experiments.
  • mice were given access to water in the testing cage and recorded on video for 20 min. Water licks were counted every minute to calculate the lick frequency per 1 min or per 5 min.
  • Overexpression of asprosin and recombinant asprosin treatment [0220] To test for overexpression of asprosin, 12–14-week-old normal chow-fed lean Ptprd ⁇ / ⁇ and Ptprd +/+ (WT) littermate male mice were i.v. injected in the tail vein with Ad5 and AAV as previously described.
  • mice injected with Ad5-empty (5 ⁇ 10 10 pfu per mouse) served as controls for experimental mice that received Ad5-IL-2-asprosin (5 ⁇ 10 10 pfu per mouse) containing an amino-terminal His-tagged human asprosin coding region preceded by an IL-2 signal peptide, under control of an EF1 promoter.
  • Ad5-IL-2-asprosin 5 ⁇ 10 10 pfu per mouse
  • the 12-week-old normal chow-fed lean WT (C57BL/6J) male mice were i.v. injected in the tail vein with AAV8 as previously described.
  • mice injected with AAV8-empty (1 ⁇ 10 12 GC per mouse) served as controls for experimental mice that received AAV8-IL-2-asprosin (1 ⁇ 10 12 GC per mouse) containing an N-terminal His-tagged human asprosin coding region preceded by an IL-2 signal peptide, under control of an EF1 promoter.
  • the 12-week-old normal chow-fed lean WT littermate male mice were intranasally treated with 2 ⁇ g recombinant asprosin (in 15 ⁇ l saline; BioLegend, 761902), and water intake was manually recorded for 2 h post treatment.
  • mice Male Pcp2-cre mice were bilaterally injected with 200 nl of AAV-hSyn-DIO-mCherry into the cerebellum lobe VI–VII (anteroposterior, ⁇ 7.02 mm, mediolateral, ⁇ 1.77 mm, dorsoventral, ⁇ 2.96 mm).
  • CNO (3 mg kg ⁇ 1 ) was i.p. injected in control (mCherry), activation (hM3Dq) and inactivation (hM4Di) mice at 09:30 h and 17:00 h, respectively. Food intake and water intake were monitored using the BioDAQ system.
  • mice After a 3-day rest period, both control and experimental mice were subjected to the same procedure but received an i.p. injection of saline. After we finished all the experiments, all mice were perfused with 10% formalin, and brains were dissected, sectioned and mounted. The mCherry signals were monitored under a fluorescent microscope for validation of injection accuracy. Only those mice with mCherry signals exclusively in the cerebellum lobes were included in analyses for feeding and drinking behavior.
  • mice Male Pcp2-cre mice were bilaterally injected with 200 nl of AAV-hSyn-GFP (RRID: Addgene_50465) into the cerebellum lobe V–VI (anteroposterior, ⁇ 6.62 mm, mediolateral, ⁇ 2.07 mm, dorsoventral, ⁇ 2.70 mm).
  • RRID Addgene_50465
  • a dual-channel wireless optogenetic device was implanted over lobe V–VI (refs.24,50).
  • mice with Pcp2 expressing ChR2 and GFP were monitored for food and water intake after yellow (598 nM, 5 Hz, 3 s on and 3 s off) or blue light (473 nM, 5 Hz, 3 s on and 3 s off) stimulation, respectively.
  • Food intake and water intake measured manually. Mice were allowed to adapt to the water bottle and food in the testing cage for 2 days before experiments and given 5 min to acclimate to the environment before light stimulation. After we finished all the experiments, all mice were perfused with 10% formalin and brains were dissected, sectioned and mounted. The YFP signals were monitored under a fluorescent microscope for validation of injection accuracy.
  • Ptprd-Fwd 5'- tctgaggccaggaactgttt-3' (SEQ ID NO: 10)
  • Ptprd-Rev 5'-tggaacccttttagagcttgc-3' (SEQ ID NO: 11)
  • Gapdh-Fwd 5'-gggttcctataaatacggactgc-3' (SEQ ID NO: 12
  • Gapdh-Rev 5'- ccattttgtctacgggacga-3' (SEQ ID NO: 13).
  • Urine volume, plasma and urine osmolality were measured in 14-week-old Fbn1 +/+ and Fbn1 NPS/+ female mice, 14- month-old Ptprd +/+ and Ptprd ⁇ / ⁇ female mice and 12-week-old Pcp2-cre; Ptprd flox/flox and Pcp2-cre; Ptprd +/+ mice.
  • a custom-built sandwich ELISA was used for measuring plasma asprosin in 14- week-old lean mice subjected to overnight water deprivation.
  • Asprosin in 50 ⁇ l plasma was captured using a fully human anti-asprosin mAb (100 ng per well), developed and generated from a naive human phage display antibody library by panning against recombinant full- length human asprosin (Texas Therapeutics Institute at the University of Texas Health Science Center at Houston).
  • a mouse anti-asprosin mAb, against human asprosin amino acids 106–134 (human profibrillin amino acids 2,838–2,865) served as the capture antibody (100 ng per well).
  • mice 26-week-old Cre + control and knockout mice were subjected to the ErasmusLadder assay.
  • the constant Speed Rotarod assay was performed using a Rotarod Rotamax Machine, and 16–20- week-old mice were placed at a constant speed of 4.0 r.p.m. Time latency over the course of three trials was recorded whereby mice were placed on a constant rotating rod for 3 min (trial 1), 5 min (trial 2) or 7 min (trial 3). Mice were given a minimum of 1 h recovery between each trial. Time latency was recorded when mice fell off the rod onto soft bedding (to prevent any injury to the mouse).
  • a grip strength meter (Bioseb BIO-GS3) was used to measure the forelimb and hindlimb grip of 16-week-old mice. Grip strength testing was repeated three times for each mouse, with a 10 min gap between the readings. The average of the three values was recorded. [0229] A tail-hang assay was performed to measure core strength and mobility. For the 6-min assay, 20-week-old mice were taped by the end of their tails and recorded. The total amount of time the mouse hung passively and motionless was recorded. If mice attempted to climb their tails more than 20% of the total trial time they were removed from the analysis. All mice were observed afterward for any adverse effects during the test.
  • the pole test was used to evaluate the mouse’s ability to climb down and maneuver on a pole to descend into its home cage.
  • a 56 cm metal pole with a diameter of 1.5 cm was wrapped in parafilm to create a grippable surface.
  • 16–20-week-old mice were placed facing upwards on the pole, with their head 5 cm from the top of the pole. Mice then must orient themselves to face the downward direction and descend the pole. The time taken to descend two consecutive trials was recorded and averaged. The number of falls was also recorded and considered a ‘failure’ by the mouse.
  • a single trial consisted of a mouse moving from one goalbox to the other, with a comprehensive list of metrics quantified during the trial including different gait patterns and cerebellum-dependent learning dynamics.
  • Control and Pcp2-cre; Ptprd flox/flox mice 26 weeks old were trained on the ErasmusLadder for sessions one and two and then subjected to a challenge paradigm over three sessions (sessions three to five).
  • a challenge paradigm over three sessions (sessions three to five).
  • one obstacle randomly activated from a series of obstacles
  • conditioning tone conditioning stimulus
  • Absolute learning is determined by the post-perturbation step-time; that is, the time between the rungs just preceding the obstacle presented and the rung just after the obstacle during the paired trials (conditioning stimulus + unconditioned stimulus). Pre- perturbation step times are also measured during trials.
  • the ErasmusLadder also recorded locomotor stepping or discrete gait dynamics, including short steps (step on a subsequent rung on the same side; that is n + 1), long steps (step over one rung on the same side; that is, n + 2) or missteps (step on lower, non-optimal stepping rungs).
  • Electrophysiology [0235] Purkinje neuron labeling and electrophysiology experiments were performed.
  • Rosa26-LSL-tdTOMATO mice or Rosa26-eGFP/Rpl10a were mated with the above-described Pcp2-cre and Pcp2-cre; Ptprd flox/flox mice for the generation of Pcp2-cre; Rosa26-LSL-tdTOMATO mice, Pcp2-cre- eGFP-Rpl10a mice and Pcp2-cre; Ptprd flox/flox ; Rosa26-LSL-tdTOMATO, respectively.
  • mice On the day of electrophysiology recording experiment, mice were euthanized under fed or water deprived conditions.
  • mice were removed and immediately submerged in an ice-cold sucrose-based cutting solution (adjusted to pH 7.3) containing (in mM): 10 NaCl, 25 NaHCO3, 195 Sucrose, 5 Glucose, 2.5 KCl, 1.25 NaH 2 PO 4 , 2 Na pyruvate, 0.5 CaCl 2 , 7 MgCl2 bubbled continuously with 95% O 2 and 5% CO2.
  • the slices were cut with a Microm HM 650V vibratome (Thermo Scientific) or VT1200 S vibratome (Leica) and recovered for 1 h at 34°C and then maintained at room temperature in artificial cerebrospinal fluid pH 7.3) containing: 126 mM NaCl, 2.5 mM KCl, 2.4 mM CaCl 2 , 1.2 mM NaH 2 PO 4 , 1.2 mM MgCl 2 , 11.1 mM glucose, and 21.4 mM NaHCO 3 saturated with 95% O2 and 5% CO2.
  • TOMATO(+) neurons were visualized using epifluorescence and IR-DIC imaging on an upright microscope equipped with a moveable stage (MP-285, Sutter Instrument).
  • brain slices were superfused at 34 °C in oxygenated aCSF at a flow rate of 1.8–2 ml min ⁇ 1 .
  • Patch pipettes with resistances of 3–5 M ⁇ were filled with intracellular solution (pH 7.3) containing 128 mM k-gluconate, 10 mM KCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl2, 0.05 mM Na-GTP and 0.05 mM Mg-ATP.
  • the aCSF solution also contained 1 ⁇ M TTX and a cocktail of fast synaptic inhibitors, namely bicuculline (50 ⁇ M; a GABA receptor antagonist) DAP-5 (30 ⁇ M; an NMDA receptor antagonist) and CNQX (30 ⁇ M; an NMDA receptor antagonist) to block the majority of presynaptic inputs.
  • bicuculline 50 ⁇ M; a GABA receptor antagonist
  • DAP-5 (30 ⁇ M; an NMDA receptor antagonist
  • CNQX ⁇ M; an NMDA receptor antagonist
  • Tungsten electrodes of 5–8 M ⁇ resistance were lowered into the brain using a motorized micromanipulator (MP- 225; Sutter Instrument) to record from neurons. Signals were amplified, bandpass- filtered from 0.3–13 kHz (ELC-03XS amplifier, NPI Electronic Instruments) and digitized (CED Power 1401, CED). Signals were recorded and spike sorted with Spike2 software (CED). Purkinje cells were identified by the presence of both complex spikes and simple spikes as well as distance from the surface of the brain. Recordings used in the analyses had a duration of about 30 s.
  • a stainless steel intracerebroventricular (i.c.v.) cannulas (RWD) were inserted into the lateral ventricles (i.c.v. coordinates without angle: AP +0.34 mm, ML -1.00 mm. and DV -2.30 mm).
  • Optical fibers and cannulas were fixed to the skull by using dental acrylic.
  • mice were individually housed for at least 3 weeks post-surgery before acclimating to the investigator's handling for 1 week prior to the recordings. [0241] Mice were allowed to adapt to the tethered patchcord for 2 days prior to experiments and given 5 minutes to acclimate to the tethered patchcord prior to any recording. Fiber photometry recordings of purkinje neurons were done in mice at fed conditions without food. All the control or Ptprd Pcp2 KO mice received i.c.v. injection of GFP (as control) and 10 ng asprosin (in 1 ⁇ l saline) on different days. There was a one-week washout period between each injection.
  • hypodipsia as a secondary consequence of compromised energy accretion and leanness cannot be ruled out in genetic loss-of-function models of the asprosin pathway.
  • hypodipsia dis- played by Fbn1 NPS/+ and Ptprd ⁇ / ⁇ mice as a ramification of leanness, low appetite and energy expenditure
  • WT wild-type mice
  • anti-asprosin mAb treatment in lean WT mice is a strategy for acute asprosin loss-of-function without the confounding effects of altered food intake and body weight.
  • Lean WT mice treated with the anti-asprosin mAb showed a significant reduction in 24-h water intake with a concomitant reduction in urine output (Fig.1O, P).
  • mice subjected to plasma asprosin elevation using adeno-associated viral vector serotype 8-mediated overexpression and secretion of human asprosin protein (AAV8-asprosin) transduction also led to a significant increase in water intake, irrespective of whether the mice were ad libitum fed or fasted (Fig.1R-T), again demonstrating asprosin- mediated increase in water intake, independent of its effects on food intake.
  • AAV8-asprosin adeno-associated viral vector serotype 8-mediated overexpression and secretion of human asprosin protein
  • AP-tagged asprosin For identification of asprosin-responsive brain regions involved in the regulation of water intake, we performed a brain-wide asprosin binding study using alkaline phosphatase (AP)-tagged asprosin. This study identified the cerebellum as a site of asprosin binding in both the human and mouse brain (Fig.2A). The binding of AP-tagged asprosin to the cerebellum could be abolished upon pre-incubation with untagged asprosin but not untagged-GFP, demonstrating competitive binding as expected for hormone–receptor interactions (Fig.2A). High expression of the asprosin receptor Ptprd has been previously documented in the mouse and human cerebellum (GTEx: ENSG00000153707.16).
  • Ptprd displays many splice variants that affect the extracellular ligand binding domain , and it is possible that the variant in granule neurons precludes asprosin responsiveness.
  • Purkinje neurons from tdTOMATO/Pcp2-cre mice responded to recombinant asprosin with increased firing frequency and resting membrane potential (Fig. 2E, F).
  • Fig. 2E, F Given the complex distribution pattern of Purkinje neuron activity and projections, we tested Purkinje neuron responsiveness to asprosin by targeting lobes II, III, IV, V, VI, VII, VIII and IX using anteroposterior sampling (Fig.2E, F) and by targeting lobes V and VI using mediolateral sampling (Fig.9C–E).
  • Fig.9C Two distinct Purkinje neuron baseline firing frequencies were noted (Fig.9C, D). Irrespective of baseline firing frequency range, sampling direction (mediolateral versus anteroposterior) or cerebellar lobes targeted, all tested Purkinje neurons responded to recombinant asprosin with increased firing frequency and resting membrane potential (Fig.2E, F and Fig.9D, E), suggesting that asprosin- mediated Purkinje neuron activation is a pan-cerebellar property. Furthermore, Purkinje neurons from both male and female mice could be activated by asprosin, suggesting that asprosin-mediated Purkinje neuron activation is not a sexually dimorphic phenomenon (Fig. 2F).
  • Purkinje neurons were activated by asprosin even when all synaptic input was blocked by treatment with bicuculline (antagonist of the GABA receptor), tetrodotoxin (sodium channel blocker), (2 R)-amino-5-phosphonovaleric acid (NMDA receptor antagonist) and cyanquixaline (a competitive AMPA/kainate receptor antagonist), suggesting that asprosin-mediated Purkinje neuron activation is probably a cell-autonomous effect (Fig. 2G, H).
  • Purkinje neuron activity modulation alters water intake [0248] Having established Purkinje neuron activation by asprosin, we sought to manipulate Purkinje neurons in a manner that is selective, rapid and reversible, and would provide insight, both qualitative and quantitative, into the function of these neurons. To achieve this goal, we used two state-of-the-art methods: chemogenetic stimulation and inhibition (using designer receptors exclusively activated by designer drugs; DREADD) and optogenetic (using channel rhodopsin (Chr2)) stimulation of Purkinje neurons.
  • chemogenetic stimulation and inhibition using designer receptors exclusively activated by designer drugs; DREADD
  • optogenetic using channel rhodopsin (Chr2) stimulation of Purkinje neurons.
  • chemogenetic manipulation we used a stereotaxic injection of Cre-recombinase-dependent AAV to selectively express hM3Dq (for stimulation) and hM4Di (for inhibition) in Purkinje (Pcp2-cre) neurons. DREADD was fused to mCherry so that receptor expression could be monitored.
  • Cre-recombinase-dependent AAV expressing Chr2 was used to target Purkinje neurons. Chr2 was fused to EYFP so that receptor expression could be monitored.
  • Purkinje neuron activation causes [0250] To elucidate the relevance of Purkinje neuron-specific Ptprd in the regulation of water intake, mice with genetic loss of Ptprd from Purkinje neurons (Pcp2-cre; Ptprd flox/flox ) were generated by crossing Ptprd flox/flox mice with mice expressing Cre recombinase under the control of the Purkinje cell protein 2 (Pcp2) promoter. To test whether this strategy led to the successful deletion of Ptprd from Purkinje neurons, Pcp2-cre; Ptprd flox/flox and Pcp2-cre; Ptprd +/+ mice were mated with Cre-responsive tdTOMATO mice.
  • Ptprd loss affects the morphology of Purkinje neurons by assessing five parameters of Purkinje neuron morphology: cell number, cell body diameter, fiber length, synapse and dendritic density. We found no impact of Ptprd loss on the morphology of Purkinje neurons (Fig.10C, D).
  • mice with Purkinje neuron-specific Ptprd deletion did not result in motor deficits
  • Purkinje neurons are well known to have pivotal roles in the coordination and control of complex movements. In particular, swallowing is a complex activity requiring a sophisticated system of neurological control from neurons within the brainstem, cerebral cortex and the cerebellum.
  • Pcp2-cre; Ptprd flox/flox mice did not show a deficit in pedestrian activity or wheel-running activity recorded over 4 days (Fig.5A, B and Fig.13A, B).
  • Pcp2-cre; Ptprd flox/flox mice did not show any neuromuscular abnormalities, with similar strength to controls on the grip test and successful completion of the inverted test despite a prolonged hold time of 5 min (Fig.5D-F). Similar immobility time in a tail-hang assay was also noted (Fig.5G).
  • Purkinje neuron-specific Ptprd deletion had no impact whatsoever induced Purkinje neuron activation (Fig.7D–F), indicating maintained health of the neurons and specificity of the perturbation for asprosin and water deprivation.
  • Fig.7D–F Purkinje neuron activation
  • SFO subfornical organ
  • OVLT organum vasculosum lamina terminalis
  • the cerebellum In addition to sensorimotor and vestibular control, the cerebellum also contributes to cognition, emotion, memory, autonomic function, satiety and meal termination. Functional imaging studies have reported increased cerebellar activity with thirst in human subjects , and a murine study has correlated pan-cerebellar and midbrain metabolic activity with water intake. However, whether the cerebellum can directly modulate fluid homeostasis has remained unknown, largely owing to the complex nature of functional parcillation, cellular diversity and spatially diverse anatomical connections of the cerebellar cortex. The present study directly implicates cerebellar purkinje neurons in the regulation of thirst.
  • Ptprd-mediated signaling seems to be central to these effects, as the genetic loss of Ptprd (Ptprd ⁇ / ⁇ ) rendered mice unresponsive to asprosin’s dipsogenic effects.
  • Ptprd ⁇ / ⁇ the genetic loss of Ptprd
  • Ptprd ⁇ / ⁇ the genetic loss of Ptprd
  • asprosin the genetic loss of Ptprd
  • Ptprd ⁇ / ⁇ rendered mice unresponsive to asprosin’s dipsogenic effects.
  • Ptprd is expressed in the cerebellum, including Purkinje neurons.
  • Purkinje neurons Although responsive to asprosin and water deprivation, were found to be unresponsive to acute hyperosmolality or hypovolemia on a time scale at par with the CVO. Similarly, Purkinje neuron Ptprd signaling was found to be dispensable for the dipsogenic effects of hypertonic saline, mannitol and PEG. This suggests that Purkinje neurons, which are protected by the blood–brain barrier, are triggered for thirst mediation by hormonal signals like asprosin that are generated outside the central nervous system and cross the blood–brain barrier rather than relying on direct sampling of plasma.
  • hypodipsia in mice with neuron-specific deletion of Ptprd and the inability of asprosin to rescue this hypodipsia demonstrates the necessity of Purkinje neuron Ptprd signaling for asprosin’s dipsogenic function.
  • Asprosin regulates thirst by modulation of Purkinje neuron activity, independent of its role in the modulation of appetite by hypothalamic AgRP neurons. Specifically, asprosin activates hypothalamic AgRP neurons, leading to enhanced appetite and body weight without affecting thirst.
  • asprosin activates Purkinje neurons, leading to enhanced thirst without affecting food intake or body weight.
  • Purkinje neuron-specific deletion of Ptprd while causing hypodipsia, did not result in Purkinje neuron morphological abnormalities, or deficits in movement, coordination or motor learning. This suggests that Purkinje neurons can regulate distinct behaviors completely independently of each other.
  • the Purkinje neuron response to asprosin and water deprivation was completely abolished with Ptprd loss, the response to norepinephrine was unaltered, exemplifying the specificity of the perturbation for asprosin and water deprivation.

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Abstract

Une méthode de régulation de la soif chez un sujet dont l'état le nécessite consiste à administrer au sujet une quantité thérapeutiquement efficace d'un agent qui module la signalisation ou l'activité du récepteur de type δ de la tyrosine phosphatase protéique médiée par l'asprosine (PTPRD).
PCT/US2024/055510 2023-11-10 2024-11-12 Modulation de la soif Pending WO2025102068A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120225940A1 (en) * 1999-06-04 2012-09-06 Diatex, Inc. Use of (-) (3-Trihalomethylphenoxy) (4-Halophenyl) Acetic Acid Derivatives for Treatment of Insulin Resistance, Type 2 Diabetes, Hyperlipidemia and Hyperuricemia
WO2023023588A2 (fr) * 2021-08-18 2023-02-23 Case Western Reserve University Méthodes et compositions d'inhibition de l'orexigenèse et/ou de la glucogenèse induite par asprosine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120225940A1 (en) * 1999-06-04 2012-09-06 Diatex, Inc. Use of (-) (3-Trihalomethylphenoxy) (4-Halophenyl) Acetic Acid Derivatives for Treatment of Insulin Resistance, Type 2 Diabetes, Hyperlipidemia and Hyperuricemia
WO2023023588A2 (fr) * 2021-08-18 2023-02-23 Case Western Reserve University Méthodes et compositions d'inhibition de l'orexigenèse et/ou de la glucogenèse induite par asprosine

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