CN111778323A

CN111778323A - AchR, sleep and wake

Info

Publication number: CN111778323A
Application number: CN202010476337.5A
Authority: CN
Inventors: 戴熙慧敏; 周恩兴; 毛仁波; 杨威; 刘源; 余腾辉; 张文霞; 饶毅
Original assignee: Beijing Yuanji Huayi Biotechnology Co ltd
Current assignee: Beijing Yuanji Huayi Biotechnology Co ltd
Priority date: 2019-10-09
Filing date: 2020-05-29
Publication date: 2020-10-16
Anticipated expiration: 2040-05-29
Also published as: WO2021068900A1; CN111778323B

Abstract

The present application provides a method for selecting an agent for modulating sleep comprising determining the effect of the candidate agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2. The agents are useful for treating, preventing or delaying the progression of sleep disorders. In addition, the present application provides methods for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder. The present application also provides a non-human organism or a living portion thereof.

Description

AchR, sleep and wake

Background

Both sleep and arousal are important in animals ranging from insects, fish, to mammals. Sleep deprivation can lead to negative consequences such as impaired memory, metabolic disturbances, and even death. The molecular mechanisms and neural circuits that control sleep and arousal are being actively studied.

Although cholinergic neurons have long been recognized as key sleep regulation modulators in both mammals and flies, the specific function of cholinergic neurons in sleep and wakefulness is still controversial, and little is known about the role of AChR in sleep regulation. In both mammals and flies, ACh is contradictory in its ability to promote both sleep and arousal. Although ACh has long been known to play an important role in sleep regulation, the molecular basis of cholinergic signaling remains elusive and little is known about the role of AChR in sleep regulation.

Summary of The Invention

The present application provides a method for selecting an agent for use in modulating sleep, the method comprising: providing a candidate agent; determining the effect of the candidate agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2; and selecting said candidate agent as an agent for modulating sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is altered by said candidate agent. The medicament may be for the treatment, prevention or delay of progression of sleep disorders. Further, the present application provides a method for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder, the method comprising: assessing the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject. The present application also provides a non-human organism or a living portion thereof.

In one aspect, the present application provides a method for selecting an agent for use in modulating sleep, the method comprising: providing a candidate agent; determining the effect of the candidate agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2; and selecting said candidate agent as an agent for modulating sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is altered by said candidate agent.

In some embodiments, if the activity and/or expression of nAChR α 2 and/or nAChR β 2 is increased by the candidate agent, then the candidate agent is selected as an agent for promoting sleep.

In some embodiments, if the activity and/or expression of nAChR α 2 and/or nAChR β 2 is decreased by the candidate agent, then the candidate agent is selected as an agent for reducing sleep.

In some embodiments, nAChR α 2 is Drosophila melanogaster (Drosophila melanogaster) nAChR α 2 or ortholog thereof.

In some embodiments, nAChR β 2 is drosophila melanogaster nAChR β 2 or an ortholog thereof.

In some embodiments, the assay comprises: determining the effect of said candidate agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 in an octopamine-capable cell.

In some embodiments, the octopamine-competent cell comprises an octopamine-competent neuronal cell.

In some embodiments, the activity of the nAChR α 2 and/or nAChR β 2 comprises one or more of: the ability to form a functional nAChR α 2 β 2 receptor complex; the ability to increase the activity, release and/or amount of octopamine; the ability to activate octopamine signaling.

In some embodiments, the method is an in vitro method or an ex vivo method.

In some embodiments, sleep includes daytime sleep and/or nighttime sleep.

In some embodiments, the agent does not substantially affect sleep recovery, circadian rhythm, or wakefulness following deprivation.

In some embodiments, the agent comprises a small molecule, protein, and/or polynucleotide.

In some embodiments, the agent acts directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acids encoding the nAChR α 2 protein and/or nAChR β 2 protein.

In another aspect, the present application provides a system for selecting an agent for modulating sleep, wherein the system comprises a substance capable of determining the effect of the agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2.

In some embodiments, the agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acid.

In some embodiments, the agent capable of determining the effect of said agent on the activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2 comprises: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

In some embodiments, the agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein.

In some embodiments, the agent capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprises: an agent capable of specifically recognizing nAChR α 2 and/or nAChR β 2 proteins and/or an agent capable of determining the activity of nAChR α 2 and/or nAChR β 2 proteins.

In another aspect, the present application provides a method for treating, preventing or delaying the progression of a sleep disorder, the method comprising: administering to a subject in need thereof a therapeutically effective amount of an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

In some embodiments, the sleep disorder is associated with insufficient sleep, and the agent is capable of increasing activity and/or expression of nAChR α 2 and/or nAChR β 2 in the subject.

In some embodiments, the sleep disorder associated with insufficient sleep includes daytime insufficient sleep and/or nighttime insufficient sleep.

In some embodiments, the sleep disorder associated with insufficient sleep includes insomnia (insomnia) and/or sleep loss (sleep loss) associated with a cardiovascular disorder and/or a neurodegenerative disease.

In some embodiments, the agent comprises a nucleic acid molecule encoding nAChR α 2 and/or nAChR β 2 or an expression product thereof.

In some embodiments, the agent comprises a nucleic acid sequence as set forth in any one of SEQ ID nos. 1-20.

In some embodiments, the sleep disorder is associated with hypersomnia, and the agent is capable of decreasing activity and/or expression of nAChR α 2 and/or nAChR β 2 in the subject.

In some embodiments, the disorder associated with hypersomnia includes daytime hypersomnia and/or nighttime hypersomnia.

In some embodiments, the sleep disorder associated with hypersomnia includes narcolepsy (narcolepsy), hypersomnia (hypersomnolence), NREM/REM-related parasomnia (NREM/REM-related parasymias), and/or hypersomnia (oversleeping)/difficult to wake (hard-to-be-awaken) associated with cardiovascular disorders and/or neurodegenerative diseases.

In some embodiments, the agent is an agent for promoting sleep in that the activity and/or expression of nAChR α 2 and/or nAChR β 2 is increased by the agent.

In some embodiments, the agent is an agent for reducing sleep in that the activity and/or expression of nAChR α 2 and/or nAChR β 2 is reduced by the agent.

In some embodiments, nAChR α 2 is drosophila melanogaster nAChR α 2 or an ortholog thereof.

In some embodiments, the alteration in activity and/or expression of nAChR α 2 and/or nAChR β 2 is in an octopamine-capable cell.

In some embodiments, the activity of nAChR α 2 and/or nAChR β 2 comprises one or more of: the ability to form a functional nAChR α 2 β 2 receptor complex; the ability to increase the activity, release and/or amount of octopamine; the ability to activate octopamine signaling.

In some embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method.

In another aspect, the present application provides the use of an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 in the manufacture of a medicament for the treatment, prevention or delay of progression of a sleep disorder.

In some embodiments, the agent is capable of determining the effect of said agent on the activity and/or expression of said nucleic acid of nAChR α 2 and/or nAChR β 2.

In some embodiments, an agent capable of determining the effect of said agent on the activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2 comprises: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

In some embodiments, the agent that determines the effect of the agent on the activity and/or expression of the nAChR α 2 and/or nAChR β 2 protein comprises: an agent capable of specifically recognizing nAChR α 2 and/or nAChR β 2 proteins and/or an agent capable of determining the activity of nAChR α 2 and/or nAChR β 2 proteins.

In another aspect, the present application provides an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 for use in treating, preventing or delaying the progression of a sleep disorder.

In another aspect, the present application provides a method for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder, the method comprising: assessing the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

In some embodiments, the activity and/or expression of nAChR α 2 and/or nAChR β 2 comprises the activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2, and/or the activity and/or expression of a protein of said nAChR α 2 and/or nAChR β 2.

In some embodiments, the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

In some embodiments, sleep disorders associated with insufficient sleep include insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.

In some embodiments, the sleep disorder associated with hypersomnia includes daytime hypersomnia and/or nighttime hypersomnia.

In some embodiments, the sleep disorder associated with hypersomnia includes narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

In another aspect, the present application provides a system for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder, the system comprising: an agent capable of indicating the activity and/or level of expression of nAChR α 2 and/or nAChR β 2 in said subject.

In another aspect, the present application provides the use of an agent capable of indicating the activity and/or expression level of nAChR α 2 and/or nAChR β 2 in a subject in the manufacture of an indicator of the likelihood that said subject suffers from, and/or is at risk of suffering from, a sleep disorder.

In some embodiments, an agent capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprises: an agent capable of specifically recognizing nAChR α 2 and/or nAChR β 2 proteins and/or an agent capable of determining the activity of nAChR α 2 and/or nAChR β 2 proteins.

In another aspect, the present application provides a non-human organism, or living portion thereof, comprising functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2.

In some embodiments, the non-human organism is drosophila melanogaster.

In some embodiments, the non-human organism or living portion thereof does not comprise any functional nAChR α 2.

In some embodiments, the non-human organism or living portion thereof does not comprise any functional nAChR β 2.

In some embodiments, the non-human organism or living portion thereof is homozygous for functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2.

In some embodiments, the non-human organism has a reduced sleep duration compared to a corresponding wild-type non-human organism.

In some embodiments, sleep includes daytime sleep and/or nighttime sleep.

In some embodiments, the nAChR α 2 gene and/or nAChR β 2 gene in the organism is knocked-down or knocked-out.

In some embodiments, the nAChR α 2 gene and/or nAChR β 2 gene in the organism is knocked down by RNAi.

In some embodiments, the nAChR α 2 gene in the organism is knocked down by α 2KIGal 4.

In some embodiments, the nAChR β 2 gene in the organism is knocked down by β 2KIGal 4.

In some embodiments, the fourth, fifth, and sixth exons of the nAChR α 2 gene in the organism are deleted.

In some embodiments, the first to eighth exons of the nAChR β 2 gene in the organism are deleted.

In another aspect, the present application provides a cell, cell line or primary cell culture derived from a non-human organism or living part thereof according to the present application.

In another aspect, the present application provides a tissue derived from a non-human organism or living portion thereof according to the present application.

In some embodiments, the tissue is derived from neural tissue.

In some embodiments, the tissue is derived from neural tissue comprising octopamine-competent cells.

In another aspect, the present application provides a method of screening for a substance, device and/or composition suitable for use in the treatment, prevention or delay of progression of a sleep disorder, the method comprising applying a candidate substance, device and/or composition to a non-human organism or a living part, cell line or primary cell culture, or tissue thereof, and determining the effect of the candidate substance, device and/or composition on one or more of: a sleep duration of the non-human organism; activity, amount and/or release of octopamine; and activation of octopamine signaling.

In some embodiments, the assay comprises: determining the effect of said candidate substance, device and/or composition on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 in octopamine-capable cells.

In some embodiments, the method is an in vitro method or an ex vivo method.

In some embodiments, candidate substances and/or compositions include small molecules, proteins, and/or polynucleotides.

In some embodiments, the candidate substances, devices and/or compositions act directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acids encoding the nAChR α 2 protein and/or nAChR β 2 protein.

In another aspect, the present application provides a method of screening for biomarkers suitable for diagnosing and/or monitoring sleep disorders, the method comprising: determining a disease value for a substance, wherein the disease value is the presence and/or level of the substance in a sample obtained from a non-human organism or a living part thereof, a cell, cell line or primary cell culture, or a tissue; determining a wild-type value for said agent, wherein said wild-type value is the presence and/or level of said agent in a sample obtained from a corresponding wild-type non-human organism or a corresponding living part, cell or tissue thereof; and identifying the substance as the biomarker when the disease value is different from the wild-type value.

In some embodiments, the disease value is greater than the wild-type value, and the biomarker is a biomarker indicative of promoting sleep.

In some embodiments, the disease value is less than the wild-type value, and the biomarker is a biomarker indicative of decreased sleep.

In another aspect, the present application provides the use of a non-human organism or a living part thereof, a cell, cell line or primary cell culture, or a tissue in the manufacture of a system for screening for substances, devices, compositions and/or biomarkers suitable for the treatment, diagnosis, prevention, monitoring and/or prognosis of sleep disorders.

In some embodiments, the substance, composition and/or biomarker comprises a small molecule, protein and/or polynucleotide.

In some embodiments, the substances, devices, compositions and/or biomarkers act directly on nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acids encoding nAChR α 2 protein and/or nAChR β 2 protein.

In another aspect, the present application provides a non-human organism or living part thereof, a cell, cell line or primary cell culture, or a tissue for screening for substances, devices, compositions and/or biomarkers suitable for the treatment, diagnosis, prevention, monitoring and/or prognosis of sleep disorders.

Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present application are shown and described. As will be realized, the application is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the application. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Is incorporated by reference

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention and the drawings thereof (also referred to herein as "figures") are utilized:

FIGS. 1A-1C illustrate the strategy for constructing AChR mutants and knock-in lines;

FIG. 2 illustrates a schematic representation of the nAChR α 2 and nAChR β 2 genes;

FIG. 3 illustrates schematic genotypes of the KOGal4 strain for rescue and the knock-in strain for marker;

figure 4 illustrates the sleep phenotype of nAChR α 2 and nAChR β 2 mutants;

FIG. 5A illustrates α 2^-/-、α2^+/-And wild type (α 2)^+/+) Sleep profile of;

FIG. 5B illustrates α 2^-/-、α2^+/-And wild type (α 2)^+/+) Daytime and nighttime duration of time;

FIG. 5C illustrates β 2^-/-、β2^+/-And wild type (β 2)^+/+) Sleep profile of;

FIG. 5D illustrates β 2^-/-、β2^+/-And wild type (β 2)^+/+) Daytime and nighttime duration of time;

FIGS. 6A-6D illustrate sleep in the context of nAChR α 2KO and nAChR β 2KO androgens;

figures 7A-7F illustrate the sleep in vivo balance (A, B), circadian phase (C, D), and stimulus-induced wakefulness (E, F) phenotypes of nAChR α 2KO and nAChR β 2 KO;

figures 8A-8B illustrate the sleep phenotype achieved by RNAi with genetic knockdown of nAChR α 2 and nAChR β 2;

FIGS. 9A-9F illustrate the expression patterns of nAChR α 2 and nAChR β 2 in the brain and the Ventral Nerve Cord (VNC);

fig. 10A to fig. 10I illustrate expression patterns of nAChR α 2 and nAChR β 2 in brain;

figure 11A illustrates a schematic representation of nAChR α 2 and nAChR β 2 in the genome of drosophila;

fig. 11B illustrates a graphical representation of the crossover strategy between nAChR α 2 and X;

FIG. 11C illustrates β 2KIGal4/α 2KILexA, LexAop-Flp, UAS-FRT-terminator Sequence (STOP) -FRT-mCD8: GFP expression pattern;

11D-11E illustrate that nAChR α 2 and β 2 act together to promote sleep;

FIGS. 12A-12B illustrate the expression pattern of T β HKIGAL4/α 2KILexA, LexAop-Flp, UAS-FRT-terminator sequence-FRT-mCD 8: GFP;

figure 12C illustrates that reintroduction of nAChR α 2 into nAChR α 2-expressing cells rescues the nAChR α 2 mutant from nighttime sleep loss;

figure 12D illustrates that reintroduction of nAChR α 2 into octopamine-capable cells rescues the nAChR α 2 mutant from sleep deficits;

figure 12E illustrates that knockdown of nAChR α 2 in octopamine-capable cells significantly reduced the duration of nighttime sleep;

figure 12F illustrates that knockdown of T β H in nAChR α 2-expressing cells significantly reduced night sleep duration;

fig. 13A-13B illustrate that reintroduction of nAChR α 2 in serotonergic (serotonergic) neurons failed to rescue sleep deficits.

Detailed Description

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. nAChR alpha 2 and nAChR beta 2

There are two types of receptors for the neurotransmitter acetylcholine (ACh): nicotinic (nicotinic) AChR (nachr) and muscarinic (muscarinic) AChR. nachrs are ligand-gated ion channels composed of five subunits. In mammals, the nAChR subunits include 16 nAChR subunits (nAChR α 1 to α 7, nAChR α 9, nAChR α 10, nAChR β 1 to β 4, nAChR γ, nAChR) and 5nAChR (CHRM1 to M5). In insects, the nAChR subunits include 10 nAChR subunits (nAChR α 1 to α 7, nAChR β 1 to β 3) and 3 machrs (mAChRA, mAChRB, mAChRC).

As used herein, the term "nAChR α 2" generally refers to a gene encoding a nicotinic acetylcholine receptor α 2 subunit (nAChR α 2). nAChR α 2 includes homologs, fragments, derivatives, variants, or orthologs of nAChR α 2 having activity of nAChR α 2. The nAChR α 2 gene is conserved in humans, chimpanzees, rhesus monkeys, dogs, cows, mice, rats, chickens, mosquitoes and frogs. In some cases, nAChR α 2 can be Drosophila melanogaster (Drosophila melanogaster) nAChR α 2, such as the nucleotide sequence of the nAChR α 2 protein in the NCBI database with accession numbers NP _524482 and NP _ 733001. The nAChR α 2 protein is encoded by the nucleotide sequence of CHRNA2 (nicotinic cholinergic receptor α 2) gene in humans, such as CHRNA2 with accession No. NM — 000742.4 in NCBI database.

As used herein, the term "nAChR β 2" generally refers to a gene encoding the β 2 subunit of nicotinic acetylcholine receptor (nAChR β 2). The term includes homologs, fragments, derivatives, variants or orthologs of nAChR β 2 having activity of nAChR β 2. In some cases, nAChR β 2 can be drosophila melanogaster nAChR β 2, such as the nucleotide sequence of nAChR β 2 with accession number NM — 079759.4 in the NCBI database.

A polynucleotide sequence or polypeptide sequence that is a "homolog" or "ortholog" of another sequence refers to a protein that performs substantially the same function in another species under test and shares substantial sequence identity, such that they are recognized in the art as being different forms of the same protein, differing primarily in the species in which they are found. Thus, for example, human nAChR α 2, mouse nAChR α 2, rat nAChR α 2, and fly nAChR α 2 are all considered homologs or orthologs of each other. Two polynucleotide or polypeptide sequences are considered to have substantial identity if, when optimally aligned (gaps are tolerated), they share at least about 50% sequence identity, or if the sequences share a defined functional motif (motifs). In alternative embodiments, optimally aligned sequences may be considered substantially identical (i.e., have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over a particular region.

The terms "identity" and "identity" refer to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each site in the aligned sequences. The degree of identity of an amino acid sequence or a nucleotide sequence is a function of the number of identical or matching amino acids or nucleotides at sites shared by the sequences, e.g., in a particular region. Alignment for the purpose of determining percent nucleotide sequence identity may be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or megalign (dnastar) software. One skilled in the art can determine parameters suitable for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

In some embodiments, the nAChR α 2 and/or nAChR β 2 of the present application can be drosophila melanogaster nAChR α 2 and/or nAChR β 2 or orthologs thereof. For example, nAChR α 2 and/or nAChR β 2 can be derived from Drosophila suzuki (Drosophila kii), Drosophila (Drosophila simulans), Drosophila seoula (Drosophila secellia), Homo sapiens (Homo sapiens), mouse (Mus musculus), limous (Rattus norvegicus), xenopus laevis (xenopus tropicalis), zebrafish (Danio rerio), as long as they have the same activity in those species. The nAChR α 2 protein and/or nAChR β 2 protein can be expressed in the central and peripheral nervous system, muscle, and many other tissues of many organisms.

Activity of nAChR alpha 2 and/or nAChR beta 2

As used herein, the term "functional" generally refers to having, being associated with, or being a function of regulating sleep.

As used herein, the term "octopamine" generally refers to an organic chemical. It can act as a neurotransmitter in many types of invertebrates. Octopamine performs a function similar to that of norepinephrine (norepinephrine) in mammals. The functions may include mobilizing the body and nervous system to achieve an action. In drosophila melanogaster, octopamine is synthesized from Tyramine (Tyramine) by Tyramine (Tyramine) β hydroxylase or T β H.

As used herein, the term "octopamine-capable cell" generally refers to a cell that expresses octopamine and/or an octopamine synthase (T β H). Octopamine is the insect equivalent of noradrenaline in mammals. Octopamine-capable cells can be in the invertebrate nervous system. In some embodiments, octopamine-capable cells may be widely expressed in the brain and Ventral Nerve Cord (VNC), such as Antenna Lobe (AL), ventral unpaired intermediate (VUM) cells of occipital gyrus (SOG), anterolateral superior temporal preconcephalon (ASM) cells of superior nerve fibers, anterior brain bridge (PB) cells, and ventral preconcephalon (VL) cells of ventral nerve fibers (VLNP).

As used herein, the term "neuronal cell" generally refers to a nervous system cell. In mammals, neuronal cells may include cholinergic (cholinergic) neurons, GABAergic (GABAergic) neurons, glutamatergic (glutamatergic) neurons, dopaminergic (dopaminergic) neurons, noradrenergic (noradrenergic) neurons, and serotonergic (serologic) neurons in a plurality of regions, including brainstem (brain stem), anterior hypothalamus (anterior hypothalamus), lateral hypothalamus (lateral hypothalamus), and basal forebrain (basal forebrain). In insects, neuronal cells may include cholinergic neurons, gabaergic neurons, glutamatergic neurons, dopaminergic neurons, octopaminergic neurons, and serotonergic neurons in a variety of regions, including dorsal sector (dorsal fan-shaped body), ellipsoid (ellipsoid body), mycosis (mushroom body), and interphalangeal (parsnterebrilis).

The nAChR α 2 and/or nAChR β 2 subunits may be capable of forming functional receptor complexes with themselves or with other subunits, such as nAChR α 1- α 7, nAChR α 9, nAChR α 10, nAChR β 1- β 4, nAChR γ, nAChR, CHRM 1-M5, mAChRA, mAChRB, mAChRC. In some embodiments, nAChR α 2 and/or nAChR β 2 can form a functional nAChR α 2 β 2 receptor complex. In some embodiments, the functional nAChR α 2 β 2 receptor complex may be a heteromer, such as a heteromentamer. In some embodiments, a functional nAChR α 2 β 2 receptor complex can comprise at least one nAChR α 2 (e.g., at least two nAChR α 2, at least three nAChR α 2, at least four nAChR α 2) and at least one nAChR β 2 (e.g., at least two nAChR β 2, at least three nAChR β 2, at least four nAChR β 2). In some embodiments, functional nAChR α 2 β 2 receptor complexes can comprise other nAChR subunits, such as nAChR α 1, nAChR α 3, nAChR α 4, nAChR α 5nAChR α 6, nAChR α 7, nAChR α 9, nAChR α 10, nAChR β 1, nAChR β 4, nAChR β 3, nAChR γ, nAChR. In some embodiments, the functional nAChR α 2 β 2 receptor complex is functional in sleep.

In some embodiments, nAChR α 2 and/or nAChR β 2 may be capable of increasing the activity, release, and/or quantity of octopamine, e.g., increasing the activity, release, and/or quantity of an octopamine synthase.

In some embodiments, nAChR α 2 and/or nAChR β 2 may be capable of activating octopamine-capable signaling. As used herein, the term "octopaminergic signaling" refers to signaling involving octopamine. In some embodiments, nAChR α 2 and/or nAChR β 2 can activate signaling pathways involving octopamine.

Activity and/or expression of nAChR α 2 and/or nAChR β 2 can be associated with sleep. Sleep may include daytime sleep and nighttime sleep. Relationships can be assessed using knock-out and/or knock-in mutants obtained by any genetic engineering method in the art (e.g., CRISPR, RNAi, homologous reorganization), such as nAChR α 2 knock-out and/or nAChR β 2 knock-out mutant flies. In some embodiments, an increase in activity and/or expression of nAChR α 2 and/or nAChR β 2 can promote sleep. In some embodiments, a decrease in activity and/or expression of nAChR α 2 and/or nAChR β 2 can reduce sleep. In some embodiments, nAChR α 2 activity and/or expression may be associated with nocturnal and daytime sleep. In some embodiments, nAChR α 2 activity and/or expression may be associated with nocturnal sleep.

Detection method

The activity and/or expression of nAChR α 2 and/or nAChR β 2 can be determined in a quantitative manner using methods known in the art including, but not limited to, immunohistochemical analysis, PCR, RT-PCR, in situ hybridization, southern blot, western blot, northern blot, spectrophotometry, gene chip, flow cytometry (FACS), protein chip, DNA sequencing, and ELISA. In some cases, methods can include primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2. The primer may be a pair of primers. In addition, the methods can include probes that are capable of specifically recognizing nAChR α 2 and/or nAChR β 2. The probe may be capable of binding to a nAChR α 2 and/or nAChR β 2 nucleotide sequence or a fragment thereof, but not another nucleotide sequence. The probe may have a detectable signal. In other instances, methods can include agents capable of specifically recognizing nAChR α 2 and/or nAChR β 2 proteins and/or agents capable of determining the activity of nAChR α 2 and/or nAChR β 2 proteins, such as antibodies and/or ligands and/or fragments thereof to nAChR α 2 and/or nAChR β 2 proteins.

In some cases, expression of nAChR α 2 and/or nAChR β 2 can be detected by methods known in the art, such as immunofluorescence, immunohistochemical analysis, and confocal imaging. Detection may be facilitated by coupling the target to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups (prosthetic groups), fluorescent substances (fluorescent materials), luminescent substances (luminescent materials), bioluminescent substances (bioluminescent materials), and radioactive substances. Examples of the aptamer include horseradish peroxidase (horse radish peroxidase), alkaline phosphatase (alkaline phosphatase), β -galactosidase (β -galactosidase), or acetylcholinesterase (acetylcholinesterase); examples of suitable prosthetic group complexes include streptavidin/biotin (streptavidin/biotin) and avidin/biotin (avidin/biotin); examples of suitable fluorescent substances include umbelliferone (umbelliferone), fluorescein (fluorescein), fluorescein isothiocyanate (fluorescein isothiocyanate), rhodamine (rhodamine), dichlorotriazinylamine fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride (dansyl chloride) or phycoerythrin (phytoerythrin); an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase (luciferase), luciferin (luciferin) and aequorin (aequorin). Examples of antibodies for immunohistochemical analysis may be GFP, GRP, nc82, AlexaFluor488 chicken antibody, AlexaFluor633 mouse antibody.

Regulating sleep

As used herein, the term "adjust sleep" generally refers to regulating the time and duration of sleep. Sleep can be divided into Rapid Eye Movement (REM) sleep and non-REM (nrem) sleep that differ in electroencephalogram (EEG), Electromyogram (EMG), and levels of arousal. Adjusting sleep includes promoting sleep and/or reducing sleep. For example, adjusting sleep may include extending daytime sleep time, extending nighttime sleep time, shortening daytime sleep time, shortening nighttime sleep time, increasing duration of daytime sleep, increasing duration of nighttime sleep, decreasing duration of daytime sleep, decreasing duration of nighttime sleep, enhancing the degree of nighttime sleep, decreasing the degree of daytime sleep, and/or decreasing the degree of nighttime sleep.

As used herein, the term "deprivation" generally refers to a condition where there is insufficient sleep. Sleep deprivation techniques in the art include light touch, single platform, multiple platforms, modified multiple platforms, and swing. For example, sleep deprivation may be performed by random shaking throughout the night, where a rebound rate (rebound rate) is calculated to assess the degree of sleep deprivation.

As used herein, the term "circadian rhythm" (circadian rhythm) generally refers to a period of approximately 24 hours in the physiological process of organisms including plants, animals, fungi and cyanobacteria. In some cases, the circadian rhythm may be endogenously produced. In other cases, the circadian rhythm may be modulated by external cues such as sunlight and temperature. Circadian rhythms can be analyzed by transitional activity in constant darkness.

As used herein, the term "arousal" refers to a state of waking or a state in which a sensory organ is stimulated to reach a certain point of perception. The arousal analysis method can include applying a brief and specific intensity of light or a brief and specific intensity of mechanical stimulation during sleep at a specific time of administration factor, and video recording the response after the stimulation.

As used herein, the term "does not substantially affect (do not substantially affect) generally means that, after treatment with an agent, the phenotype does not differ significantly from the wild type.

In one aspect, the present application provides a method for selecting an agent for use in modulating sleep. The method comprises the following steps: providing a candidate agent; determining the effect of the candidate agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2; and selecting said candidate agent as an agent for modulating sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is altered by said candidate agent.

In some embodiments, the method may be an in vitro (in vitro) method or an ex vivo (ex vivo) method. The methods can include using cells, cell lines, or primary cell cultures that express nAChR α 2 and/or nAChR β 2. The cells may be from a human, such as stem cells or human neural cells. In some cases, the methods can be performed using tissues and/or cells that contain or correspond to nAChR α 2 and/or nAChR β 2 expression, such as neuronal cells, brain regions, or other tissues. In some cases, the cell may be a neural cell from any suitable species, such as a fly neural cell, a mouse neural cell, and/or a zebrafish neural cell. The method may comprise contacting the agent with the tissue and/or cells. The cells can be incubated with the agent and transfected with a vector comprising the agent. For example, tissues and/or cells can be cultured in vitro or ex vivo, and then candidate substances can be applied to the cultured tissues and/or cells, and after an appropriate period of incubation (e.g., hours, days, weeks, or months), the expression levels of nAChR α 2 and/or nAChR β 2 can be examined using the methods described herein.

Subsequently, the expression level or activity of nAChR α 2 and/or nAChR β 2 is determined. Assay techniques can be performed as described herein. A candidate agent can be an agent for promoting sleep if nAChR α 2 and/or nAChR β 2 activity is increased as compared to a control; if the activity of nAChR α 2 and/or nAChR β 2 decreases, then the candidate agent can be an agent for reducing sleep.

In some embodiments, the agent can affect the activity and/or expression of nAChR α 2 and/or nAChR β 2 in an octopamine-capable cell. In a certain embodiment, the octopamine-competent cell comprises an octopamine-competent neuronal cell.

In some embodiments, the agents of the present application do not substantially affect sleep recovery after deprivation. For example, the cumulative rebound rate of knock-out flies is about 80% to about 120%, e.g., about 90% to about 110%, about 95% to about 105%, or about 100% of the cumulative rebound rate of wild type, as measured in a behavioral analysis.

In some embodiments, the agents of the present application do not substantially affect circadian rhythms. For example, the cycle length of the knockout fly in constant darkness is about 80% to about 120%, e.g., about 90% to about 110%, about 95% to about 105%, or about 100% of the length of that period of the wild type, as measured in a circadian analysis.

In some embodiments, the agents of the present application do not substantially affect wakefulness. For example, the wake rate of a knockout fly is about 80% to about 120%, e.g., about 90% to about 110%, about 95% to about 105%, or about 100% of that of the wild type, as measured in a wake assay.

In some embodiments, the agent may include a small molecule, protein, and/or polynucleotide. In some embodiments, the agent acts directly on a nucleic acid encoding a nAChR α 2 protein and/or a nAChR β 2 protein. The nucleic acids encoding nAChR α 2 protein and/or nAChR β 2 protein can be natural or synthetic nucleic acids, including DNA and RNA, such as cDNA, antisense (antisense), and mRNA.

Sleep disorders

In another aspect, the present application provides a method for treating, preventing or delaying the progression of a sleep disorder.

As used herein, the term "sleep disorder" refers generally to any condition that would benefit from treatment with an agent of the present invention, including any sleep disease or disorder that can be treated by an effective amount of an agent described herein. Sleep disorders may include endogenous sleep disorders (intrinsic sleep disorders), exogenous sleep disorders (extrinsic sleep disorders), and circadian rhythm sleep disorders (circadian rhythm sleeping disorders). Examples of endogenous sleep disorders include psychophysiological insomnia (psychophysiological malfunction), sleep state perception error (sleep state perturbation), idiopathic insomnia (idiopathetic insomnia), narcolepsy (narcolepsy), recurrent hypersomnia (recurrent hypersomnia), idiopathic hypersomnia (idiopathetic hypersomnia), post-traumatic hypersomnia (post-traumatic hypersomnia), obstructive sleep apnea syndrome (acquired sleep apnea syndrome), central sleep apnea syndrome (central sleep apnea syndrome), central alveolar hypoventilation (central alveolar hypoplasia), periodic limb movement disorder (peripheral movement disorder), restless leg syndrome (syndrome, moisture s), and the like. Examples of the extrinsic sleep disorder include sleep hygiene disorder (acquired sleep disorder), environmental sleep disorder (environmental sleep disorder), insomnia at high altitude (acquired insomnia), sleep disorder adjustment (acquired sleep disorder), insufficient sleep syndrome (acquired sleep syndrome), restricted sleep disorder (limit-sleeping disorder), sleep-on-associated disorder (sleep-on-association disorder), food-allergic insomnia (food-induced insomnia), night-eating/night-drinking syndrome (nociceptive/drinking syndrome), hypnotic-dependent sleep disorder (sleeping-dependent sleep disorder), irritant-dependent sleep disorder (sleep-dependent disorder), sleep disorder (sleep-induced toxin-induced disorder), and the like. Examples of circadian rhythm sleep disorders include time-zone change (jet lag) syndrome, shift work sleep disorder (shift work sleep disorder), irregular sleep-wake pattern (irregular sleep/wake pattern), delayed sleep phase syndrome (delayed sleep-phase syndrome), advanced sleep phase syndrome (advanced sleep-phase syndrome), non-24-hour sleep-wake disorder (non-24-home sleep/wake disorder), and the like.

As used herein, the term "insomnia" (insomnia) generally refers to a sleep disorder in which people have sleep distress.

As used herein, the term "sleep loss" generally refers to a sleep disorder that does not have sufficient sleep.

As used herein, the term "narcolepsy" generally refers to a sleep disorder characterized by excessive drowsiness, sleep paralysis, hallucinations, and, in some cases, the occurrence of cataplexy events.

As used herein, the term "NREM" generally refers to a sleep stage that can be distinguished by little or no eye movement.

As used herein, the term "REM" generally refers to a sleep stage that is discernable by random/rapid eye movements.

As used herein, the term "parasomnias" generally refers to sleep disorders that involve abnormal movement, behavior, mood, perception, and dreaminess that occur while falling asleep, sleeping, between sleep stages, or during arousal from sleep.

In a certain embodiment, sleep disorders may include under-sleep and over-sleep. In some embodiments, sleep disorders may include daytime sleepiness deficiency, nighttime sleepiness deficiency, daytime sleepiness excess, and nighttime sleepiness excess. In some embodiments, the lack of sleep may include insomnia and loss of sleep associated with other diseases such as cardiovascular disorders and neurodegenerative diseases, and the like. In other embodiments, hypersomnia may include narcolepsy, NREM/REM related drowsiness, and hypersomnia/difficult to wake associated with other diseases such as cardiovascular disorders and neurodegenerative diseases.

The term "treatment" as used herein refers to curative therapy (curative therapy), prophylactic therapy (therapeutic therapy) and prophylactic therapy (prophylactic therapy). Continuous treatment or administration refers to treatment on at least a daily basis, with no interruption in treatment for one or more days. Intermittent treatment or administration or treatment or administration in an intermittent manner means that the treatment is not continuous, but periodic in nature. Treatment according to the methods of the invention may result in complete remission or cure of the disease or condition, or partial amelioration of one or more symptoms of the disease or condition, and may have a temporary or substantially permanent effect.

As used herein, the term "prevention" means alleviation of the symptoms of the mentioned disorders. In particular, the term encompasses the full range of therapeutic positive effects of administering the agents of the present application to a subject, including alleviating, moderating, and relieving sleep disorders, such as insufficient or excessive sleep. The term "preventing" includes preventing or delaying the development of a disease, preventing or delaying the development of a symptom, and/or lessening the severity of the symptom that will develop or is expected to develop. These further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing latent causes of symptoms.

Methods for sleep disorders

The term "effective amount" as used herein generally refers to a dosage sufficient to provide a concentration high enough to impart a beneficial effect to its recipient. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease or condition being treated, the severity of the disease or condition, the activity of the specific components, the route of administration, the rate of clearance, the duration of treatment, the age, weight, sex, diet, and general health of the subject, among other relevant factors.

In some embodiments, a method may comprise administering to a subject in need thereof a therapeutically effective amount of an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject. The agents (and any additional therapeutic agents) used in the methods of the invention may be administered by any suitable means, including parenterally, intrapulmonary and intranasally, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

In some embodiments, the agent may be administered to a non-human organism or a living portion thereof. In some embodiments, the agent can be administered to a tissue derived from a non-human organism or a living portion thereof. In some embodiments, the method may be a gene therapy method. In some cases, primary cells are obtained from a subject, a vector is administered to the cells to produce transduced, infected or transfected recombinant cells, and the recombinant cells are re-administered into the same or a different subject.

In some embodiments, administration may comprise delivering a vector for recombinant protein expression into the cultured cell or cells and/or into a cell or organ of the subject. Vectors for recombinant protein or polypeptide expression may be introduced into cells by: transfection (transfection), which generally means the insertion of heterologous DNA into cells by physical means (e.g. calcium phosphate transfection, electroporation, microinjection or lipofection); infection (infection), which generally refers to introduction by an infectious agent, i.e., a virus; or transduction, which generally means stable infection of cells with a virus or transfer of genetic material from one microorganism to another by viral agents, such as bacteriophages. In a clinical setting, the agent delivery system can be introduced into the subject by any of a number of methods, each of which is well known in the art. For example, the drug formulation of the agent delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the target cells occurs primarily due to transfection specificity provided by the agent delivery vector, cell-type or tissue-type expression due to transcriptional regulatory sequences that control expression of the nucleic acid molecule, or a combination thereof.

In addition, the agent may be delivered in an acceptable diluent, or the delivery system may comprise a slow release matrix in which the delivery vehicle is embedded. Alternatively, where the complete agent delivery system can be produced intact from recombinant cells, such as a retroviral package, the pharmaceutical preparation may comprise one or more cells which produce the agent delivery system. In the latter case, the method of introducing viral packaging cells (viral packaging cells) may be provided by, for example, a refillable device or a biodegradable device. Various slow release polymeric devices have been developed in recent years and tested in vivo for controlled delivery of drugs including protein biopharmaceuticals, and by manipulating the polymeric composition and form, the devices may be adapted to release viral particles. A variety of biocompatible polymers (including hydrogels), including both biodegradable polymers and non-degradable polymers, can be used to form implants for the sustained release of viral particles from cells implanted at a particular target site. The embodiments of the invention may be used to deliver exogenous purified virus that has been incorporated into a polymerization device, or to deliver viral particles produced by cells encapsulated in a polymerization device.

In some embodiments, an agent can include a nucleic acid molecule encoding nAChR α 2 and/or nAChR β 2 or an expression product thereof. In some embodiments, the agent may comprise a nucleic acid sequence as set forth in any one of SEQ ID nos. 1-20. In some embodiments, administration of an agent to a subject in need thereof can increase the activity and/or expression of nAChR α 2 and/or nAChR β 2.

In another aspect, the present application provides a system for selecting an agent for modulating sleep, wherein the system comprises a substance capable of determining the effect of the agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2. The agent may be capable of determining the activity and/or expression of nAChR α 2 and/or nAChR β 2 nucleic acids. In some embodiments, the agent can include primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2. The agent may be capable of determining the activity and/or expression of a protein of nAChR α 2 and/or nAChR β 2. In some embodiments, the agent can include an agent capable of specifically recognizing nAChR α 2 and/or nAChR β 2 proteins and/or an agent capable of determining the activity of nAChR α 2 and/or nAChR β 2 proteins.

Possibility of sleep disorder

In another aspect, the present application provides a method of determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder. Methods can include assaying nAChR α 2 and/or nAChR β 2 for activity and/or expression. The assay technique may be a method as described herein. If the activity and/or expression level of nAChR α 2 and/or nAChR β 2 is higher in a subject, then the subject may have a sleep disorder, and/or be at risk of having a sleep disorder, e.g., sleep inefficiency, as compared to a control. A subject may have, and/or be at risk of having, a sleep disorder, e.g., hypersomnia, if the activity and/or expression level of nAChR α 2 and/or nAChR β 2 in the subject is low, as compared to a control. As used herein, the term "at risk of having a sleep disorder" generally refers to a higher likelihood of having a sleep disorder than a control.

Non-human model

The term "interfering RNA (RNAi)" is used herein to refer to double-stranded RNA that causes catalytic degradation of a particular mRNA and, thus, can be used to inhibit/reduce the expression of a particular gene.

In another aspect, the present application provides a non-human organism, or living portion thereof, comprising functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2. The non-human organism may be an insect, such as a marine centipede (Strigamia maritima), a scleroderma scapulae (Ixodes scapularis), a silkworm (Bombyx mori), a butterfly asterias (Danaus plexippus), a housefly (Musca domestica), a tsetse fly (Glossina morsitians) and/or a Drosophila species. In some embodiments, the non-human organism may be a Drosophila species, such as Drosophila melanogaster (Drosophila melanogaster), Drosophila suzukii (Drosophila suzukii), Drosophila melanogaster (Drosophila simulans), Drosophila ficus (Drosophila necta), Drosophila seolus (Drosophila secellia), samba pela (Drosophila yakuba), Drosophila pepromia (Drosophila anhuba), Drosophila ananas (Drosophila ananasosa), Drosophila melanogaster (Drosophila pseudoscia), Drosophila melanogaster (Drosophila persimilimilis), Drosophila willstonia (Drosophila willingsoni), Drosophila willebrand (Drosophila willebra), Drosophila melanogaster (Drosophila javanica), Drosophila griffii (Drosophila girophila griffii). In some embodiments, the non-human organism is drosophila melanogaster, or a western bee (Apis mellifera).

In some embodiments, the non-human organism or living portion thereof may not comprise any functional nAChR α 2 and/or nAChR β 2. The non-human organism of the present application may be produced by: the nAChR α 2 and/or nAChR β 2-free heterologous nucleic acid sequence is introduced, for example, into a fertilized egg, unfertilized egg, sperm, primordial germ cell, oogonium, oocyte, spermatogonium, spermatocyte and/or sperm cell of a non-human organism, for example, at an initial stage in embryonic development of the fertilized egg (e.g., prior to the 8-cell stage). Heterologous nucleic acid sequences can be introduced by gene transfer methods such as calcium phosphate co-precipitation, electroporation, lipofection, agglutination, microinjection, gene gun (particle gun) and/or DEAE-dextran methods. The heterologous nucleic acid sequence can also be introduced into somatic cells, tissues and/or organs of flies (e.g., by gene transfer methods), which can then be further cultured and/or maintained in the engineered somatic cells, tissues and/or organs. The engineered cells can also be fused to an embryo or another cell (such as a cell from the germline of a non-human organism) by a cell fusion method to produce a non-human organism of the present application.

In the methods for generating model animals of the present application, nuclease agents can be used to help modify a target gene locus (target gene loci). Such nuclease agents can promote homologous recombination between the donor nucleic acid molecule and the target genomic locus. In some embodiments, the nuclease agent can comprise an endonuclease agent.

As used herein, the term "recognition site for an a nucleic acid agent" generally refers to a DNA sequence in which a nick or double-strand break can be induced by a nuclease agent. The recognition site for the nuclease agent can be endogenous to the cell (or native), or the recognition site can be exogenous to the cell. In some embodiments, the recognition site may be exogenous to the cell, and thus not naturally present in the genome of the cell. In other embodiments, the exogenous or endogenous recognition site may be present only once in the genome of the host cell. In particular embodiments, endogenous or native sites that occur only once within the genome may be identified. Such sites can then be used to design nuclease reagents that will create nicks or double strand breaks at the endogenous recognition sites.

The length of the recognition site can vary, and includes, for example, recognition sites that are at least 4, 6, 8, 10, 12, 14, 16, 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 or more nucleotides in length. In one embodiment, each monomer of the nuclease agent can recognize a recognition site having at least 9 nucleotides. In other embodiments, the recognition site can be about 9 to about 12 nucleotides, about 12 to about 15 nucleotides, about 15 to about 18 nucleotides, or about 18 to about 21 nucleotides in length and any combination of the sub-ranges (e.g., 9-18 nucleotides). The recognition site may be palindromic, that is to say the sequence on one strand is read identically to the complementary strand in the opposite direction. It will be recognized that a given nuclease agent can bind to a recognition site and cleave that binding site, or alternatively, a nuclease agent can bind to a sequence different from the recognition site. Furthermore, the term recognition site may include both nuclease agent binding sites and nicking/cleavage sites regardless of whether the nicking/cleavage site is inside or outside of the nuclease agent binding site. In another variation, cleavage by nuclease agents may occur at nucleotide positions that are closely opposed to each other to create blunt end cuts (or in other cases, the cuts may be staggered to create single-stranded overhangs also known as "sticky ends," which may be 5' -end overhangs (5 ' -overhangids) or 3' -end overhangs.

Any nuclease agent that induces a nick or double strand break in the desired recognition site can be used in the methods of the present application. Naturally occurring or natural nuclease agents can be employed, so long as the nuclease agent induces a nick or double-strand break in the desired recognition site. Alternatively, modified or engineered nuclease agents may be employed. An "engineered nuclease agent" includes a nuclease that is engineered (modified or derived) from its native form to achieve specific recognition and induce nicks or double strand breaks in the desired recognition sites. Thus, the engineered nuclease agent can be derived from a naturally occurring nuclease agent, or it can be artificially created or synthesized. The nuclease agent can be modified by as little as one amino acid in a protein cleavage agent or one nucleotide in a nucleic acid cleavage agent. In some embodiments, the engineered nuclease may induce nicks or double-strand breaks in a recognition site, wherein the recognition site is not a sequence that would have been recognized by a native (non-engineered or non-modified) nuclease agent. Creating nicks or double-strand breaks in a recognition site or other DNA may be referred to herein as "cleaving" or "cleaving" the recognition site or other DNA.

In some embodiments, the Nuclease agent can be a transcription activator-Like Effector Nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genome of prokaryotic or eukaryotic organisms. TAL effector nucleases can be created by fusing a natural or engineered transcription activator-like (TAL) effector, or functional portion thereof, to an endonuclease, such as, for example, a catalytic domain like FokI. Unique modular TAL effector DNA binding domains allow for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domain of TAL effector nucleases can be engineered to recognize specific DNA target sites, thus serving to create a double-strand break at a desired target sequence. See WO 2010/079430; morbitzer et al (2010) PNAS 10.1073/pnas.1013133107; scholze and Boch (2010) Virulence 1: 428-; christian et al Genetics (2010)186: 757-; li et al (2010) Nuc. acids Res. (2010) doi:10.1093/nar/gkq 704; and Miller et al (2011) Nature Biotechnology 29: 143-148; all of which are incorporated herein by reference.

In some embodiments, the nuclease agent can be a Zinc Finger Nuclease (ZFN). For example, each monomer of a ZFN may comprise 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain may bind a 3 base pair (bp) subsite. In other embodiments, the ZFNs may be chimeric proteins comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In some embodiments, the independent endonuclease can be a fokl endonuclease. In some embodiments, the nuclease agent can comprise a first ZFN and a second ZFN, wherein the first ZFN and the second ZFN are each operably linked to a fokl nuclease, wherein the first ZFN and the second ZFN recognize two consecutive target DNA sequences separated by about 6 base pairs to about 40 base pair cleavage sites or about 5 base pairs to about 6 base pair cleavage sites in each strand of the target DNA sequence, and wherein the fokl nucleases dimerize and create a double-strand break. See, e.g., US 20060246567; US 20080182332; US 20020081614; US 20030021776; WO/2002/057308A 2; US 20130123484; US 20100291048; and WO/2011/017293A2, each of which is incorporated herein by reference.

In some embodiments, the nuclease agent can be a megabase meganuclease (meganuclease). Megabase meganucleases have been classified into four families based on conserved sequence motifs, the families being the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in coordination of metal ions and hydrolysis of phosphodiester bonds. HE enzymes are known for their long recognition sites and for allowing some sequence polymorphisms (sequence polymorphisms) in their DNA substrates. Megabase meganuclease domains, structures and functions are known, see, e.g., Guhan and Muniyappa (2003) Crit. Rev Biochem Mol Biol 38: 199-248; lucas et al, (2001) Nucleic Acids Res29: 960-9; jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; stoddard, (2006) Q Rev biophyls 38: 49-95; and Moure et al, (2002) Nat Struct Biol 9: 764.

In some embodiments, the nuclease agent employed in the methods of the present application can employ a CRISPR/Cas system. The system may employ, for example, Cas9 nuclease, which in some cases may be codon-optimized for the desired cell type in which it is to be expressed. The system may further employ a fusion crRNA-tracrRNA construct that functions with a codon-optimized Cas 9. This single RNA may often be referred to as a small guide RNA or sgRNA. Briefly, a short DNA fragment containing the target sequence can be inserted into the sgRNA expression plasmid. The sgRNA expression plasmid may comprise a target sequence (in some embodiments, about 20 nucleotides), some form of tracrRNA sequence (backbone), and a suitable promoter active in the cell and elements necessary for proper processing in eukaryotic cells, such as fly cells. The sgRNA expression cassette and Cas9 expression cassette can then be introduced into the cell. See, e.g., Mali P et al (2013) Science 2013, 2, 15; 339(6121) 823-6; jinek M et al Science 2012 8 month 17; 337(6096) 816-21; hwang W Y et al NatBiotechnol 2013 for 3 months; 31(3) 227-9; jiang W et al Nat Biotechnol 2013 for 3 months; 31(3) 233-9; and Cong L et al Science 2013, 2 months and 15 days; 339(6121) 819-23, each of which is incorporated herein by reference.

In some embodiments, the nAChR α 2 gene and/or nAChR β 2 gene in the organism is knocked down by RNAi. Double-stranded rna (dsrna) can be introduced into the cell (e.g., using short oligomeric small double-stranded interfering rna (siRNA) or a DNA plasmid from which siRNA can be transcribed). In practicing the methods, an effective amount of an RNAi agent is administered to a non-human organism to modulate the expression of a target gene in a desirable manner, e.g., to achieve an expected reduction in gene expression in the target cell. The RNAi agent employed in the present application is a small ribonucleic acid molecule, i.e., oligoribonucleotides (oligoribonucleotides), in a duplex structure, e.g., two different oligoribonucleotides hybridized to each other or a single ribonucleotide in a small hairpin configuration to produce a duplex structure. In some embodiments, wherein the RNA agent is a duplex structure of two different ribonucleic acids hybridized to each other, e.g., an siRNA. In some cases, the siRNA is introduced into the cytoplasm (e.g., a neuronal cell). In some embodiments, the siRNA may be derived from the inside of a cell. In other embodiments, the siRNA can be exogenously introduced into the cell.

The RNAi agent can be administered to the non-human organism using any suitable protocol, typically a nucleic acid administration protocol, wherein many different such protocols are known in the art.

In some embodiments, the nAChR α 2 gene in the organism is knocked down by α 2KIGal 4. nAChR α 2 was knocked down by expression of α 2RNAi under the control of α 2KIGal 4. In some embodiments, the nAChR β 2 gene in the organism is knocked down by β 2KIGal 4. nAChR β 2 was knocked down by expression of α 2RNAi under the control of β 2KIGal 4. In some embodiments, the fourth, fifth, and sixth exons of the nAChR α 2 gene in the organism are deleted. In some embodiments, the first to eighth exons of the nAChR β 2 gene in the organism are deleted.

In another aspect, the present application provides a cell, cell line or primary cell culture derived from a non-human organism or a living part thereof.

In another aspect, the present application provides a tissue derived from a non-human organism or living portion thereof. In some embodiments, the tissue is derived from neural tissue. In some embodiments, the tissue is derived from neural tissue comprising octopamine-competent cells.

In some embodiments, the non-human organism or living portion may be used in a method of selecting an agent for modulating sleep. In some embodiments, the methods can comprise administering an agent to a non-human organism or a living body part, and detecting the activity and/or expression of nAChR α 2 and/or nAChR β 2I. In some embodiments, the non-human organism or living portion may be used to screen for biomarkers suitable for diagnosing and/or monitoring sleep disorders. In some embodiments, the non-human organism or living body part may be used for the preparation of a system for screening substances, devices, compositions and/or biomarkers suitable for the treatment, diagnosis, prevention, monitoring and/or prognosis of sleep disorders.

Screening method

In another aspect, the present application provides a method of screening for a substance, device and/or composition suitable for use in the treatment, prevention and/or delay of progression of a sleep disorder, the method comprising applying a candidate substance, device and/or composition to a non-human organism, or living portion, cell line or primary cell culture or tissue thereof, of the present application, and determining the effect of the candidate substance, device and/or composition on one or more of: the duration of sleep, the activity, amount and/or release of octopamine, and the activation of octopamine signaling of the non-human organism.

In some embodiments, the method may be an in vitro method or an ex vivo method. For example, samples (e.g., cells, tissues or other DNA or RNA containing samples, protein containing samples, and/or metabolite containing samples) can be taken from a non-human organism or living portion thereof of the present application before and after a sleep disorder (e.g., sleep inefficiency and/or hypersomnia). Then, gene transcription products (transcriptome), gene translation products (proteome), or metabolites (metabolome) derived from the sample can be comprehensively measured, and substances that change before and after the sleep disorder can be identified.

Gene transcription products (e.g., transcriptomes) can be analyzed using nucleic acid microarrays, such as DNA microarrays. The gene translation products (e.g., proteomes) can be analyzed using gel electrophoresis such as two-dimensional gel electrophoresis (two-dimensional gel electrophoresis) or mass spectrometry such as time-of-flight mass spectrometry, electrospray ionization mass spectrometry, capillary HPLC/MS, and LC/MS. Metabolites (metabolome) can be analyzed using NMR, capillary electrophoresis (capillary electrophoresis), LC/MS and/or LC/MS/MS.

When the presence/amount of a substance shows a significant difference before and after a sleep disorder, such a substance can be considered as a biomarker for a sleep disorder, which can then be used for early diagnosis (in particular preclinical diagnosis) of a sleep disorder. The identified biomarkers can be further detected using specific reagents or detection methods. For example, when the biomarker is a protein or peptide, it can be detected using an immunoassay using specific antibodies. When the biomarker is a nucleic acid molecule (such as a transcript), it can be detected by Northern blot analysis using specific probes, or by RT-PCR using specific primers.

In another aspect, the present application provides a system for selecting an agent for use in modulating sleep. In some embodiments, a system may include a sales network providing for selling a composition comprising a medicament of the present application, and providing instructional material to a patient or physician regarding the use of the medicament in regulating sleep in a subject.

In some embodiments, a system can include determining a formulation and dose of an agent of the present application suitable for administration in a subject to adjust sleep, performing a therapeutic analysis on the formulation identified as described above with respect to efficacy and toxicity in an animal; and providing a sales network for selling preparations identified as having acceptable treatment characteristics as described above.

The system may further comprise a kit (kit). In some embodiments, a kit can comprise the agents of the present application in suitable packaging, as well as instructions, clinical study assays, side effects, and the like. The kit may also contain information indicating or confirming the activity and/or advantages of the composition, such as scientific references, package inserts, clinical trial results, and/or summaries of such similar information, and/or information on dosing regimens, administration, side effects, drug interactions, or other information useful to the health care provider. The system may further comprise another agent. In some cases, the agents of the present application are provided in separate containers within the kit.

In some cases, the system may be provided, sold, and/or sold to associated personnel, including healthcare providers, physicians, nurses, pharmacists, prescribers, drug developers, drug manufacturers, and the like. In other cases, the system may be sold directly to the consumer.

The present application may also include the following embodiments:

1. a method for selecting an agent for use in modulating sleep, the method comprising:

providing a candidate agent;

determining the effect of the candidate agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2; and is

Selecting said candidate agent as an agent for modulating sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is altered by said candidate agent.

2. The method of embodiment 1, wherein said candidate agent is selected as an agent for promoting sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is increased by said candidate agent.

3. The method of any one of embodiments 1-2, wherein said candidate agent is selected as an agent for reducing sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is reduced by said candidate agent.

4. The method according to any one of embodiments 1 to 3, wherein said nAChR α 2 is Drosophila melanogaster (Drosophila melanogaster) nAChR α 2 or an ortholog thereof.

5. The method of any one of embodiments 1-4, wherein said nAChR β 2 is drosophila melanogaster nAChR β 2 or an ortholog thereof.

6. The method of any one of embodiments 1-5, wherein said determining comprises: determining the effect of said candidate agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 in an octopamine-capable cell.

7. The method of embodiment 6, wherein the octopamine-competent cells comprise octopamine-competent neuronal cells.

8. The method of any one of embodiments 1-7, wherein said activity of said nAChR α 2 and/or nAChR β 2 comprises one or more of:

the ability to form a functional nAChR α 2 β 2 receptor complex;

the ability to increase the activity, release and/or amount of octopamine;

the ability to activate octopamine signaling.

9. The method of any one of embodiments 1-8, which is an in vitro method or an ex vivo method.

10. The method according to any one of embodiments 1-9, wherein said sleep comprises daytime sleep and/or nighttime sleep.

11. The method of any one of embodiments 1-10, wherein the agent does not substantially affect sleep recovery, circadian rhythm, or wakefulness following deprivation.

12. The method of any one of embodiments 1-11, wherein the agent comprises a small molecule, a protein, and/or a polynucleotide.

13. The method of any one of embodiments 1-12, wherein the agent acts directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acid encoding the nAChR α 2 protein and/or nAChR β 2 protein.

14. A system for selecting an agent for modulating sleep, wherein said system comprises a substance capable of determining the effect of said agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2.

15. The system of embodiment 14 wherein said agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acid.

16. The system of embodiment 15 wherein said agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acids comprise: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

17. The system of any one of embodiments 14-16, wherein said agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein.

18. The system of embodiment 17, wherein said agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprise: an agent capable of specifically recognizing said nAChR α 2 and/or nAChR β 2 protein and/or an agent capable of determining said activity of said nAChR α 2 and/or nAChR β 2 protein.

19. A method for treating, preventing or delaying progression of a sleep disorder, the method comprising:

administering to a subject in need thereof a therapeutically effective amount of an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

20. The method of embodiment 19, wherein said sleep disorder is associated with insufficient sleep and said agent is capable of increasing said activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

21. The method of any one of embodiments 19-20, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

22. The method of any one of embodiments 19-21, wherein the sleep disorder associated with insufficient sleep comprises insomnia (insomnia) and/or sleep loss (sleeploss) associated with a cardiovascular disorder and/or a neurodegenerative disease.

23. The method of any one of embodiments 19-22, wherein said agent comprises a nucleic acid molecule encoding nAChR α 2 and/or nAChR β 2 or an expression product thereof.

24. The method of any one of embodiments 19-23, wherein the agent comprises a nucleic acid sequence as set forth in any one of SEQ ID nos. 1-20.

25. The method of embodiment 24, wherein said sleep disorder is associated with hypersomnia and said agent is capable of decreasing said activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

26. The method of embodiment 25, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

27. The method of any one of embodiments 25-26, wherein the sleep disorder associated with hypersomnia comprises narcolepsy (narcolepsy), hypersomnia (hypersomnolence), NREM/REM-related parasympathetic (NREM/REM-related parasymias), and/or hypersomnia (oversleeping)/difficult to wake (hard-to-be-awaken) associated with cardiovascular disorders and/or neurodegenerative diseases.

28. The method of any one of embodiments 19-27, wherein said agent is an agent for promoting sleep in that said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is increased by said agent.

29. The method of any one of embodiments 19-28, wherein said agent is an agent for reducing sleep in that said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is reduced by said agent.

30. The method of any one of embodiments 19-29, wherein said nAChR α 2 is drosophila melanogaster nAChR α 2 or an ortholog thereof.

31. The method of any one of embodiments 19-30, wherein said nAChR β 2 is drosophila melanogaster nAChR β 2 or an ortholog thereof.

32. The method of any one of embodiments 19-31, wherein said alteration of said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is in an octopamine-capable cell.

33. The method of embodiment 32, wherein the octopamine-competent cells comprise octopamine-competent neuronal cells.

34. The method of any one of embodiments 32-33, wherein said activity of said nAChR α 2 and/or nAChR β 2 comprises one or more of:

the ability to form a functional nAChR α 2 β 2 receptor complex;

the ability to increase the activity, release and/or amount of octopamine;

the ability to activate octopamine signaling.

35. The method of any one of embodiments 19-34, which is an in vitro method, an in vivo method, or an ex vivo method.

36. The method of any one of embodiments 19-35, wherein the agent comprises a small molecule, a protein, and/or a polynucleotide.

37. The method of any one of embodiments 19-36, wherein the agent acts directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acid encoding the nAChR α 2 protein and/or nAChR β 2 protein.

38. Use of an agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 in the manufacture of a medicament for treating, preventing or delaying the progression of a sleep disorder.

39. The use of embodiment 38, wherein said agent is capable of determining the effect of said agent on the activity and/or expression of said nucleic acid of nAChR α 2 and/or nAChR β 2.

40. The use of embodiment 39, wherein said agent capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acids comprises: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

41. The use according to any one of embodiments 38 to 40, wherein said agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein.

42. The use of embodiment 40, wherein said agent that determines the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprises: an agent capable of specifically recognizing said nAChR α 2 and/or nAChR β 2 protein and/or an agent capable of determining said activity of said nAChR α 2 and/or nAChR β 2 protein.

43. An agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 for use in the treatment, prevention or delay of progression of a sleep disorder.

44. A method for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder, the method comprising:

assessing the activity and/or expression of nAChR α 2 and/or nAChR β 2 in said subject.

45. The method of embodiment 44, wherein said activity and/or expression of nAChR α 2 and/or nAChR β 2 comprises activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2, and/or activity and/or expression of a protein of said nAChR α 2 and/or nAChR β 2.

46. The method of embodiment 45, wherein the agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acid comprise: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

47. The method of any one of embodiments 45 to 46, wherein the agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprise: an agent capable of specifically recognizing said nAChR α 2 and/or nAChR β 2 protein and/or an agent capable of determining said activity of said nAChR α 2 and/or nAChR β 2 protein.

48. The method of any one of embodiments 44-47, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

49. The method of any one of embodiments 44-48, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

50. The method of any one of embodiments 44-49, wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

51. The method of embodiment 48, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

52. The method of any one of embodiments 50-51, wherein the sleep disorder associated with hypersomnia comprises narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

53. A system for determining the likelihood that a subject has, and/or is at risk of having, a sleep disorder, the system comprising:

an agent capable of indicating the activity and/or level of expression of nAChR α 2 and/or nAChR β 2 in said subject.

54. The system of embodiment 53, wherein said activity and/or expression of nAChR α 2 and/or nAChR β 2 comprises the activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2, and/or the activity and/or expression of a protein of said nAChR α 2 and/or nAChR β 2.

55. The system of embodiment 54, wherein the agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acid comprise: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

56. The system of any one of embodiments 54-55, wherein the agents capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprise: an agent capable of specifically recognizing said nAChR α 2 and/or nAChR β 2 protein and/or an agent capable of determining said activity of said nAChR α 2 and/or nAChR β 2 protein.

57. The system of any one of embodiments 53-56, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

58. The system of any one of embodiments 53-57, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

59. The system of any one of embodiments 53-58, wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

60. The system of embodiment 57, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

61. The system of any one of embodiments 59-60, wherein the sleep disorder associated with hypersomnia comprises sudden sleepiness, narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

62. Use of an agent capable of indicating the activity and/or level of expression of nAChR α 2 and/or nAChR β 2 in a subject in the manufacture of an indicator of the likelihood that the subject suffers from, and/or is at risk of suffering from, a sleep disorder.

63. The use of embodiment 62, wherein said activity and/or expression of nAChR α 2 and/or nAChR β 2 comprises activity and/or expression of a nucleic acid of said nAChR α 2 and/or nAChR β 2, and/or activity and/or expression of a protein of said nAChR α 2 and/or nAChR β 2.

64. The use of embodiment 63, wherein an agent capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acids comprises: primers capable of specifically amplifying nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

65. The use according to any one of embodiments 63 to 64, wherein the agent capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 protein comprises: an agent capable of specifically recognizing said nAChR α 2 and/or nAChR β 2 protein and/or an agent capable of determining said activity of said nAChR α 2 and/or nAChR β 2 protein.

66. The use of any one of embodiments 63-65, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

67. The use of any one of embodiments 63-66, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

68. The use of any one of embodiments 63-67, wherein the sleep disorder associated with insufficient sleep includes insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.

69. The use of embodiment 66, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

70. The use according to any one of embodiments 68-69, wherein the sleep disorder associated with hypersomnia comprises sudden sleep disorder, narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

71. A non-human organism or living part thereof comprising functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2.

72. The non-human organism or living portion thereof of embodiment 71, wherein the non-human organism is drosophila melanogaster.

73. The non-human organism or living portion thereof of any one of embodiments 71-72, which does not comprise any functional nAChR α 2.

74. The non-human organism or living portion thereof of any one of embodiments 71-73, which does not comprise any functional nAChR β 2.

75. The non-human organism or living part thereof of any one of embodiments 71-74, which is homozygous for functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2.

76. The non-human organism or living portion thereof of any one of embodiments 71-75, wherein the non-human organism has a reduced sleep duration compared to a corresponding wild-type non-human organism.

77. The non-human organism or living portion thereof of any one of embodiments 71-76, wherein said sleep comprises daytime sleep and/or nighttime sleep.

78. The non-human organism or living portion thereof of any one of embodiments 71-77, wherein a nAChR α 2 gene and/or nAChR β 2 gene in said organism is knocked-down or knocked-out.

79. The non-human organism or living portion thereof of any one of embodiments 71-78, wherein said nAChR α 2 gene and/or nAChR β 2 gene in said organism is knocked down by RNAi.

80. The non-human organism or living portion thereof of any one of embodiments 71-79, wherein the nAChR α 2 gene in said organism is knocked down by α 2KIGal 4.

81. The non-human organism or living portion thereof of any one of embodiments 71-80, wherein said nAChR β 2 gene in said organism is knocked down by β 2KIGal 4.

82. The non-human organism or living portion thereof of any one of embodiments 71-81, wherein the fourth, fifth and sixth exons of the nAChR α 2 gene in said organism are deleted.

83. The non-human organism or living portion thereof of any one of embodiments 71-82, wherein the first to eighth exons of the nAChR β 2 gene in said organism are deleted.

84. A cell, cell line or primary cell culture derived from a non-human organism or a living part thereof according to any one of embodiments 71 to 83.

85. A tissue derived from the non-human organism or living portion thereof of any one of embodiments 71-83.

86. The tissue of embodiment 85, wherein the tissue is derived from neural tissue.

87. The tissue of any one of embodiments 85-86, wherein the tissue is derived from neural tissue comprising octopamine-capable cells.

88. A method of screening for a substance, device and/or composition suitable for use in the treatment, prevention or delay of progression of a sleep disorder, the method comprising applying a candidate substance, device and/or composition to a non-human organism or living part thereof according to any one of embodiments 71-83, a cell, cell line or primary cell culture according to embodiment 84, or a tissue according to any one of embodiments 85-87, and determining the effect of the candidate substance, device and/or composition on one or more of:

a sleep duration of the non-human organism;

activity, amount and/or release of octopamine; and

activation of octopamine signalling.

89. The method of embodiment 88, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

90. The method of any one of embodiments 88-89, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

91. The method of any one of embodiments 88-90, wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

92. The method of embodiment 89, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

93. The method of any one of embodiments 91-92, wherein the sleep disorder associated with hypersomnia comprises narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

94. The method of any one of embodiments 88-93, wherein said determining comprises: determining the effect of said candidate substance, device and/or composition on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 in octopamine-capable cells.

95. The method of embodiment 94, wherein said octopamine-competent cells comprise octopamine-competent neuronal cells.

96. The method of any one of embodiments 94-95, wherein said nAChR α 2 is drosophila melanogaster nAChR α 2 or an ortholog thereof.

97. The method of any one of embodiments 94-96, wherein said nAChR β 2 is drosophila melanogaster nAChR β 2 or an ortholog thereof.

98. The method of any one of embodiments 88-97, which is an in vitro method or an ex vivo method.

99. The method of any one of embodiments 88-98, wherein said candidate substance and/or composition comprises a small molecule, protein and/or polynucleotide.

100. The method of any one of embodiments 88 to 99, wherein said candidate substance, device and/or composition acts directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or nucleic acids encoding nAChR α 2 protein and/or nAChR β 2 protein.

101. A method of screening for biomarkers suitable for diagnosing and/or monitoring sleep disorders, the method comprising:

determining a disease value for a substance, wherein the disease value is the presence and/or level of the substance in a sample obtained from a non-human organism or a living part thereof according to any one of embodiments 71-83, a cell, cell line or primary cell culture according to embodiment 84, or a tissue according to any one of embodiments 85-87;

determining a wild-type value for said agent, wherein said wild-type value is the presence and/or level of said agent in a sample obtained from a corresponding wild-type non-human organism or a corresponding living part, cell or tissue thereof;

and identifying the substance as the biomarker when the disease value is different from the wild-type value.

102. The method of embodiment 101, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

103. The method of any one of embodiments 101-102, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

104. The method of any one of embodiments 101-103, wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

105. The method of embodiment 102, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

106. The method of any one of embodiments 105-106, wherein the sleep disorder associated with hypersomnia comprises narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

107. The method of any one of embodiments 101-106, wherein the disease value is greater than the wild-type value and the biomarker is a biomarker indicative of promoting sleep.

108. The method of any one of embodiments 101-107, wherein the disease value is less than the wild-type value and the biomarker is a biomarker indicative of reduced sleep.

109. Use of a non-human organism or living part thereof according to any one of embodiments 71 to 83, a cell, cell line or primary cell culture according to embodiment 84, or a tissue according to any one of embodiments 85 to 87 for the preparation of a system for screening for substances, devices, compositions and/or biomarkers suitable for the treatment, diagnosis, prevention, monitoring and/or prognosis of sleep disorders.

110. The use of embodiment 109, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

111. The use according to any one of embodiments 109-110, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

112. The use of any one of embodiments 109-111 wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

113. The use of embodiment 110, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

114. The use according to any one of embodiments 110-113, wherein the sleep disorder associated with hypersomnia comprises narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

115. The use of any one of embodiments 110-114, wherein the substance, composition and/or biomarker comprises a small molecule, protein and/or polynucleotide.

116. The use according to any one of embodiments 109 and 116, wherein the substance, device, composition and/or biomarker acts directly on the nAChR α 2 protein and/or nAChR β 2 protein, and/or a nucleic acid encoding the nAChR α 2 protein and/or nAChR β 2 protein.

117. A non-human organism or living part thereof according to any one of embodiments 71 to 83, a cell, cell line or primary cell culture according to embodiment 84, or a tissue according to any one of embodiments 85 to 87 for use in screening for a substance, device, composition and/or biomarker useful for the treatment, diagnosis, prevention, monitoring and/or prognosis of sleep disorders.

118. The use of embodiment 117, wherein the sleep disorder comprises a sleep disorder associated with insufficient sleep and/or a sleep disorder associated with excessive sleep.

119. The use of any one of embodiments 117-118, wherein the sleep disorder associated with insufficient sleep comprises insufficient daytime sleep and/or insufficient nighttime sleep.

120. The use of any one of embodiments 117-119, wherein the sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with a cardiovascular disorder and/or a neurodegenerative disease.

121. The use of embodiment 120, wherein the sleep disorder associated with hypersomnia comprises daytime hypersomnia and/or nighttime hypersomnia.

122. The use according to any one of embodiments 120-121, wherein the sleep disorder associated with hypersomnia comprises narcolepsy, NREM/REM-related drowsiness, and/or hypersomnia/difficult to wake associated with cardiovascular disorders and/or neurodegenerative diseases.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation may occur. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pair; kb, kilobases; pl, picoliter; s or sec, seconds; min, min; h or hr, hours; aa, an amino acid; nt, nucleotide; i.m., intramuscular; i.p., intraperitoneally; s.c., subcutaneous; and the like.

Statistical analysis was performed with Prism 5 (GraphPad). The Mann-Whitney test (Mann-Whitney test) was used to compare two columns of data. The Kruesky-Wallis test (Kruskal-Wallis test) using the Dengen's post hoc test (Dunn's posttest) was used to compare multiple columns of data from mutants, rescue and RNAi. Fisher's exact test (Fisher's exact test) was used to compare the arousal rates. Statistical significance (P) is indicated by an asterisk: p <0.001, P <0.01, P <0.05, n.s. P > 0.05.

Example 1 Generation of transgenic, knock-out and knock-in flies

Flies were raised in standard medium at 25 ℃ and 60% humidity and maintained in a 12hr:12hr light-dark cycle unless a constant dark assay was used. 1) UAS-. beta.2 RNAi (THU2877) and 2) UAS-T.beta.HRNAi (TH02221) were from the Drosophila qinghuaensis Center (Tsinghua Fly Center). 3) UAS-. alpha.2 RNAi (v1195) and 4) UAS-Dicer from the Drosophila Vienna RNAi Center (Vienna Drosophila RNAi Center). 5) UAS-mCD8: GFP, 6) UAS-singer: GFP, 7) UAS-Syt: GFP, 8) UAS-DenMark, 9) LexAop-Flp, UAS-FRT-terminator sequence-FRT-mCD 8: GFP, 10) LexAop-myr: GFP and 11) UAS-LexAxABDD from Bludington's Reserve center (Bloomton Stockcenter). All flies used in this study had been backcrossed to Canton-S background for at least five generations.

Total RNA from wild-type flies was isolated using TRIzol reagent (Invitrogen) followed by PrimeScript^TMII chain 1 cDNA Synthesis kit (Takara, 6210A) to prepare chain 1 cDNA the coding sequence of nAChR α was amplified from chain 1 cDNA and inserted into PACU2 vector (Jan laboratories from UCSF) to create UAS- α 2DNA construct.

The CRISPR/Cas9 system was used to generate knock-out (knockout) and knock-in (knockin) flies. Materials and protocols for the design and generation of guide rna (guide rna) and Cas9 mRNA were generously provided by Renjie Jiao (Institute of biophysics, Cas). Two grnas targeting the coding sequence were injected along with Cas9 mRNA to generate deletion (deletion) and indel (indel) strains. KO strains were obtained by injecting Cas9 RNA and two guide RNAs into wild-type embryos (fig. 1A). After cleavage of the target site, non-homologous end joining (NHEJ) repair results in randomly disrupted genes. KO-Gal4 (FIG. 1B), KO-RFP (FIG. 1B), KI-Gal4 (FIG. 1C) and KILexA (FIG. 1C) strains were obtained by injecting Cas9 RNA, two guide RNAs and a donor plasmid into wildtype embryos. After cleavage of the target site, homology-directed repair introduces the target sequence (Gal4, RFP or LexA) from the donor into a specific site in the genome, replacing the original genomic sequence between the two homology arms (homologus arms).

A schematic representation of the nAChR α 2 and nAChR β 2 genes is shown in FIG. 2, PA and PB representing the two subtypes (isofom) resulting from variable splicing. The dashed lines indicate the deletion regions in nAChR α 2KO and nAChR β 2 KO. A portion of the transmembrane domain (TMD) and Ligand Binding Domain (LBD) in nAChR α 2KO was deleted, and a majority of LBD and TMD in nAChR β 2KO were deleted. The solid lines indicate LBD and TMD, the white boxes indicate coding regions (CDS), and the gray boxes indicate untranslated regions (UTR). Via the deletion portion: nAChR alpha 2KO, NP-524482.1, 197aa-512 aa; nAChR beta 2KO, NP-524483.1, 57aa-459 aa. The gRNAs for nAChR alpha 2KO are shown in SEQ ID NO:1-2, and the gRNAs for nAChR beta 2KO are shown in SEQ ID NO: 3-4.

Another donor plasmid was co-injected with two grnas and Cas9 mRNA to generate KOGal4, KORFP, KIGal4, and KILexA flies. The 5 'homology arm (about 2.5kb) and 3' homology arm (about 2.5kb) were inserted into the pBSKII vector to generate a donor plasmid with the target sequence in between the two homology arms. After cleavage of the target site by gRNA, homology-directed repair introduces the target sequence (Gal4, RFP or LexA) from the donor into a specific site in the genome, replacing the original genomic sequence between the two homology arms. The 3 'end of the 5' homology arm was designed to be located just after the start codon in KOGal4 and KORFP (fig. 1B) so that translation was terminated by a stop codon in the target sequence. The 3 'end of the 5' homology arm was designed to be located just before the stop codon in KIGal4 and KILexA (fig. 1C) so that translation was not disrupted, and therefore KIGal4 and KILexA could represent the most native expression pattern of the target gene as possible. nAChR-KOGal4, nAChR-KORFP, nAChR-KIGal4 and nAChR-KILexA flies were generated by the above strategies. The primers of nAChR alpha 2KOGal4, nAChR alpha 2KIGal4/LexA, nAChR beta 2KIGal4 and nAChR beta 2-PB-KIGal4 are respectively shown in SEQ ID NO. 5-20.

nAChR α 2 encodes one protein, while nAChR β 2 encodes two subtypes that differ in carboxyl (C) terminus. 2A-Gal4 was fused in-frame to the C-terminus just before the stop codon to yield α 2KIGal4, β 2KIGal4 (for the long isoform of nAChR β 2) and β 2-PB-KIGal4 (for the short isoform of nAChR β 2), as shown in FIG. 3.

Example 2 behavioral determination

Within 5 hours after eclosion, male and female flies were isolated, and flies aged 5-8 days were used in the behavioral determination.

2.1 sleep

An uninterrupted period of inactivity lasting more than 5 minutes is defined as sleep. Briefly, a single fly was transferred to monitor tubes (5mm x 65mm) containing fly food, 48 monitor tubes were fixed on a recording plate, and flies were recorded for 3-5 days. The fly position was then followed and analyzed for sleep duration and speed using matlab (mathworks).

2.2 Sleep deprivation (Sleep depletion)

Sleep deprivation was achieved by random shaking throughout the night. The recording tube was fixed to a silicone holder, and then horizontally placed into a housing box. The cartridge is rotated in either a clockwise or counterclockwise direction under the control of a servo motor (TowerProTM MG995) and impacts the plastic plug to shake the flies. The flies were shaken at random intervals of 2-5 minutes. Each shake lasted 18 seconds, including 9 consecutive rotations of the cartridge. The rebound rate (rebound rate) per 30 minutes was calculated as (sleep duration after deprivation-sleep duration before deprivation within equivalent time)/(sleep loss). The cumulative rebound rate was calculated as the sum of the rebound rates since the stop of deprivation.

2.3 circadian rhythm analysis

For circadian rhythm analysis, flies were directed with a 12h:12h light dark cycle (LD cycles) for 3 days, followed by constant darkness for 9 days. The migration activity (lococolor activity) was measured and analyzed by an Actogram J plug-in to analyze cycle length.

2.4 determination of wakefulness

In the arousal assay, flies were stimulated three times at night by an eccentric vibrating motor (1.0g) (ZT16, ZT18 and ZT 20). An eccentric vibrating motor was fixed under the recording plate to stimulate flies, and the intensity of stimulation was controlled by adjusting the voltage output. The stimulation intensity was measured by an acceleration sensor (model CJMCU _ ADXL345, read by arduino (tm) plates) attached in the surface of the plates. The stimulation intensity was set to 1.0g (1.0g equals gravity at the earth's surface, 9.8m/s 2). Each stimulus contained 3 oscillations of 200 milliseconds duration with an interval of 800 milliseconds. The arousal rate was calculated as the ratio of the number of flies aroused by the stimulus to the number of flies sleeping before the stimulus.

Example 3 nAChR α 2 and nAChR β 2 promote sleep

The sleep duration of wild-type (wt) and AChR mutant strains was measured using a video-based fly recording system. Both RNAi knockdown (knockdown) on nAChR α 2 driven by α 2KIGal4 and RNAi knockdown on nAChR β 2 driven by β 2KIGal4 resulted in a significant reduction in nocturnal sleep (fig. 8). The female adult brains were dissected, fixed, and stained. The following primary antibodies (primary antibodies) were used: chicken anti-GFP antibody (1:1000) (Invitrogen), mouse nc82(1:40) (DSHB). The following secondary antibodies (secondary antibodies) were used: AlexaFluor488 anti-chicken antibodies (Life technologies), AlexaFluor633 anti-mouse antibodies (Life technologies). The brain was imaged using a Zeiss LSM710 confocal microscope and the images were processed with Imaris (biplane AG, Zurich, Switzerland) and ImageJ (National Institutes of Health, u.s.) software.

The sleep phenotypes of α2 and β2 receptor mutants are shown in Figure 3. The results showed that in the case of nAChRα2 knockout (nAChRα2KO, α2-/-) and nAChRβ2 knockout (nAChRβ2KO, β2-/-) mutant flies, sleep was significantly reduced (Figure 4). The dotted line represents the night sleep duration of wild-type (wt) flies. In the case of α2^-/-, the duration of both daytime sleep and night sleep was significantly reduced, and in the case of β2-/-, night sleep was significantly reduced (Figure 5). The phenotype was observed in both males and females (Figure 6). The sleep duration of α2^+/- and β2^+/- is similar to that of wild-type sleep, which indicates that both nAChRα2KO and nAChRβ2KO are recessive. ). In terms of sleep recovery after deprivation, in terms of circadian rhythm and wakefulness, α2^-/- and β2^-/- are phenotypically similar to wild-type flies (Figure 7 ).

mCD8:: GFP, stinger:: GFP, Syt:: GFP and Denmark are driven by α 2KIGal4, β 2KIGal4 and β 2-PB-KIGal4 to label the membrane, nucleus, axon and dendrite of neurons expressing nAChR α 2 or nAChR β 2, respectively. α 2KIGal4 and β 2KIGal4 are expressed in a plurality of brain regions including Antennal Lobe (AL), subgsophageal ganglion (SOG) and sleep control regions MB and PI, as well as in the mesothoracic neurogenic section, the postthoracic neurogenic section (MN, MtN) and the Abdominal Center (AC) of the abdominal nerve cord (VNC) (FIGS. 9A-9D, 10A-10F), while β 2-PB-KIGal4 shows no expression in the brain and VNC other than dendrites in the optic lobe (FIGS. 9E-9F, 10G-10I).

Example 4 nAChR α 2 and nAChR β 2 together produce sleep-promoting effects

The nAChR α 2 and nAChR β 2 genes are closely related: separated from each other only by-18 kb in the Drosophila genome. nAChR α 2 and nAChR β 2 were crossed (interleaved) by simultaneous expression of UAS-FRT-terminator sequence-FRT-GFP driven by β 2KIGal4 in β 2 expressing neurons and LexAop-Flp driven by α 2kilex in α 2 expressing neurons. In nAChR α 2+ nAChR β 2+ neurons, the termination (STOP) cassette between UAS and GFP was removed by Flp recombinase, thereby labeling the neurons with GFP (fig. 11B).

Intersections of nAChR α 2 and nAChR β 2 were identified in multiple brain regions (fig. 11C). Furthermore, RNAi knockdown of nAChR β 2 in nAChR α 2-expressing cells and knockdown of nAChR α 2 in nAChR β 2-expressing cells both decreased the duration of nighttime sleep (fig. 11D-11E). Results of expression, crossover and RNAi experiments show that nAChR α 2 and nAChR β 2 together produce sleep-promoting effects.

Example 5nAChR α 2 produces sleep-promoting effects in octopamine neurons

Subsequently, nAChR α 2 was crossed with serotonin synthase (Tryptophan hydroxylase, Tryptophan hydroxylase or Trh) and octopamine synthase (Tyramine β hydroxylase, Tyramine β hydroxylase or T β H). T β HKIGAL4 was generated by the strategy described in example 1, with the forward and reverse primers of the T β HKIGAL 45 'arm shown as SEQ ID Nos. 21-22 and the forward and reverse primers of the T β HKIGAL 43' arm shown as SEQ ID Nos. 23-24, respectively.

Trh and nAChR α 2 were found to have overlapping expression in the SOG (FIG. 13A). T β H and nAChR α 2 were found to overlap extensively in the brain and VNC (fig. 12A-12B), including AL, Ventral Unpaired Medial (VUM) cells of SOG, anterolateral anterior brain (ASM) cells of the superior neural networks (SNPs), anterior superior lateral forebrain bridge (PB) cells, and ventral forebrain (VL) cells of the ventral neural networks (VLNPs).

UAS-nAChR α 2 was reintroduced into different types of neurons in an α 2-/-background. The nighttime sleep duration of α 2-/-was saved by reintroducing UAS-nAChR α 2 into α 2 expressing cells labeled with α 2KOGal4 (fig. 12C), where the 2A-Gal 4-stop sequence was fused to the initiation codon of nAChR α 2.

Expression of nAChR α 2 rescued the duration of nocturnal sleep in octopamine cells labeled by T β HKIGal4 (fig. 12D), whereas expression of nAChR α 2 failed to rescue in serotonergic cells labeled by TrhKIGal4 (fig. 13B), suggesting that nAChR α 2 produces sleep-promoting effects in octopamine neurons. RNAi knockdown of nAChR α 2 in octopamine-competent cells, driven by T β HKIGal4, also decreased the duration of nocturnal sleep (fig. 12E), suggesting that nAChR α 2 in octopamine-competent cells is essential for proper sleep duration. Furthermore, RNAi knockdown of T β H in nAChR α 2-expressing cells decreased the duration of nocturnal sleep (fig. 12F), suggesting that octopamine in α 2-expressing cells is also essential for sleep. Taken together, these data indicate that nAChR α 2 produces sleep-promoting effects in octopamineergic neurons most likely through octopamineergic signaling.

While certain embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited to the particular embodiments provided within the specification. While the invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention herein. Further, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Claims

providing a candidate agent;

2. The method of claim 1, wherein said candidate agent is selected as an agent for promoting sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is increased by said candidate agent.

3. The method of any one of claims 1-2, wherein said candidate agent is selected as an agent for reducing sleep if said activity and/or expression of said nAChR α 2 and/or nAChR β 2 is reduced by said candidate agent.

4. The method of any one of claims 1 to 3, wherein said nAChR α 2 is Drosophila melanogaster (Drosophila melanogaster) nAChR α 2 or an orthologue thereof.

5. A system for selecting an agent for modulating sleep, wherein said system comprises a substance capable of determining the effect of said agent on the activity and/or expression of nAChR α 2 and/or nAChR β 2.

6. The system of claim 5, wherein said agent is capable of determining the effect of said agent on the activity and/or expression of said nAChR α 2 and/or nAChR β 2 nucleic acids.

7. The system of claim 6, wherein said agent can be assayed for said nAChR α 2 and/or said nAChR α 2

Said substances of influence of the activity and/or expression of nAChR β 2 nucleic acids include: capable of specific amplification

Primers for nAChR α 2 and/or nAChR β 2, and/or probes capable of specifically recognizing nAChR α 2 and/or nAChR β 2.

8. An agent capable of altering the activity and/or expression of nAChR α 2 and/or nAChR β 2 for use in the treatment, prevention or delay of progression of a sleep disorder.

9. Use of an agent capable of indicating the activity and/or level of expression of nAChR α 2 and/or nAChR β 2 in a subject in the manufacture of an indicator of the likelihood that the subject suffers from, and/or is at risk of suffering from, a sleep disorder.

10. A non-human organism or living part thereof comprising functionally impaired nAChR α 2 and/or functionally impaired nAChR β 2.