WO2021068900A1

WO2021068900A1 - Agent and method for adjusting sleep

Info

Publication number: WO2021068900A1
Application number: PCT/CN2020/120031
Authority: WO
Inventors: Xihuimin DAI; Enxing ZHOU; Renbo MAO; Wei Yang; Yuan Liu; Tenghui YU; Wenxia Zhang; Yi Rao
Original assignee: Gritscience Biopharmaceuticals Co Ltd
Current assignee: Gritscience Biopharmaceuticals Co Ltd
Priority date: 2019-10-09
Filing date: 2020-10-09
Publication date: 2021-04-15
Anticipated expiration: 2022-04-09
Also published as: CN111778323A; CN111778323B

Abstract

Provided is a method for selecting an agent for adjusting sleep. The method comprises determining an effect of said candidate agent on an activity and/or expression of nAChRα2 and/or nAChRβ2. The agent can be used for the treatment, prevention or delay of progression of a sleep disorder. Furthermore, provided is a method for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder. Also provided is a non-human organism or a living part thereof.

Description

Agent and method for adjusting sleep

BACKGROUND OF THE INVENTION

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, disturbed metabolism, and even death. Molecular mechanisms and neural circuits controlling sleep and arousal are under active research.

Although the cholinergic neurons have long been identified as a key modulator in sleep regulation in both mammals and flies, the specific function of cholinergic neurons in sleep and arousal remains controversial, and little is known about the AChRs’ role in sleep regulation. In both mammals and flies, ACh is paradoxical in being able to promote both sleep and arousal. Although ACh have long been known to play important roles in sleep regulation, the molecular basis of cholinergic signaling remain elusive, and little is known about the roles of AChRs in sleep regulation.

SUMMARY OF THE INVENTION

The present disclosure provides a method for selecting an agent for adjusting sleep, the method comprising: providing a candidate agent; determining an effect of said candidate agent on an activity and/or expression of nAChRα2 and/or nAChRβ2; and if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is altered by said candidate agent, then selecting said candidate agent as an agent for adjusting sleep. The agent can be used for the treatment, prevention or delay of progression of a sleep disorder. Furthermore, the present disclosure provides method for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder, the method comprising: evaluating an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject. The present also provides a non-human organism or a living part thereof.

In one aspect, the present disclosure provides a method for selecting an agent for adjusting sleep, the method comprising: providing a candidate agent; determining an effect of said candidate agent on an activity and/or expression of nAChRα2 and/or nAChRβ2; and if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is altered by said candidate agent, then selecting said candidate agent as an agent for adjusting sleep.

In some embodiments, if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is increased by said candidate agent, then selecting said candidate agent as an agent for promoting sleep.

In some embodiments, if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is decreased by said candidate agent, then selecting said candidate agent as an agent for reducing sleep.

In some embodiments, the nAChRα2 is Drosophila melanogaster nAChRα2, or an ortholog thereof.

In some embodiments, the nAChRβ2 is Drosophila melanogaster nAChRβ2, or an ortholog thereof.

In some embodiments, the determining comprises: determining an effect of said candidate agent on an activity and/or expression of said nAChRα2 and/or nAChRβ2 in an octopaminergic cell.

In some embodiments, the octopaminergic cell comprises an octopaminergic neuronal cell.

In some embodiments, the said activity of said nAChRα2 and/or nAChRβ2 comprises one or more of the following: an ability to form a functional nAChRα2β2 receptor complex; an ability to increase an activity, releasing, and/or amount of octopamine; an ability to activate an octopaminergic signaling.

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

In some embodiments, the sleep comprises daytime sleep and/or nighttime sleep.

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

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

In some embodiments, the agent directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.

In another aspect, the present disclosure provides a system for selecting an agent for adjusting sleep, wherein the system comprises a substance capable of determining an effect of said agent on an activity and/or expression of nAChRα2 and/or nAChRβ2.

In some embodiments, the substance is capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2.

In some embodiments, the substance capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.

In some embodiments, the substance is capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.

In some embodiments, the substance capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.

In another aspect, the present disclosure provides a method for the treatment, prevention or delay of 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 an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.

In some embodiments, the sleep disorder is associated with insufficient sleep, and said agent is capable of increasing the activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.

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

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

In some embodiments, the agent comprises a nucleic acid molecule encoding nAChRα2 and/or nAChRβ2 or an expression product thereof.

In some embodiments, the agent comprises a nucleic acid sequence as set forth in any one of SEQ ID No. 1-20.

In some embodiments, the sleep disorder is associated with oversleeping, and said agent is capable of decreasing the activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.

In some embodiments, the disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.

In some embodiments, the sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.

In some embodiments, the agent is an agent for promoting sleep, as the activity and/or expression of said nAChRα2 and/or nAChRβ2 is increased by said agent.

In some embodiments, the agent is an agent for reducing sleep, as the activity and/or expression of said nAChRα2 and/or nAChRβ2 is decreased by said agent.

In some embodiments, the altering of the activity and/or expression of said nAChRα2 and/or nAChRβ2 is in an octopaminergic cell.

In some embodiments, the activity of said nAChRα2 and/or nAChRβ2 comprises one or more of the following: an ability to form a functional nAChRα2β2 receptor complex; an ability to increase an activity, releasing, and/or amount of octopamine; an ability to activate an octopaminergic 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 disclosure provides use of an agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2 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 an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2.

In some embodiments, the agent capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.

In some embodiments, the agent is capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.

In some embodiments, the substance agent of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.

In another aspect, the present disclosure provides an agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2, for use in the treatment, prevention or delay of progression of a sleep disorder.

In another aspect, the present disclosure provides a method for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder, the method comprising: evaluating an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.

In some embodiments, the activity and/or expression of nAChRα2 and/or nAChRβ2 comprise an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2, and/or an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.

In some embodiments, a substance capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.

In some embodiments, a substance capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.

In some embodiments, the sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.

In some embodiments, the sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.

In another aspect, the present disclosure provides a system for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder, the system comprising: an agent capable of indicating an activity and/or expression level of nAChRα2 and/or nAChRβ2 in said subject.

In another aspect, the present disclosure provides use of an agent capable of indicating an activity and/or expression level of nAChRα2 and/or nAChRβ2 of a subject in the manufacture of an indicator of a likelihood of said subject to have a sleep disorder, and/or at a risk of having a sleep disorder.

In some embodiments, an agent capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.

In some embodiments, an agent capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.

In another aspect, the present disclosure provides a non-human organism or a living part thereof, comprising a functionally impaired nAChRα2 and/or a functionally impaird nAChRβ2.

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

In some embodiments, the non-human organism or living part thereof does not comprise any functional nAChRα2.

In some embodiments, the non-human organism or living part thereof does not comprise any functional nAChRβ2.

In some embodiments, the non-human organism or living part thereof is homozygous for the functionally impaired nAChRα2 and/or the functionally impaired nAChRβ2.

In some embodiments, the non-human organism has reduced duration of sleep comparing to a corresponding wildtype non-human organism.

In some embodiments, the sleep comprises daytime sleep and/or nighttime sleep.

In some embodiments, the nAChRα2 gene and/or nAChRβ2 gene in said organism is knocked down or knocked-out.

In some embodiments, the nAChRα2 gene and/or nAChRβ2 gene in said organism is knocked down by RNAi.

In some embodiments, the nAChRα2 gene in said organism is knocked down by α2KIGal4.

In some embodiments, the nAChRβ2 gene in said organism is knocked down by β2KIGal4.

In some embodiments, the fourth, the fifth and the sixth exons of the nAChRα2 gene in said organism is deleted.

In some embodiments, the first to the eighth exons of the nAChRβ2 gene in said organism is deleted.

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

In another aspect, the present disclosure provides a tissue derived from the non-human organism or a living part thereof of the present disclosure.

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

In some embodiments, the tissue is derived from a neural tissue comprising octopaminergic cells.

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

In some embodiments, the determining comprises: determining an effect of said candidate substance, device and/or composition on an activity and/or expression of said nAChRα2 and/or nAChRβ2 in an octopaminergic cell.

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

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

In some embodiments, the candidate substance, device and/or composition directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.

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

In some embodiments, the disease value is greater than said wildtype value, and said biomarker is a biomarker indicating promoting sleep.

In some embodiments, the disease value is less than said wildtype value, and said biomarker is a biomarker indicating reducing sleep.

In another aspect, the present disclosure provides use of the non-human organism or living part thereof, the cell, cell line or primary cell culture, or the tissue in the preparation of a system of screening for a substance, a device, a composition and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a sleep disorder.

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

In some embodiments, the substance, device, composition and/or biomarker directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.

In another aspect, the present disclosure provides the non-human organism or living part thereof the cell, cell line or primary cell culture, or the tissue, for use in screening for a substance, a device, a composition and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a sleep disorder.

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

INCORPORATION 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 THE 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 are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1A-1C illustrate strategy of constructing AChR mutants and knockin lines;

FIG. 2 illustrates schematic representations of nAChRα2 and nAChRβ2 genes;

FIG. 3 illustrate schematic genotypes of KOGal4 lines used for rescue and knockin lines for labeling;

FIG. 4 illustrates sleep phenotypes of nAChRα2 and nAChRβ2 mutants;

FIG. 5A illustrates sleep profiles of α2 ^-/-, α2 ^+/-and wt (α2 ^+/+) , wherein ZT is time (hour) ; FIG. 5B illustrates daytime and nighttime duration of α2 ^-/-, α2 ^+/-and wt (α2 ^+/+) ; FIG. 5C illustrates sleep profiles of β2 ^-/-, β2 ^+/-and wt (β2 ^+/+) , wherein ZT is time (hour) ; FIG. 5D illustrates daytime and nighttime duration of β2 ^-/-, β2 ^+/-and wt (β2 ^+/+) ;

FIG. 6A illustrates sleep profiles of nAChRα2KO male flies, wherein ZT is time (hour) ; FIG. 6B illustrates daytime and nighttime duration of nAChRα2KO male flies; FIG. 6C illustrates sleep profiles of nAChRβ2KO male flies, wherein ZT is time (hour) ; and FIG. 6D illustrates daytime and nighttime duration of nAChRβ2KO male flies;

FIG. 7A-7F illustrate sleep homeostasis (A, B) , circadian period (C, D) and stimuli-induced arousal (E, F) phenotypes of nAChRα2KO and nAChRβ2KO;

FIG. 8A-8B illustrate sleep phenotypes of nAChRα2 and nAChRβ2 genetic knockdown by RNAi;

FIG. 9A-9F illustrate expression patterns of nAChRα2 and nAChRβ2 in the brain and the VNC;

FIG. 10A-10I illustrate expression patterns of nAChRα2 and nAChRβ2 in the brain;

FIG. 11A illustrates schematic representation of nAChRα2 and nAChRβ2 in the genome of Drosophila; FIG. 11B illustrates schematic representation of intersection strategy between nAChRα2 and X; FIG. 11C illustrates expression patterns of β2KIGal4/α2KILexA, LexAop-Flp, UAS-FRT-STOP-FRT-mCD8: : GFP; FIG. 11D-11E illustrate that nAChRα2 and nAChRβ2 function together to promote sleep;

FIG. 12A-12B illustrate expression patterns of TβHKIGal4/α2KILexA, LexAop-Flp, UAS-FRT-STOP-FRT-mCD8: : GFP; FIG. 12C illustrates that reintroduction of nAChRα2 into nAChRα2-expressing cells rescues nighttime sleep loss of nAChRα2 mutants; FIG. 12D illustrates that reintroduction of nAChRα2 in octopaminergic cells rescues the sleep defect of nAChRα2 mutants; FIG. 12E illustrates that knockdown of nAChRα2 in octopaminergic cells significantly reduces nighttime sleep duration; FIG. 12F illustrates that knockdown of TβH in nAChRα2-expressing cells significantly reduces nighttime sleep duration;

FIG. 13A-13B illustrate that reintroduction of nAChRα2 in serotonergic neurons fails to rescue the sleep defect.

DETAILED DESCRIPTION

While various embodiments of the 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 may 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.

nAChRα2 and nAChRβ2

There are two types of neurotransmitter acetylcholine (ACh) receptors: nicotinic AChRs (nAChRs) and muscarinic AChRs. nAChRs are ligand-gated ion channels composed of five subunits. In mammals, nAChRs submit comprises 16 nAChR subunits (nAChRα1～α7, nAChRα9, nAChRα10, nAChRβ1～β4, nAChRδ, nAChRγ, nAChRε) and 5 mAChRs (CHRM1～M5) . In insects, nAChRs submit comprises 10 nAChR subunits (nAChRα1～α7, nAChRβ1～β3) and 3 mAChRs (mAChRA, mAChRB, mAChRC) .

As used herein, the term “nAChRα2” generally refers to a gene encoding nicotinic acetylcholine receptor α2 subunit (nAChRα2) . The nAChRα2 includes homologs, fragments, derivatives, variants or ortholog of nAChRα2 that possess the activity of nAChRα2. The nAChRα2 gene is conserved in human, chimpanzee, rhesus monkey, dog, cow, mouse, rat, chicken, mosquito, and frog. In some cases, the nAChRα2 may be a Drosophila melanogaster nAChRα2, such as the nucleotide sequence of nAChRα2 protein having Accession No. NP_524482 and NP_733001 in NCBI database. The nAChRα2 protein is encoded by the CHRNA2 (cholinergic receptor nicotinic alpha 2) gene in humans, such as the nucleotide sequence of CHRNA2 having Accession No. NM_000742.4 in NCBI database.

As used herein, the term “nAChRβ2” generally refers to a gene encoding nicotinic acetylcholine receptor β2 subunit (nAChRβ2) . The term includes homologs, fragments, derivatives, variants or ortholog of nAChRβ2 that possess the activity of nAChRβ2. In some cases, the nAChRβ2 may be Drosophila melanogaster nAChRβ2, such as the nucleotide sequence of nAChRβ2 having Accession No. NM_079759.4 in NCBI database.

A polynucleotide sequence or polypeptide sequence is a “homolog” of, a “ortholog” of another sequence refers to a protein that performs substantially the same function in another subject species and shares substantial sequence identity, to the extent that they are recognized in the art as being different versions of the same protein, differing primarily in the species in which they are found. Thus, for example, human nAChRα2, mouse nAChRα2, rat nAChRα2 and fly nAChRα2 are all considered a homolog or ortholog to each other. Two polynucleotide sequences or polypeptide sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted) , they share at least approximately 50%sequence identity, or if the sequences share defined functional motifs. In alternative embodiments, optimally aligned sequences may be considered to be substantially identical (i.e. to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity over a specified region.

The terms “identity” and “identical” refer to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences or nucleotide sequences is a function of the number of identical or matching amino acids or nucleotides at positions shared by the sequences, i.e. over a specified region. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In some embodiments, the nAChRα2 and/or nAChRβ2 of the present disclosure may Drosophila melanogaster nAChRα2 and/or nAChRβ2, or an ortholog thereof. For example, the nAChRα2 and/or nAChRβ2 may be derived from Drosophila suzukii, Drosophila simulans, Drosophila sechellia, Homo sapiens, Mus musculus, Rattus norvegicus, Xenopus tropicalis, Danio rerio, as long as they have the same activity in those species. The nAChRα2 protein and/or nAChRβ2 protein may express in the central and peripheral nervous system, muscle, and many other tissues of many organisms.

Activity of nAChRα2 and/or nAChRβ2

As used herein, the term “functional” generally refers to of, connected with, or being a function of regulating sleep.

As used herein, the term “octopamine” generally refers to an organic chemical. In many types of invertebrates, it may function as a neurotransmitter. Octopamine performs functions similar to those of norepinephrine in mammals. The functions may comprise mobilizing the body and nervous system for action. In Drosophila melanogaster, octopamine is synthesized from tyramine by Tyramine β hydroxylase, or TβH.

As used herein, the term “octopaminergic cell” generally refers to a cell expressing octopamine and/or synthases of octopamine (TβH) . Octopamine is insect equivalent of norepinephrine in mammals. The octopaminergic cell may be in invertebrate nervous systems. In some embodiments, the octopaminergic cell may express widely in the brain and the ventral nerve cord (VNC) , such as the antenna lobe (AL) , the ventral unpaired median (VUM) cells of the SOG, the anterior superior medial protocerebrum (ASM) cells of the superior neuropils (SNP) , the protocerebral bridge (PB) cells, and the ventrolateral protocerebrum (VL) cells of the ventrolateral neuropils (VLNP) .

As used herein, the term “neuronal cell” generally refers to nervous system cell. In mammals, the neuronal cell may comprise a cholinergic neuron, a GABAergic neuron, a glutamatergic neuron, a dopaminergic neuron, a norepinephrinergic neuron and a serotonergic neuron in multiple regions including the brain stem, the preoptic hypothalamus, the lateral hypothalamus and the basal forebrain. In insect, the neuronal cell may comprise a cholinergic neuron, a GABAergic neuron, a glutamatergic neuron, a dopaminergic neuron, a octopaminergic neuron and a serotonergic neuron in multiple regions including the dorsal fan-shaped body, the ellipsoid body, the mushroom body, and the pars intercerebralis.

The nAChRα2 and/or nAChRβ2 subunit may capable of forming a functional receptor complex itself or with others (such as, nAChRα1～α7, nAChRα9, nAChRα10, nAChRβ1～β4, nAChRδ, nAChRγ, nAChRε, CHRM1～M5, mAChRA, mAChRB, mAChRC) . In some embodiments, the nAChRα2 and/or nAChRβ2 may form a functional nAChRα2β2 receptor complex. In some embodiments, functional nAChRα2β2 receptor complex maybe a heteropolymer, for example, hetero-pentamer. In some embodiments, functional nAChRα2β2 receptor complex may comprise at least one nAChRα2 (e.g. at least two nAChRα2, at least three nAChRα2, at least four nAChRα2) and at least one nAChRβ2 (e.g. at least two nAChRβ2, at least three nAChRβ2, at least four nAChRβ2) . In some embodiments, functional nAChRα2β2 receptor complex may comprise other nAChR subunits, such as nAChRα1, nAChRα3, nAChRα4, nAChRα5 nAChRα6, nAChRα7, nAChRα9, nAChRα10, nAChRβ1, nAChRβ4, nAChRβ3, nAChRδ, nAChRγ, nAChRε. In some embodiments, the functional nAChRα2β2 receptor complex is functional in sleep.

In some embodiments, the nAChRα2 and/or nAChRβ2 may process an ability to increase an activity, releasing, and/or amount of octopamine, for example, to increase activity, releasing, and/or amount of synthases of octopamine.

In some embodiments, the nAChRα2 and/or nAChRβ2 may process an ability to activate an octopaminergic signaling. As used herein, the term “octopaminergic signaling” refers to a signaling involving octopamine. In some embodiments, the nAChRα2 and/or nAChRβ2 may activate a signaling pathway involving octopamine.

The activity and/or expression of nAChRα2 and/or nAChRβ2 may be related with sleep. The sleep may comprise daytime sleep and nighttime sleep. The relationship may be evaluated using a knockout and/or knockin mutant by any gene engineering methods in the art (e.g. CRISPR, RNAi, homologous reorganization) , for example, a nAChRα2 knockout and/or nAChRβ2 knockout mutant flies. In some embodiments, the increasing of activity and/or expression of nAChRα2 and/or nAChRβ2 may promote sleep. In some embodiments, the decreasing of activity and/or expression of nAChRα2 and/or nAChRβ2 may reduce sleep. In some embodiments, the activity and/or expression of nAChRα2 may be related with nighttime sleep and day time sleep. In some embodiments, the activity and/or expression of nAChRα2 may be related with nighttime sleep.

Methods of detection

The activity and/or expression of nAChRα2 and/or nAChRβ2 may be quantifiably determined with methods known in the art, including but not limit to, immunohistochemistry, 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, the method may comprise a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2. The primer may be a pair of primers. Furthermore, the method may comprise a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2. The probe may be capable of binding to nAChRα2 and/or nAChRβ2 nucleotides sequence or fragment thereof, rather than another nucleotides sequence. The probe may have a detectable signal. In other cases, the methods may comprise an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein, such as an antibody and/or ligand of nAChRα2 and/or nAChRβ2 protein, and/or fragment thereof.

In some cases, the expression of nAChRα2 and/or nAChRβ2 may be detected by methods known in the arts, such as immunofluorescence, immunohistochemistry and confocal imaging. Detection can be facilitated by coupling the target to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of antibodies for immunohistochemistry may be GFP, GRP, nc82, AlexaFluor488 chicken antibody, AlexaFluor 633 mouse antibody.

Adust sleep

As used herein, the term “adjust sleep” generally refers to regulate time and duration of sleep. Sleep may be divided into rapid eye movement (REM) sleep and non-REM (NREM) sleep, which are different in electroencephalogram (EEG) , electromyogram (EMG) , and arousal levels. Adjust sleep comprise promoting sleep and/or reduce sleep. For example, adjust sleep may comprise lengthening the daytime sleep time, lengthening the nighttime sleep time, shortening the daytime sleep time, shortening the nighttime sleep time, increasing the duration of daytime sleep, increasing the duration of nighttime sleep, decreasing the duration of daytime sleep, decreasing the duration of nighttime sleep, , enforcing the degree of nighttime sleep, lightening the degree of daytime sleep and/or lightening the degree of nighttime sleep.

As used herein, the term “deprivation” generally refers to the condition of not having enough sleep. The sleep deprivation techniques in the art comprise gentle handling, single platform, multiple platform, modified multiple platform and pendulum. For example, sleep deprivation may be conducted by random shaking in the whole night, in which a rebound rate was calculated to evaluate the degree of sleep deprivation.

As used herein, the term “circadian rhythm” generally refers to a roughly 24-hour cycle in the physiological processes of living beings, including plants, animals, fungi and cyanobacteria. In some cases, circadian rhythm may be endogenously generated. In other cases, circadian rhythm may be modulated by external cues such as sunlight and temperature. The circadian rhythms may be analyzed by locomotor activity in constant darkness.

As used herein, the term “arousal” refers to a state of being awoken or of sense organs stimulated to a point of perception. The methods analysis arousal may comprise transient and specific intensity of light or transient and specific intensity of mechanical stimulations applied during sleep at specific zeitgeber time, and video recording of responses following stimulations.

As used herein, the term “does not substantially affect” generally refers to phenotypically does not significant different from wide type after treatment of an agent.

In one aspect, the present disclosure provides a method for selecting an agent for adjusting sleep. The method comprises: providing a candidate agent; determining an effect of said candidate agent on an activity and/or expression of nAChRα2 and/or nAChRβ2; and if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is altered by said candidate agent, then selecting said candidate agent as an agent for adjusting sleep.

In some embodiments, the method may be an in vitro method, or an ex vivo method. the method may comprise using a cell, a cell line or primary cell culture expressing nAChRα2 and/or nAChRβ2. The cell may be from human, such as a stem cell or a human neural n cell. In some cases, the method may be performed using tissues and/or cells, such as neuronal cells, brain regions or other tissues comprising or corresponding to the nAChRα2 and/or nAChRβ2 expression. In some cases, the cell may be a neural cell from any suitable species, e.g., 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 cell. The cell may be incubated with the agent, transfected with a vector comprising the agent. For example, the tissues and/or cells may be cultured in vitro or ex vivo, then, the candidate substance, may be applied to the cultured tissues and/or cells, after being incubated for an appropriate period of time (e.g., a few hours, a few days, a few weeks or a few months) , the amount of expression of nAChRα2 and/or nAChRβ2 may be examined with a method as described in the present disclosure.

Then the expression amount or activity of nAChRα2 and/or nAChRβ2 is determined. The determined technique may be conducted as described in the present disclosure. Compared to a control, if the activity of the nAChRα2 and/or nAChRβ2 is increased, then candidate agent may be an agent for promoting sleep; if the activity of the nAChRα2 and/or nAChRβ2 is decreased, then candidate agent may be an agent for reducing sleep.

In some embodiments, the agent may affect the activity and/or expression of the nAChRα2 and/or nAChRβ2 in an octopaminergic cell. In some embodiment, octopaminergic cell comprises an octopaminergic neuronal cell.

In some embodiments, the agent of the present disclosure does not substantially affect sleep recovery after deprivation. For example, the accumulated rebound rate of knockout flies is from about 80%to about 120%of that rate of widetype, e.g., from about 90%to about 110%, from about 95%to about 105%or about 100%, as measured in behavior analysis.

In some embodiments, the agent of the present disclosure does not substantially affect circadian rhythm. For example, the period length in constant dark of knockout flies is from about 80%to about 120%of that rate of widetype, e.g., from about 90%to about 110%, from about 95%to about 105%or about 100%, as measured in circadian analysis.

In some embodiments, the agent of the present disclosure does not substantially affect arousal. For example, the arousal rate of knockout flies is from about 80%to about 120%of that rate of widetype, e.g., from about 90%to about 110%, from about 95%to about 105%or about 100%, as measured in arousal assay

In some embodiments, the agent may comprise a small molecule, a protein and/or a polynucleotide. In some embodiments, the agent directly acts on a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein. The nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein may natural or synthetic nucleic acids, including DNA and RNA, e.g., cDNAs, antisense and mRNA.

Sleep disorder

In another aspect, the present disclosure provides a method for the treatment, prevention or delay of progression of a sleep disorder.

As used herein, the term “sleep disorder” in general refers to any condition that would benefit from treatment with the agents of the present invention, including any sleep disease or disorder that can be treated by effective amounts of agents described herein. Sleep disorder may comprise intrinsic sleep disorders, extrinsic sleep disorders, and circadian rhythm sleep disorders. Examples of intrinsic sleep disorders include psychophysiological insomnia, sleep state misperception, idiopathic insomnia, narcolepsy, recurrent hypersomnia, idiopathic hypersomnia, posttraumatic hypersomnia, obstructive sleep apnea syndrome, central sleep apnea syndrome, central alveolar hypoventilation, periodic limb movement disorder, restless leg syndrome (RLS) , etc. Examples of extrinsic sleep disorders include inadequate sleep hygiene, environmental sleep disorder, altitude insomnia, adjustment sleep disorder, insufficient sleep syndrome, limit-setting sleep disorder, sleep-onset association disorder, food allergy insomnia, nocturnal eating/drinking syndrome, hypnotic-dependent sleep disorder, stimulant-dependent sleep disorder, alcohol-dependent sleep disorder, toxin-induced sleep disorder, etc. Examples of circadian rhythm sleep disorders include time-zone change (jet lag) syndrome, shiftwork sleep disorder, irregular sleep/wake pattern, delayed sleep-phase syndrome, advanced sleep-phase syndrome, non-24-hour sleep/wake disorder, etc.

As used herein, the term “insomnia” in general refers to a sleep disorder in which people have trouble sleeping.

As used herein, the term “sleep loss” in general refers to a sleep disorder of not having enough sleep.

As used herein, the term “narcolepsy” in general refers to a sleep disorder characterized by excessive sleepiness, sleep paralysis, hallucinations, and in some cases episodes of cataplexy.

As used herein, the term “NREM” in general refers to a sleep phase distinguishable by little or no eye movement.

As used herein, the term “REM” in general refers to a sleep phase distinguishable by random/rapid movement of the eyes.

As used herein, the term “parasomnias” in general refers to a sleep disorder that involved abnormal movements, behaviors, emotions, perceptions, and dreams that occur while falling asleep, sleeping, between sleep stages, or during arousal from sleep.

In some embodiment, the sleep disorder may comprise insufficient sleep and oversleeping. In some embodiments, the sleep disorder may comprise daytime insufficient sleep, nighttime insufficient sleep, daytime oversleeping and nighttime oversleeping. In some embodiments, the insufficient sleep may comprise insomnia and sleep loss associated with other diseases such as cardiovascular disorders and neurodegenerative diseases, etc. In other embodiments, the oversleeping may comprise narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and oversleeping/hard-to-be-awaken associated with other diseases such as cardiovascular disorders and neurodegenerative diseases, etc.

The terms “treatment” as used herein refer to curative therapy, prophylactic therapy, and preventative therapy. Consecutive treatment or administration refers to treatment on at least a daily basis without interruption in treatment by one or more days. Intermittent treatment or administration, or treatment or administration in an intermittent fashion, refers to treatment that is not consecutive, but rather cyclic in nature. Treatment according to the methods of the invention can result in complete relief or cure from a disease or condition, or partial amelioration of one or more symptoms of the disease or condition, and can be temporary or permanent substantially affect.

As used herein, the term “prevention” means to mitigate a symptom of the referenced disorder. In particular, said term encompasses the complete range of therapeutically positive effects of administrating an agent of the present disclosure to a subject including reduction of, alleviation of, and relief from, a sleep disorder, e.g. insufficient sleep or oversleeping thereof. The term “prevention” includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing additional symptoms and ameliorating or preventing the underlying causes of symptoms.

Methods for sleep disorder

The term “effective amount” , as used herein, generally refers to a dose sufficient to provide concentrations high enough to impart a beneficial effect on the recipient thereof. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease or disorder being treated, the severity of the disease or disorder, the activity of the specific component, the route of administration, the rate of clearance, the duration of treatment, the age, body weight, sex, diet, and general health of the subject, and other related factors.

In some embodiments, the method may comprise administering to a subject in need thereof a therapeutically effective amount of an agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject. An agent for use in the methods of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

In some embodiments, the agent may be administrated to a non-human organism or living part thereof. In some embodiments, the agent may be administrated to a tissue derived from non-human organism or living part thereof. In some embodiments, the mothed may be a method of gene therapy. In some cases, a primary cell is taken from a subject, a vector is administered to the cells to produce transduced, infected or transfected recombinant cells and the recombinant cells are readministered to the same or a different subject.

In some embodiments, administrating may comprise delivering a vector for recombinant protein expression to a cell or to cells in culture and or to cells or organs of a subject. A vector for recombinant protein or polypeptide expression may be introduced into a cell by transfection, which typically means insertion of heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection or lipofection) ; infection, which typically refers to introduction by way of an infectious agent, i.e. a virus; or transduction, which typically means stable infection of a cell with a virus or the transfer of genetic material from one microorganism to another by way of a viral agent (e.g., a bacteriophage) . In clinical settings, the agent delivery systems can be introduced into a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the agent delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the target (cells occurs predominantly from specificity of transfection provided by the agent delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the nucleic acid molecule, or a combination thereof.

Moreover, the agent may be delivered in an acceptable diluent, or the delivery system can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete agent delivery system can be produced in tact from recombinant cells, e.g., retroviral packages, the pharmaceutical preparation can comprise one or more cells which produce the agent delivery system. In the case of the latter, methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals, and can be adapted for release of viral particles through the manipulation of the polymer composition and form. A variety of biocompatible polymers (including hydrogels) , including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of the viral particles by cells implanted at a particular target site. Such embodiments of the present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.

In some embodiments, the agent may comprise a nucleic acid molecule encoding nAChRα2 and/or nAChRβ2 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 No. 1-20. In some embodiments, administrating the agent to a subject in need thereof may increase the activity and/or expression of nAChRα2 and/or nAChRβ2.

In another aspect, the present disclosure provides a system for selecting an agent for adjusting sleep, wherein the system comprises a substance capable of determining an effect of said agent on an activity and/or expression of nAChRα2 and/or nAChRβ2. The substance may be capable to determine the activity and/or expression of nucleic acid of the nAChRα2 and/or nAChRβ2. In some embodiments, the substance may comprise a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2. The substance may be capable to determine the activity and/or expression of protein of the nAChRα2 and/or nAChRβ2. In some embodiments, the substance may comprise an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.

On another aspect, the present disclosure provides use of an agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2 in the manufacture of a medicament for the treatment, prevention or delay of progression of a sleep disorder.

Likelihood of sleep disorder

In another aspect, the present disclosure provides a method determining a likelihood of a subject to have a sleep disorder, and/or at risk of having a sleep disorder. The method may comprise determining the activity and/or expression of nAChRα2 and/or nAChRβ2. The determining technique may be method described in the present disclosure. Compared with a control, if the activity and/or expression level of nAChRα2 and/or nAChRβ2 in a subject is higher, the subject may have a sleep disorder, and/or at risk of having a sleep disorder, e.g., inefficient sleep. Compared with a control, if the activity and/or expression level of nAChRα2 and/or nAChRβ2 in a subject is lower, the subject may have a sleep disorder, and/or at risk of having a sleep disorder, e.g. oversleeping. As used herein, the term “at risk of having a sleep disorder” generally refers to possibility of having a sleep disorder is higher than a control.

Non-human model

The term “interfering RNA (RNAi) ” is used herein to refer to a double-stranded RNA that results in catalytic degradation of specific mRNAs, and thus can be used to inhibit/lower expression of a particular gene.

In another aspect, the present disclosure provides the non-human organism or living part thereof comprising a functionally impaired nAChRα2 and/or a functionally impaird nAChRβ2. The non-human organism may be an insect, such as Strigamia maritima, Ixodes scapularis, Bombyx mori, Danaus plexippus, Musca domestica, Glossina morsitans and/or Drosophila species. In some embodiments, the non-human organism may be a Drosophila species, such as Drosophila melanogaster, Drosophila suzukii, Drosophila simulans, Drosophila erecta, Drosophila sechellia, Drosophila yakuba, Drosophila ananassae, Drosophila pseudoobscura pseudoobscura, Drosophila persimilis, Drosophila willistoni, Drosophila virilis, Drosophila mojavensis, Drosophila grimshawi. in some embodiments, the non-human organism is a Drosophila melanogaster, Drosophila simulans, Apis mellifera.

In some embodiments, the non-human organism or living part thereof may not comprise any functional nAChRα2 and/or nAChRβ2. The non-human organism of the present disclosure may be generated by introducing a heterologous nucleic acid sequence without nAChRα2 and/or nAChRβ2 into, for example, a fertilized egg, an unfertilized egg, a spermatozoon, a primordial germ cell, an oogonium, an oocyte, a spermatogonium, a spermatocyte and/or a sperm cell of the non-human organism, for example, at an initial stage in the embryonic development of the fertilized egg (e.g., before 8-cell stage) . The heterologous nucleic acid sequence may be introduced by a gene transfer method, such as calcium phosphate co-precipitation, electroporation, lipofection, agglutination, microinjection, gene gun (particle gun) and/or DEAE-dextran method. The heterologous nucleic acid sequence may also be introduced into a somatic cell, a tissue and/or an organ of a flies (e.g., by a gene transfer method) and then, the engineered somatic cell, tissue and/or organ may be further cultured and/or maintained. The engineered cells may also be fused with an embryo or another cell (such as a cell from the germline of the non-human organism) by cell fusion methods to produce a non-human organism of the present disclosure.

In the method of the present disclosure for generating the model animal, nuclease agents may be utilized to aid in the modification of the target gene locus. Such a nuclease agent may promote homologous recombination between the donor nucleic acid molecule and the target genomic locus. In some embodiments, the nuclease agent may comprise an endonuclease agent.

As used herein, the term “recognition site for a nuclease agent” generally refers to a DNA sequence at which a nick or double-strand break may be induced by a nuclease agent. The recognition site for a nuclease agent can be endogenous (or native) to the cell or the recognition site can be exogenous to the cell. In some embodiments, the recognition site may be exogenous to the cell and thereby is not naturally occurring in the genome of the cell. In further embodiments, the exogenous or endogenous recognition site may be present only once in the genome of the host cell. In specific embodiments, an endogenous or native site that occurs only once within the genome may be identified. Such a site can then be used to design nuclease agents that will produce a nick or double-strand break at the endogenous recognition site.

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 may recognize a recognition site of at least 9 nucleotides. In other embodiments, the recognition site may be from about 9 to about 12 nucleotides in length, from about 12 to about 15 nucleotides in length, from about 15 to about 18 nucleotides in length, or from about 18 to about 21 nucleotides in length, and any combination of such subranges (e.g., 9-18 nucleotides) . The recognition site could be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. It is recognized that a given nuclease agent can bind the recognition site and cleave that binding site or alternatively, the nuclease agent can bind to a sequence that is the different from the recognition site. Moreover, the term recognition site may comprise both the nuclease agent binding site and the nick/cleavage site irrespective whether the nick/cleavage site is within or outside the nuclease agent binding site. In another variation, the cleavage by the nuclease agent can occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions can be staggered to produce single-stranded overhangs, also called “sticky ends” , which can be either 5’ overhangs, or 3’ overhangs.

Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used in the methods of the present disclosure. A naturally-occurring or native nuclease agent can be employed so long as the nuclease agent induces a nick or double-strand break in a desired recognition site. Alternatively, a modified or engineered nuclease agent can be employed. An “engineered nuclease agent” comprises a nuclease that is engineered (modified or derived) from its native form to specifically recognize and induce a nick or double-strand break in the desired recognition site. Thus, an engineered nuclease agent can be derived from a native, naturally-occurring nuclease agent or it can be artificially created or synthesized. The modification of the nuclease agent can be 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 comprise a nick or double-strand break in a recognition site, wherein the recognition site was not a sequence that would have been recognized by a native (non-engineered or non-modified) nuclease agent. Producing a nick or double-strand break in a recognition site or other DNA can be referred to herein as “cutting” or “cleaving” the recognition site or other DNA.

In some embodiments, the nuclease agent may be a Transcription Activator-Like Effector Nuclease (TALEN) . TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases may be created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073/pnas. 1013133107; Scholze &Boch (2010) Virulence 1: 428-432; Christian et al. Genetics (2010) 186: 757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi: 10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143-148; all of which are herein incorporated by reference.

In some embodiments, the nuclease agent may be a zinc-finger nuclease (ZFN) . For example, each monomer of the ZFN may comprise 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain may bind to a 3 bp subsite. In other embodiments, the ZFN may be a chimeric protein comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In some embodiments, the independent endonuclease may be a FokI endonuclease. In some embodiments, the nuclease agent may comprise a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably linked to a FokI nuclease, wherein the first and the second ZFN recognize two contiguous target DNA sequences in each strand of the target DNA sequence separated by about 6 bp to about 40 bp cleavage site or about a 5 bp to about 6 bp cleavage site, and wherein the FokI nucleases dimerize and make a double strand break. See, for example, US20060246567; US20080182332; US20020081614; US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and, WO/2011/017293A2, each of which is herein incorporated by reference.

In some embodiments, the nuclease agent may be a meganuclease. Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are known, see for example, 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 Biophys 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 disclosure may employ a CRISPR/Cas system. Such systems can employ, for example, a Cas9 nuclease, which in some instances, may be codon-optimized for the desired cell type in which it is to be expressed. The system may further employ a fused crRNA-tracrRNA construct that functions with the codon-optimized Cas9. This single RNA may be often referred to as a small guide RNA or sgRNA. Briefly, a short DNA fragment containing the targeting sequence may be inserted into an sgRNA expression plasmid. The sgRNA expression plasmid may comprise the targeting sequence (in some embodiments around 20 nucleotides) , a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells (such as flies cells) . The sgRNA expression cassette and the Cas9 expression cassette may then be introduced into the cell. See, for example, Mali P et al. (2013) Science 2013 Feb. 15; 339 (6121) : 823-6; Jinek M et al. Science 2012 Aug. 17; 337 (6096) : 816-21; Hwang W Y et al. Nat Biotechnol 2013 March; 31 (3) : 227-9; Jiang W et al. Nat Biotechnol 2013 March; 31 (3) : 233-9; and, Cong L et al. Science 2013 Feb. 15; 339(6121) : 819-23, each of which is herein incorporated by reference.

In some embodiments, the nAChRα2 gene and/or nAChRβ2 gene in said organism is knocked down by RNAi. A double stranded RNA (dsRNA) may be introduced into the cell (e.g., using a short oligo small double-stranded interfering RNAs (siRNA) or a DNA plasmid from which a siRNA can be transcribed ) . In practicing methods, an effective amount of an RNAi agent is administered to the non-human organism to modulate expression of a target gene in a desirable manner, e.g., to achieve the desired reduction in target cell gene expression. The RNAi agents employed in the present disclosure are small ribonucleic acid molecules, i.e., oligoribonucleotides, that are present in duplex structures, e.g., two distinct oligoribonucleotides hybridized to each other or a single ribooligonucleotide that assumes a small hairpin formation to produce a duplex structure. In some embodiments, Where the RNA agent is a duplex structure of two distinct ribonucleic acids hybridized to each other, e.g., an siRNA. In some cases, siRNAs are introduced into a cytoplasm (e.g. neuronal cell) . In some embodiments, the siRNA may originate from inside the cell. In other embodiments, the siRNA may be exogenously introduced into the cell.

The RNAi agent can be administered to the non-human organism using any convenient protocol, where the protocol employed is typically a nucleic acid administration protocol, where a number of different such protocols are known in the art.

In some embodiments, the nAChRα2 gene in said organism is knocked down by α2KIGal4. Knockdown of nAChRα2 by expression of α2RNAi under the control of α2KIGal4. In some embodiments, the nAChRβ2 gene in said organism is knocked down by β2KIGal4. Knockdown of nAChRβ2 by expression of α2RNAi under the control of β2KIGal4. In some embodiments, the fourth, the fifth and the sixth exons of the nAChRα2 gene in said organism is deleted. In some embodiments, the first to the eighth exons of the nAChRβ2 gene in said organism is deleted.

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

In another aspect, the present disclosure provides a tissue derived from the non-human organism or a living part thereof. In some embodiments, the tissue is derived from a neural tissue. In some embodiments, the tissue is derived from a neural tissue comprising octopaminergic cells.

In some embodiments, the non-human organism or living part may be used in a mothed of selecting an agent for adjusting sleep. In some embodiments, the mothed may comprise administrating an agent to the non-human organism or living part and detecting the activity and/or expression of said nAChRα2 and/or nAChRβ2. In some embodiments, the non-human organism or living part may be used in screening for a biomarker useful in the diagnosis and/or monitoring of a sleep disorder. In some embodiments, the non-human organism or living part may be used in the preparation of a system of screening for a substance, a device, a composition and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a sleep disorder.

Method of screening

In another aspect, the present disclosure provides a method of screening for a substance, a device, and/or a composition useful in the treatment, prevention and/or delay of progression of a sleep disorder, comprising applying a candidate substance, device and/or composition to the non-human organism or living part thereof, the cell, the cell line or primary cell culture or the tissue according to the present disclosure, and determining an effect of said candidate substance, device and/or composition on one or more of the following: a sleep duration of said non-human organism, an activity, amount and/or releasing of octopamine, and an activate of an octopaminergic signaling.

In some embodiments, the method may be a in vitro method, or an extro method. For example, a sample (e.g., cells, tissues, or other DNA-or RNA-containing sample, protein-containing sample and/or metabolite-containing sample) may be taken from the non-human organism or the living part thereof according to the present disclosure before and after a sleep disorder (e.g., inefficient sleep and/or oversleeping) . Then, a gene transcription product (transcriptome) , a gene translation product (proteome) or a metabolite (metabolome) derived from the sample may be comprehensively assayed and a substance that changes before and after the sleep disorder may be identified.

Gene transcription products (e.g., transcriptome) may be analyzed using nucleic acid microarray, such as a DNA microarray. Gene translation products (e.g., proteome) may be analyzed using gel electrophoresis (such as a two-dimensional gel electrophoresis) , or mass spectrometry (such as time-of-flight mass spectrometry, electronspray ionization mass spectrometry, capillary HPLC/MS and LC/MS/MS) . Metabolites (metabolome) may be analyzed using NMR, capillary electrophoresis, LC/MS and/or LC/MS/MS.

When the presence/amount of a substance shows a significant difference before and after the sleep disorder, such a substance may be considered as a biomarker of sleep disorder, which may then be used in early diagnosis (particularly a preclinical diagnosis) of sleep disorder. The identified biomarker may be further detected with a specific agent or a detection method. For example, when the biomarker is a protein or a peptide, it may be detected with an immunoassay using a specific antibody. When the biomarker is a nucleic acid molecule (such as a transcription product) , it may be detected with Northern blot analysis using a specific probe, or with RT-PCR using specific primers.

In another aspect, the present disclosure provides a system for selecting an agent for adjusting sleep. In some embodiments, the system may comprise providing a distribution network for selling a composition comprising an agent of the present disclosure and providing instruction material to patients or physicians for using the agent for adjust sleep in a subject.

In some embodiments, the system may comprise determining an appropriate formulation and dosage of an agent of the present disclosure to be administered to adjust sleep in a subject, conducting therapeutic profiling of formulations identified as described above for efficacy and toxicity in animals; and providing a distribution network for selling a preparation identified as described above as having an acceptable therapeutic profile.

The system may further comprise a kit. In some embodiments, the kit may comprise an agent of the present disclosure in suitable package, as well as instruction, clinical research analysis, side effects and the like. The kit may also comprise information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of such similar information indicating or confirming the activity and/or advantages of the composition, and/or information of dosing regimen, administration, side effects, drug interactions, or other information useful to the healthcare provider. The system may further comprise another agent. In some cases, an agent of the present disclosure is provided in separate container within a kit.

In some cases, the system may be provided, sold, and/or marketed to relevant 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.

Examples

The following examples are set 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 present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Statistical analyses were performed with Prism 5 (GraphPad) . Mann-Whitney test was used to compare two columns of data. Kruskal-Wallis test with Dunn’s posttest was used to compare multiple columns of data from mutants, rescue and RNAi. Fisher’s exact test was used to compare arousal rates. Statistical significance was denoted by asterisks: ***P<0.001, **P<0.01, *P<0.05, n. s., P>0.05.

Example 1 Generation of transgenic, knockout and knockin flies

Flies were raised in standard medium at 25℃ and 60%humidity and kept in a 12hr: 12hr light: dark cycle, unless constant darkness assay were used. 1) UAS-β2RNAi (THU2877) , and 2) UAS- TβHRNAi (TH02221) were obtained from TsingHua Fly Center. 3) UAS-α2RNAi (v1195) and 4) UAS-Dicer were from Vienna Drosophila RNAi Center. 5) UAS-mCD8: : GFP, 6) UAS-stinger: : GFP, 7) UAS-Syt: : GFP, 8) UAS-DenMark, 9) LexAop-Flp, UAS-FRT-STOP-FRT-mCD8: : GFP, 10) LexAop-myr: : GFP and 11) UAS-LexADBD were obtained from the Bloomington Stock Center. All the flies used in this study have been backcrossed into a Canton-Sbackground for at least five generations.

Total RNA of wt flies was isolated using TRIzol reagent (Invitrogen) , 1st strand cDNA was then made by the PrimeScript ^TM II 1st strand cDNA synthesis kit (Takara, 6210A) . The coding sequences of nAChRα2 were amplified from the 1st strand cDNA and inserted into the PACU2 vector (from the Jan Lab at UCSF) , resulting in the UAS-α2 DNA construct. The construct was inserted in attP2 site.

The CRISPR/Cas9 system was used to generate knockout and knockin flies. The materials and protocols for designing and generating the guide RNA and Cas9 mRNA were generously provided by Renjie Jiao (Institute of Biophysics, CAS) . Two gRNAs targeting the coding sequence were injected together with Cas9 mRNA to generate deletion and indel lines. KO lines were obtained by injecting Cas9 RNA and two guide RNAs into wt embryos (FIG. 1A) . After the target sites were cut, nonhomologous end joining (NHEJ) repair led to randomly disrupted gene. KO-Gal4 (FIG. 1B) , KO-RFP (FIG. 1B) , KI-Gal4 (FIG. 1C) and KILexA (FIG. 1C) lines were obtained by injecting Cas9 RNA, two guide RNAs and a donor plasmid into wt embryos. After the target sites were cut, homology-directed repair introduced target sequence (Gal4, RFP, or LexA) from the donor into specific site in the genome, replacing the original genome sequence between two homologous arms.

Schematic representations of nAChRα2 and nAChRβ2 genes were shown in FIG. 2 (PA and PB are two isoforms) . Dotted lines indicated the deleted regions in nAChRα2KO and nAChRβ2KO. Transmembrane domain (TMD) and part of ligand-binding domain (LBD) were deleted in nAChRα2KO, most part of LBD and TMD were deleted in nAChRβ2KO. Solid lines denote LBD and TMD, white boxes denote CDS, grey boxes denote UTR. Deleted part: nAChRα2KO, NP_524482.1, 197aa-512aa. nAChRβ2KO, NP_524483.1, 57aa-459aa. The gRNAs of nAChRα2KO were set forth in SEQ ID NO: 1-2, and the gRNAs of nAChRβ2KO were set forth in SEQ ID NO: 3-4.

An additional donor plasmid was co-injected with two gRNAs and the Cas9 mRNA to generate KOGal4, KORFP, KIGal4 and KILexA flies. The 5’ homologous arm (～2.5kb) and 3’ homologous arm (～2.5kb) were inserted into the pBSKII vector to generate the donor plasmids, with the target sequence lying between the two homologous arms. After the target sites were cut by gRNAs, homology-directed repair introduced target sequence (Gal4, RFP or LexA) from the donor into specific site in the genome, replacing the original genome sequence between two homologous arms. The 3’ end of the 5’ homologous arm was designed to locate right behind the start codon in KOGal4 and KORFP (FIG. 1B) , so that the translation was ended by the stop codon in the target sequence. The 3’ end of the 5’ homologous arm was designed to locate right before the stop codon in KIGal4 and KILexA (FIG. 1C) , so that the translation was not disrupted, and hence the KIGal4 and KILexA could represent the expression pattern of the target gene as natural as possible. nAChR-KOGal4, KORFP, KIGal4 and KILexA flies were generated by the above strategies. The primes of nAChRα2KOGal4, nAChRα2KIGal4/LexA, nAChRβ2KIGal4, nAChRβ2-PB-KIGal4 were set forth in SEQ ID NO: 5-20, respectively.

nAChRα2 encodes one protein while nAChRβ2 encodes two isoforms differing in the carboxyl (C) terminal. 2A-Gal4 was fused in-frame to the C terminal precisely before the stop codon, to produce α2KIGal4, β2KIGal4 (for the long isoform of nAChRβ2) and β2-PB-KIGal4 (for the short isoform of nAChRβ2) , as shown in FIG. 3.

Example 2 Behavioral assays

Male and female flies were separated in 5 hours after eclosion, and flies aged 5～8 days were used in behavioral assays.

2.1 Sleep

Periods of uninterrupted behavioral immobility lasting for more than 5 minutes were defined as sleep. Briefly, a single fly was transferred into the monitor tube (5mm x 65mm) containing fly food, 48 monitor tubes were fixed on a recording plate, and the flies were recorded for 3～5 days. Then the position of the flies were tracked, sleep duration and speed were analyzed using Matlab (Mathworks) .

2.2 Sleep deprivation

Sleep was deprived by random shaking in the whole night. The recording tubes were fixed to a silica gel holder, and then placed horizontally into a holding box. The box was rotated clock-wise or counter clock-wise and bumped to plastic stoppers under the control of a servo motor (TowerProTM MG995) to shake the flies. The flies were shaken with random intervals of 2～5 minutes. Each shaking lasts 18 seconds, including 9 times of continuous rotation of the box. Rebound rate for every 30 minutes was calculated as (sleep duration after the deprivation –sleep duration for the equivalent time before the deprivation) / (sleep loss) . Accumulated rebound rate was calculated as the sum of rebound rate since the deprivation stopped.

2.3 Circadian analyses

For circadian analysis, flies were entrained to 12h: 12h LD cycles for 3 days and then entrained to constant darkness for 9 days. Locomotor activity was measured and analyzed by the Actogram J plugin, to analyze the period length.

2.4 Arousal assay

In arousal assay, flies were stimulated three times at night (ZT16, ZT18, and ZT20) by eccentric vibration motors (1.0g) . Eccentric vibration motors were fixed underneath the recording plates to stimulate the flies, and the strength of the stimuli was controlled by modulating the voltage output. The stimulation strength was measured by an acceleration sensor (model CJMCU_ADXL345, read by an ArduinoTM plate) attached in the surface of the plate. The stimulation strength was set to 1.0g (1.0g equals the gravitational force at the surface of the earth, 9.8m/s2) . Each stimulation contains 3 times of a vibration lasting 200ms, with an interval of 800ms. Arousal rate was calculated as the ratio of the number of flies awaken by the stimuli to the number of flies slept before the stimuli.

Example 3 nAChRα2 and nAChRβ2 promotes sleep

The sleep durations of wild type (wt) and AChR mutant lines were measured using a video-based fly recording system. RNAi knockdown of nAChRα2 driven by α2KIGal4 and RNAi knockdown of nAChRβ2 driven by β2KIGal4 both significantly reduced nighttime sleep (FIG. 8) . Adult female brains were dissected, fixed, and stained. The following primary antibodies were used: chicken anti-GFP (1: 1000) (Invitrogen) , mouse nc82 (1: 40) (DSHB) . The following secondary antibodies were used: AlexaFluor488 anti-chicken (Life technologies) , AlexaFluor633 anti-mouse (Life technologies) . Brains were imaged using a Zeiss LSM710 confocal microscope, and images were processed with Imaris (Bitplane AG, Zurich, Switzerland) and ImageJ (National Institutes of Health, U.S. ) softwares.

Sleep phenotypes of α2 and β2 receptor mutants were shown in FIG. 3. The results showed that sleep was significantly reduced in nAChRα2 knockout (nAChRα2KO, α2-/-) and nAChRβ2 knockout (nAChRβ2KO, β2-/-) mutant flies (FIG. 4) . Dash line stands for the nighttime sleep duration of wildtype (wt) flies. Durations of both daytime sleep and nighttime sleep were significantly reduced in α2 ^-/-, and nighttime sleep was significantly reduced in β2-/- (FIG. 5) . The phenotypes were observed in both males and females (Fig. 6) . The sleep duration of α2 ^+/-and β2 ^+/-were comparable with that of wt, suggesting that both nAChRα2KO and nAChRβ2KO are recessive. α2 ^-/-and β2 ^-/-flies were phenotypically similar to the wt in sleep recovery after deprivation, in circadian rhythm and arousal (FIG. 7) .

mCD8: : GFP, stinger: : GFP, Syt: : GFP and DenMark were driven by α2KIGal4, β2KIGal4 and β2-PB-KIGal4 to label the membrane, the nuclei, the axons and the dendrites of neurons expressing nAChRα2 or nAChRβ2, respectively. α2KIGal4 and β2KIGal4 was expressed in multiple brain regions, including the antenna lobe (AL) , the subesophageal ganglion (SOG) , and the sleep-regulating regions MBs and PI , and in the mesothoracic, metathoracic neuromere (MN, MtN) and the abdominal center (AC) of the ventral nerve cord (VNC) (Fig. 9A-D, Fig. 10A-F) , while β2-PB-KIGal4 showed no expression in the brain and the VNC except for dendrites in the optic lobe (Fig. 9E-F, Fig. 10G-I) .

Example 4 nAChRα2 and β2 function together to promote sleep

The nAChRα2 and nAChRβ2 genes are tightly linked: only ～18kb apart from each other in the Drosophila genome (Fig. 11A) . nAChRα2 and nAChRβ2 were intersected by simultaneously expressing UAS-FRT-STOP-FRT-GFP in β2-expressing neurons driven by β2KIGal4, and LexAop-Flp in α2-expressing neurons driven by α2KILexA. In nAChRα2 ⁺ nAChRβ2 ⁺ neurons, the STOP cassette between the UAS and GFP was removed by the Flp recombinase, thus labeling the neurons with GFP (Fig. 11B) .

Intersection of nAChRα2 and nAChRβ2 was identified in multiple brain regions (Fig. 11C) . Also, RNAi knockdown of nAChRβ2 in nAChRα2-expressing cells and knockdown of nAChRα2 in nAChRβ2-expressing cells both reduced nighttime sleep durations (Fig. 11D-E) . The results of expression, intersection, and RNAi experiments is that nAChRα2 functions with nAChRβ2 to promote sleep.

Example 5 nAChRα2 functions in octopaminergic neurons to promote sleep

Then intersect nAChRα2 with synthases of serotonin (Tryptophan hydroxylase, or Trh) and octopamine (Tyramine β hydroxylase, or TβH) . TβHKIGal4 was generated by the strategies described in Example 1, wherein the forward and reverse primes of TβHKIGal4 5’arm were set forth in SEQ ID NO: 21-22, and the forward and reverse primes of TβHKIGal4 3’arm were set forth in SEQ ID NO: 23-24, respectively.

Trh and nAChRα2 were found to have overlapping expression in the SOG (Fig. 13A) . TβH and nAChRα2 were found to be overlapped widely in the brain and the VNC (FIG. 12A-B) , including the AL, the ventral unpaired median (VUM) cells of the SOG, the anterior superior medial protocerebrum (ASM) cells of the superior neuropils (SNP) , the protocerebral bridge (PB) cells, and the ventrolateral protocerebrum (VL) cells of the ventrolateral neuropils (VLNP) .

UAS-nAChRα2 was reintroduced into different types of neurons in the α2-/-background. The nighttime sleep duration of α2-/-was rescued by reintroduction of UAS-nAChRα2 into α2-expressing cells (Fig. 12C) labeled by α2KOGal4 in which 2A-Gal4-STOP was fused to the start codon of nAChRα2.

Expression of nAChRα2 in the octopaminergic cells labeled by TβHKIGal4 rescued the nighttime sleep duration (Fig. 12D) , whereas expression in the serotonergic cells labeled by TrhKIGal4 failed to rescue (Fig. 13B) , indicating that nAChRα2 functions in octopaminergic neurons to promote sleep. RNAi knockdown of nAChRα2 in octopaminergic cells driven by TβHKIGal4 also reduced nighttime sleep duration (Fig. 12E) , suggesting that nAChRα2 in octopaminergic cells was necessary for proper sleep duration. Furthermore, RNAi knockdown of TβH in nAChRα2-expressing cells reduced nighttime sleep duration (Fig. 12F) , indicating that octopamine in α2-expressing cells was also necessary for sleep. Taken together, these data suggest that nAChRα2 functions in octopaminergic neurons to promote sleep, most likely through octopaminergic signaling.

While preferred 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. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the 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 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.

Claims

A method for selecting an agent for adjusting sleep, the method comprising:

providing a candidate agent;

determining an effect of said candidate agent on an activity and/or expression of nAChRα2 and/or nAChRβ2; and

if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is altered by said candidate agent, then selecting said candidate agent as an agent for adjusting sleep.
The method of claim 1, wherein if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is increased by said candidate agent, then selecting said candidate agent as an agent for promoting sleep.
The method of any one of claims 1-2, wherein if the activity and/or expression of said nAChRα2 and/or nAChRβ2 is decreased by said candidate agent, then selecting said candidate agent as an agent for reducing sleep.
The method of any one of claims 1-3, wherein said nAChRα2 is Drosophila melanogaster nAChRα2, or an ortholog thereof.
The method of any one of claims 1-4, wherein said nAChRβ2 is Drosophila melanogaster nAChRβ2, or an ortholog thereof.
The method of any one of claims 1-5, wherein said determining comprises: determining an effect of said candidate agent on an activity and/or expression of said nAChRα2 and/or nAChRβ2 in an octopaminergic cell.
The method of claim 6, wherein said octopaminergic cell comprises an octopaminergic neuronal cell.
The method of any one of claims 1-7, wherein said activity of said nAChRα2 and/or nAChRβ2 comprises one or more of the following:

an ability to form a functional nAChRα2β2 receptor complex;

an ability to increase an activity, releasing, and/or amount of octopamine;

an ability to activate an octopaminergic signaling.
The method of any one of claims 1-8, which is an in vitro method, or an ex vivo method.
The method of any one of claims 1-9, wherein said sleep comprises daytime sleep and/or nighttime sleep.
The method of any one of claims 1-10, wherein said agent does not substantially affect sleep recovery after deprivation, circadian rhythm or arousal.
The method of any one of claims 1-11, wherein said agent comprises a small molecule, a protein and/or a polynucleotide.
The method of any one of claims 1-12, wherein said agent directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.
A system for selecting an agent for adjusting sleep, wherein the system comprises a substance capable of determining an effect of said agent on an activity and/or expression of nAChRα2 and/or nAChRβ2.
The system of claim 14, wherein said substance is capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2.
The system of claim 15, wherein said substance capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.
The system of any one of claims 14-16, wherein said substance is capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.
The system of claim 17, wherein said substance capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.
A method for the treatment, prevention or delay of 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 an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.
The method of claim 19, wherein said sleep disorder is associated with insufficient sleep, and said agent is capable of increasing the activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.
The method of any one of claims 19-20, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The method of any one of claims 19-21, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of any one of claims 19-22, wherein said agent comprise a nucleic acid molecule encoding nAChRα2 and/or nAChRβ2 or an expression product thereof.
The method of any one of claims 19-23, wherein said agent comprise a nucleic acid sequence as set forth in any one of SEQ ID No. 1-20.
The method of claim 24, wherein said sleep disorder is associated with oversleeping, and said agent is capable of decreasing the activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.
The method of claim 25, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The method of any one of claims 25-26, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of any one of claims 19-27, wherein said agent is an agent for promoting sleep, as the activity and/or expression of said nAChRα2 and/or nAChRβ2 is increased by said agent.
The method of any one of claims 19-28, wherein said agent is an agent for reducing sleep, as the activity and/or expression of said nAChRα2 and/or nAChRβ2 is decreased by said agent.
The method of any one of claims 19-29, wherein said nAChRα2 is Drosophila melanogaster nAChRα2, or an ortholog thereof.
The method of any one of claims 19-30, wherein said nAChRβ2 is Drosophila melanogaster nAChRβ2, or an ortholog thereof.
The method of any one of claims 19-31, wherein said altering of the activity and/or expression of said nAChRα2 and/or nAChRβ2 is in an octopaminergic cell.
The method of claim 32, wherein said octopaminergic cell comprises an octopaminergic neuronal cell.
The method of any one of claims 32-33, wherein said activity of said nAChRα2 and/or nAChRβ2 comprises one or more of the following:

an ability to form a functional nAChRα2β2 receptor complex;

an ability to increase an activity, releasing, and/or amount of octopamine;

an ability to activate an octopaminergic signaling.
The method of any one of claims 19-34, which is an in vitro method, an in vivo method, or an ex vivo method.
The method of any one of claims 19-35, wherein said agent comprises a small molecule, a protein and/or a polynucleotide.
The method of any one of claims 19-36, wherein said agent directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.
Use of an agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2 in the manufacture of a medicament for the treatment, prevention or delay of progression of a sleep disorder.
The use of claim 38, wherein said agent is capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2.
The use of claim 39, wherein said agent capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.
The use of any one of claims 38-40, wherein said agent is capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.
The use of claim 40, wherein said substance agent of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.
An agent capable of altering an activity and/or expression of nAChRα2 and/or nAChRβ2, for use in the treatment, prevention or delay of progression of a sleep disorder.
A method for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder, the method comprising:

evaluating an activity and/or expression of nAChRα2 and/or nAChRβ2 in said subject.
The method of claim 44, wherein said activity and/or expression of nAChRα2 and/or nAChRβ2 comprise an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2, and/or an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.
The method of claim 45, wherein a substance capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.
The method of any one of claims 45-46, wherein a substance capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.
The method of any one of claims 44-47, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The method of any one of claims 44-48, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The method of any one of claims 44-49, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of claim 48, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The method of any one of claims 50-51, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
A system for determining a likelihood of a subject to have a sleep disorder, and/or at a risk of having a sleep disorder, the system comprising:

an agent capable of indicating an activity and/or expression level of nAChRα2 and/or nAChRβ2 in said subject.
The system of claim 53, wherein said activity and/or expression of nAChRα2 and/or nAChRβ2 comprise an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2, and/or an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.
The system of claim 54, wherein a substance capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.
The system of any one of claims 54-55, wherein a substance capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.
The system of any one of claims 53-56, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The system of any one of claims 53-57, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The system of any one of claims 53-58, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The system of claim 57, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The system of any one of claims 59-60, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
Use of an agent capable of indicating an activity and/or expression level of nAChRα2 and/or nAChRβ2 of a subject in the manufacture of an indicator of a likelihood of said subject to have a sleep disorder, and/or at a risk of having a sleep disorder.
The use of claim 62, wherein said activity and/or expression of nAChRα2 and/or nAChRβ2 comprise an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2, and/or an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2.
The use of claim 63, wherein an agent capable of determining an effect of said agent on an activity and/or expression of nucleic acid of said nAChRα2 and/or nAChRβ2 comprises: a primer capable of specifically amplifying nAChRα2 and/or nAChRβ2, and/or a probe capable of specifically recognizing nAChRα2 and/or nAChRβ2.
The use of any one of claims 63-64, wherein an agent capable of determining an effect of said agent on an activity and/or expression of protein of said nAChRα2 and/or nAChRβ2 comprises: an agent capable of specifically recognizing the nAChRα2 and/or nAChRβ2protein and/or an agent capable of determining the activity of the nAChRα2 and/or nAChRβ2 protein.
The use of any one of claims 63-65, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The use of any one of claims 63-66, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The use of any one of claims 63-67, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The use of claim 66, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The use of any one of claims 68-69, wherein said sleep disorder associated with oversleeping sleep comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
A non-human organism or a living part thereof, comprising a functionally impaired nAChRα2 and/or a functionally impaired nAChRβ2.
The non-human organism or living part thereof of claim 71, wherein said non-human organism is a Drosophila melanogaster.
The non-human organism or living part thereof of any one of claims 71-72, which does not comprise any functional nAChRα2.
The non-human organism or living part thereof of any one of claims 71-73, which does not comprise any functional nAChRβ2.
The non-human organism or living part thereof of any one of claims 71-74, which is homozygous for the functionally impaired nAChRα2 and/or the functionally impaired nAChRβ2.
The non-human organism or living part thereof of any one of claims 71-75, wherein said non- human organism has reduced duration of sleep comparing to a corresponding wildtype non-human organism.
The non-human organism or living part thereof of any one of claims 71-76, wherein said sleep comprises daytime sleep and/or nighttime sleep.
The non-human organism or living part thereof of any one of claims 71-77, wherein the nAChRα2 gene and/or nAChRβ2 gene in said organism is knocked down or knocked-out.
The non-human organism or living part thereof of any one of claims 71-78, wherein the nAChRα2 gene and/or nAChRβ2 gene in said organism is knocked down by RNAi.
The non-human organism or living part thereof of any one of claims 71-79, wherein the nAChRα2 gene in said organism is knocked down by α2KIGal4.
The non-human organism or living part thereof of any one of claims 71-80, wherein the nAChRβ2 gene in said organism is knocked down by β2KIGal4.
The non-human organism or living part thereof of any one of claims 71-81, wherein the fourth, the fifth and the sixth exons of the nAChRα2 gene in said organism is deleted.
The non-human organism or living part thereof of any one of claims 71-82, wherein the first to the eighth exons of the nAChRβ2 gene in said organism is deleted.
A cell, a cell line or a primary cell culture derived from the non-human organism or living part thereof of any one of claims 71-83.
A tissue derived from the non-human organism or a living part thereof of any one of claims 71-83.
The tissue of claim 85, wherein the tissue is derived from a neural tissue.
The tissue of any one of claims 85-86, wherein the tissue is derived from a neural tissue comprising octopaminergic cells.
A method of screening for a substance, a device, and/or a composition useful in the treatment, prevention or delay of progression of a sleep disorder, comprising applying a candidate substance, device and/or composition to the non-human organism or living part thereof of any one of claims 71-83, the cell, cell line or primary cell culture of claim 84, or the tissue of any one of claims 85-87, and determining an effect of said candidate substance, device and/or composition on one or more of the following:

a sleep duration of said non-human organism;

an activity, amount and/or releasing of octopamine; and

an activate of an octopaminergic signaling.
The method of claims 88, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The method of any one of claims 88-89, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The method of any one of claims 88-90, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of claim 89, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The method of any one of claims 91-92, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of any one of claims 88-93, wherein said determining comprises: determining an effect of said candidate substance, device and/or composition on an activity and/or expression of said nAChRα2 and/or nAChRβ2 in an octopaminergic cell.
The method of claim 94, wherein said octopaminergic cell comprises an octopaminergic neuronal cell.
The method of any one of claims 94-95, wherein said nAChRα2 is Drosophila melanogaster nAChRα2, or an ortholog thereof.
The method of any one of claims 94-96, wherein said nAChRβ2 is Drosophila melanogaster nAChRβ2, or an ortholog thereof.
The method of any one of claims 88-97, which is an in vitro method, or an ex vivo method.
The method of any one of claims 88-98, wherein said candidate substance and/or composition comprises a small molecule, a protein and/or a polynucleotide.
The method of any one of claims 88-99, wherein said candidate substance, device and/or composition directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.
A method of screening for a biomarker useful in the diagnosis and/or monitoring of a sleep disorder, comprising:

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

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

and identifying said substance as the biomarker when said disease value is different from said wildtype value.
The method of claim 101, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The method of any one of claims 101-102, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The method of any one of claims 101-103, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of claim 102, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The method of claim 105, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
The method of any one of claims 101-106, wherein said disease value is greater than said wildtype value, and said biomarker is a biomarker indicating promoting sleep.
The method of any one of claims 101-107, wherein said disease value is less than said wildtype value, and said biomarker is a biomarker indicating reducing sleep.
Use of the non-human organism or living part thereof of any one of claims 71-83, the cell, cell line or primary cell culture of claim 84, or the tissue of any one of claims 85-87 in the preparation of a system of screening for a substance, a device, a composition and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a sleep disorder.
The use of claim 109, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The use of any one of claims 109-110, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The use of any one of claims 109-111, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The use of claim 110, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The use of any one of claims 110-113, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.
The use of any one of claims 110-114, wherein said substance, composition and/or biomarker comprises a small molecule, a protein and/or a polynucleotide.
The use of any one of claims 109-115, wherein said substance, device, composition and/or biomarker directly acts on a nAChRα2 protein and/or a nAChRβ2 protein, and/or a nucleic acid encoding a nAChRα2 protein and/or a nAChRβ2 protein.
The non-human organism or living part thereof of any one of claims 71-83, the cell, cell line or primary cell culture of claim 84, or the tissue of any one of claims 85-87, for use in screening for a substance, a device, a composition and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a sleep disorder.
The use of claim 117, wherein said sleep disorder comprise sleep disorder associated with insufficient sleep and/or sleep disorder associated with oversleeping.
The use of any one of claims 117-118, wherein said sleep disorder associated with insufficient sleep comprises daytime insufficient sleep and/or nighttime insufficient sleep.
The use of any one of claims 117-119, wherein said sleep disorder associated with insufficient sleep comprises insomnia and/or sleep loss associated with cardiovascular disorders and/or neurodegenerative diseases.
The use of claim 120, wherein said sleep disorder associated with oversleeping comprises daytime oversleeping and/or nighttime oversleeping.
The use of any one of claims 120-121, wherein said sleep disorder associated with oversleeping comprises narcolepsy, hypersomnolence, NREM/REM-related parasomnias, and/or oversleeping/hard-to-be-awaken associated with cardiovascular disorders and/or neurodegenerative diseases.