Patent Application
20230358770
A Sequence Listing is provided herewith as a text file, “Sequence listing_ST25” created on Sep. 22, 201, and having a size of 160 kb. The contents of the text file are incorporated by reference herein in their entirety.
The present disclosure provides biosensors and methods for detecting one or more odorants associated with a level or a change in the levels of one or more neurotransmitters, in particular dopamine deficiency, in the central nervous system of a subject. More particularly, the disclosure relates to biosensors comprising one or more populations of olfactory sensory neurons, or cilia derived therefrom, where each population preferentially expresses an odorant receptor.
Neurotransmitters are chemical agents released by neurons to stimulate neighboring neurons or muscle or gland cells, thus allowing electrical impulses to be passed from one cell to the next throughout the nervous system. For example, the neurotransmitter dopamine is responsible for transmitting signals within the brain that allow for coordination of movement. Accordingly, a dopamine imbalance in the brain can negatively affect mood, sleep, memory, learning, concentration, and motor control. Dopamine deficiency is related to a number of diseases and conditions, including, but not limited to, Parkinson's disease (PD), depression, schizophrenia, dystonia, and restless leg syndrome.
PD is a neurodegenerative disorder affecting at least one million people in the United States, and more than five million worldwide. PD is associated with disruption of dopamine-producing (“dopaminergic”) neurons in the brain, in particular in an area called the substantia nigra. Alteration of dopamine production causes neurons to fire without normal control, leaving patients less able to direct or control their movement. Symptoms of dopamine alteration in PD generally develop slowly over years and include movement-related (“motor”) symptoms such as tremor; slowness of movements (bradykinesia); stiffness or rigidity of the arms, legs or trunk; and gait and balance problems. In addition, some PD symptoms may be unrelated to movement (“non-motor” symptoms), and can include apathy, depression, constipation, sleep behavior disorders, anosmia (loss of sense of smell), and cognitive impairment.
There is no objective test or biomarker (such as a blood test, brain scan, or electroencephalogram) to make a definitive diagnosis of PD or the associated dopamine alteration. Rather, a diagnosis of PD is made following ascertainment of the patient's medical history and a thorough neurological examination, looking in particular for two or more of the cardinal late onset symptoms—tremor, bradykinesia, rigidity of the limbs or trunk, and balance trouble. Additionally, a doctor may also look for responsiveness to PD medications as further evidence that PD is the correct diagnosis. Unfortunately, the rates for misdiagnosing PD or failing to diagnose PD are high, because there is no definitive test for PD, and because PD symptoms are similar to those of other neurological conditions. Importantly, current standard methods for the diagnosis of PD are limited to the identification of PD after symptoms of PD have already manifested in the patient. Conversely, these PD symptoms occur only after a significant amount of the substantia nigra neurons have already been lost or impaired.
Accordingly, new devices and methods that facilitate a determination of whether or not a patient exhibits a change in the levels of one or more neurotransmitters (including, but not limited to dopamine) in the central nervous system (CNS) are urgently needed.
In one aspect, provided is a biosensor comprising one or more populations of olfactory sensory neurons (OSNs), or cilia derived therefrom; wherein each population of OSNs preferentially expresses an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1-40. In embodiments, provided is a biosensor comprising one or more populations of OSNs, or cilia derived therefrom; wherein each population of OSNs preferentially expresses an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1 and 6-18. In embodiments, provided is a biosensor comprising one or more populations of OSNs, or cilia derived therefrom; wherein each population of OSNs preferentially expresses an amino acid sequence comprising an OR described in any of Tables 1-5. In one aspect, provided is a biosensor comprising one or more populations of OSNs, or cilia derived therefrom; wherein each population of OSNs preferentially expresses an OR comprising an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any of the amino acid sequences of an OR described in any of Tables 1-5. In embodiments, the one or more populations of OSNs, or cilia derived therefrom, are attached to a solid support. In some embodiments, the solid support is selected from the group consisting of silicon, glass, polystyrene, and polymers.
In embodiments, the one or more populations of OSNs further express a marker for detecting activation or lack of activation of the OR. In embodiments, the one or more populations of OSNs further express one or more markers for detecting activation or lack of activation of the OR, wherein the activation, if any, occurs upon exposure of the one or more populations of OSNs, or cilia derived therefrom, to a sample, or and extract of the sample, from a subject. In some embodiments, the markers is a calcium-sensitive fluorescent dye selected from the group consisting of fura-2, fluo-3, fluo-4, fluo-5F, indo-1, and Oregon Green BAPTA. In some embodiments, the marker is selected from the group consisting of GECO2.1, GCaMP6, Flamindo, Flamindo2, and Pink Flamindo. In some embodiments, the marker for detecting activation or lack of activation of the OR is co-expressed with the preferentially expressed odorant receptor (OR).
In embodiments, the OSNs comprise an enhancer operably linked to the sequence encoding the preferentially expressed OR. In embodiments, the enhancer is a singular gene choice enhancer. In some embodiments, the enhancer comprises at least four sequential repeats of a 21 base pair (bp) sequence wherein each 21 bp sequential repeat comprises the sequence AACTTTTTAATGA (SEQ ID NO: 81). In some embodiments, the singular gene choice enhancer sequence comprises at least four sequential repeats of a 21 bp sequence wherein each 21 bp sequential repeat comprises the sequence AACTTTTTAATGA (SEQ ID NO: 81). In some embodiments, the enhancer comprises at least four sequential repeats of ACATAACTTTTTAATGAGTCT (SEQ ID NO: 82). In some embodiments, the singular gene choice enhancer sequence comprises at least four sequential repeats of ACATAACTTTTTAATGAGTCT (SEQ ID NO: 82). In some embodiments, the enhancer comprises ten or fewer of the 21 bp sequential repeats. In some embodiments, the singular gene choice enhancer comprises ten or fewer of the 21 bp sequential repeats. In some embodiments, the enhancer comprises one or more TetO sequences. In some embodiments, the singular gene choice enhancer comprises one or more TetO sequences.
In aspects, provided is a biosensor comprising a cell or population of cells engineered to express an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1-40. In aspects, provided is a biosensor comprising a cell or population of cells engineered to express an OR comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, (ii) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1 and 6-18, (iii) an amino acid sequence of an OR described in any of Tables 1-5, and/or (iv) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to an amino acid sequence of an OR described in any of Tables 1-5. In some embodiments, the cell is a eukaryotic cell or the population of cells is a population of eukaryotic cells. In some embodiments, the cell or the cells in the population of eukaryotic cells are yeast cells or OSNs. In some embodiments, the cell or the population of cells further expresses one or more markers for detecting activation or lack of activation of the OR. In some embodiments, the cell or population of cells further expresses one or more markers for detecting activation or lack of activation of the OR, wherein the activation occurs upon exposure of the cell or population of cells to a sample from a subject with a change in the levels of one or more neurotransmitters in the CNS (e.g., as compared to the control levels for the one or more neurotransmitters). In some embodiments, the marker is a-sensitive fluorescent dye selected from the group consisting of fura-2, fluo-3, fluo-4, fluo-5F, indo-1, and Oregon Green BAPTA. In some embodiments, the marker is selected from the group consisting of GECO2.1, GCaMP6, Flamindo, Flamindo2, and Pink Flamindo. In some embodiments, the marker for detecting activation or lack of activation of the OR is co-expressed with the expressed OR.
In aspects, the biosensors described herein are for use in detecting one or more odorants, or a change in the levels of one or more odorants (e.g., as compared to control levels of the one or more odorants), in a sample from a subject. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, is associated with the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to control levels of the one or more neurotransmitters), in the CNS of a subject. Thus, in embodiments, the biosensors described herein are for use in detecting the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to control levels of the one or more neurotransmitters), in the CNS of a subject. The one or more neurotransmitters can include, for example, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and/or serotonin. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, is associated with a disease characterized by a change in the levels of one or more neurotransmitters in the CNS (e.g., as compared to control levels of the one or more neurotransmitters), including, e.g., a disease associated with a dopamine deficiency in the CNS such as PD, depression, schizophrenia, dystonia, or restless leg syndrome. Thus, in embodiments, the biosensors described herein are for use in detecting a disease characterized by a change in the levels of one or more neurotransmitters in the CNS including, e.g., a disease associated with a dopamine deficiency in the CNS such as PD, depression, schizophrenia, dystonia, or restless leg syndrome. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, detected by the biosensors disclosed herein are associated with a neurodegenerative disease in the subject. Thus, in embodiments, the biosensors described herein are for use in detecting neurodegenerative disease in the subject. In embodiments, the neurodegenerative disease is PD.
In one aspect, provided is a transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal, wherein the transgenic animal comprises an olfactory epithelium in which the OSNs preferentially express an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1-40. In one aspect, provided is a transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal, wherein the transgenic animal comprises an olfactory epithelium in which the OSNs preferentially express an OR comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1 and 6-18, (ii) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1 and 6-18, (iii) an amino acid sequence of an OR described in any of Tables 1-5, and/or (iv) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to an amino acid sequence of an OR described in any of Tables 1-5. In embodiments, the isolated tissue is an olfactory epithelium. In embodiments, the isolated cell or population of cells is an olfactory epithelium cell or population of olfactory epithelium cells (e.g., an OSN or a population of OSNs that preferentially express an OR described herein).
In one aspect, provided is a transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal, wherein the transgenic animal, or the tissue, cell, or population of cells isolated from the transgenic animal, comprises: (a) a transgene sequence encoding an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1-40; and (b) an enhancer operably linked to the transgene sequence. In one aspect, provided is a transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal, wherein the transgenic animal, or the tissue, cell, or population of cells isolated from the transgenic animal, comprises: (a) a transgene sequence encoding an OR comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NOs: 1 and 6-18, (ii) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1 and 6-18, (iii) an amino acid sequence of an OR described in any of Tables 1-5, and/or (iv) an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to an amino acid sequence of an OR described in any of Tables 1-5; and (b) an enhancer operably linked to the transgene sequence. In embodiments, the enhancer is a singular gene choice enhancer. In embodiments, the enhancer comprises at least four sequential repeats of a 21 base pair (bp) sequence wherein each 21 bp sequential repeat comprises the sequence AACTTTTTAATGA (SEQ ID NO: 81). In some embodiments, the enhancer comprises at least four sequential repeats of ACATAACTTTTTAATGAGTCT (SEQ ID NO: 82). In embodiments, the enhancer comprises ten or fewer of the 21 bp sequential repeats. In embodiments, the singular gene choice enhancer comprises ten or fewer of the 21 bp sequential repeats. In some embodiments, the enhancer comprises one or more TetO sequences. In some embodiments, the singular gene choice enhancer comprises one or more TetO sequences.
In some embodiments, the transgenic animal is a non-human mammal. In some embodiments, the non-human mammal belongs to the family of Bovidae, Canidae, or Muridae. In some embodiments, the non-human mammal is a rat, a mouse, a dog, cat, goat, chicken, sheep, pig, or primate.
In aspects, the transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal, described herein are for use in detecting one or more odorants, or a change in the levels of one or more odorants (e.g., as compared to control levels of the one or more odorants), in a sample from the subject. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, is associated with the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to control levels of the one or more neurotransmitters), in the CNS of a subject. Thus, in embodiments, the transgenic animals, or a tissue, cell, or population of cells isolated from the transgenic animal described herein are for use in detecting the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to control levels of the one or more neurotransmitters), in the CNS of a subject. The one or more neurotransmitters can include, for example, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and/or serotonin.
In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, detected using a transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal disclosed herein are associated with a neurodegenerative disease in the subject. Thus, in embodiments, the transgenic animal, or a tissue, cell, or population of cells isolated from the transgenic animal described herein are for use in detecting a neurodegenerative disease in the subject. In embodiments, the neurodegenerative disease is PD.
In one aspect, provided is an expression construct comprising: an OR coding sequence, wherein the OR coding sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1-40. In embodiments, provided is an expression construct comprising an OR coding sequence, wherein the OR coding sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, or an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to any one of SEQ ID NOs: 1 and 6-18. In embodiments, provided is an expression construct comprising an OR coding sequence, wherein the OR coding sequence encodes an amino acid sequence of an OR described in any of Tables 1-5. In embodiments, provided is an expression construct comprising an OR coding sequence, wherein the OR coding sequence encodes an amino acid sequence with greater than 85% identity (e.g., greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity) to the amino acid sequences of any of the ORs described in any of Tables 1-5. In embodiments, the expression construct comprises a nucleotide sequence selected from SEQ ID NO: 41-80, or a nucleotide sequence having greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity to a nucleotide sequence selected from SEQ ID NO: 41-80.
In embodiments, the expression construct further comprises an enhancer operably linked to the OR coding sequence. In embodiments, the enhancer is a singular gene choice enhancer operably linked to the OR coding sequence. In aspects, the expression construct is for preferentially expressing in a population of OSNs an OR described herein and for the uses described herein. In some embodiments, the enhancer comprises at least four sequential repeats of a 21 bp sequence wherein each 21 bp sequential repeat comprises the sequence of AACTTTTTAATGA (SEQ ID NO: 81). In some embodiments, the enhancer comprises at least four sequential repeats of ACATAACTTTTTAATGAGTCT (SEQ ID NO: 82). In some embodiments, the enhancer comprises ten or fewer of the 21 bp sequential repeats. In some embodiments, the singular gene choice enhancer sequence comprises ten or fewer of the 21 bp sequential repeats. In some embodiments, the enhancer comprises one or more TetO sequences. In some embodiments, the enhancer is a singular gene choice enhancer. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a tTA or an rtTA protein. In some embodiments, the rTA or rtTA protein comprises a sequence derived from VP16, VP32, VP48, VP64, and/or GAL4-VP16. In some embodiments, the one or more TetO sequences are located upstream of a cytomegalovirus (CMV) promoter, such as a minimal CMV promoter.
In one aspect, provided is a method for detecting one or more odorants, the method comprising: (a) obtaining a sample from the subject; (b) exposing a biosensor disclosed herein to the sample or to an extract from the sample; and (c) measuring the activation or lack of activation of the one or more ORs, described herein by one or more odorant molecules in the sample obtained from said subject. In embodiments, the levels of the one or more odorants is associated with the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to the control levels for the one or more neurotransmitters) in the CNS of a subject.
In one aspect, provided is a method for detecting a change in the levels of one or more odorants in a sample (e.g., as compared to the control levels for the one or more odorants), the method comprising: (a) obtaining a sample comprising the one or more odorants; (b) exposing a biosensor disclosed herein to the sample or to an extract from the sample; and (c) measuring the activation or lack of activation of the one or more ORs, described herein by one or more odorant molecules in the sample. In some embodiments, the change in the levels of the one or more odorants is associated with a change in the levels of one or more neurotransmitters in the CNS of a subject from which the sample was derived.
In some embodiments of the method, one or more of the neurotransmitters are catecholamines. In some embodiments, the one or more neurotransmitters are selected from the group consisting of dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and/or serotonin. In embodiments, the one or more neurotransmitters are selected from the group of dopamine and serotonin. Thus, in embodiments, the methods disclosed herein can be used to determine if a subject has a change in the levels of one or more neurotransmitters in the CNS, including for example, if the subject has dopamine deficiency. In embodiments, the methods can be used to determine if a subject has, or is likely to develop, a disease associated with dopamine deficiency including PD, depression, schizophrenia, dystonia, or restless leg syndrome. In embodiments, the methods can be used to determine if a subject has, or is likely to develop, PD.
In some embodiments, measuring the activation of the OR comprises detecting a decrease in ATP levels or a change in action potential. In some embodiments, measuring the lack of activation of the OR comprises detecting a lack of decrease in ATP levels or a lack of change in action potential. In some embodiments, measuring the activation of the OR comprises detecting an increase in Ca2+, guanosine diphosphate (GDP) and/or cyclic adenosine monophosphate (cAMP) levels. In some embodiments, measuring the lack of activation of the OR comprises detecting a lack of increase in Ca2+, GDP and/or cAMP level.
In some embodiments, the one or more odorants associated with a change in the levels of one or more neurotransmitters in the CNS of a subject are present at elevated levels in the sample from the subject as compared to control levels. In some embodiments, the one or more odorants associated with a change in the levels of one or more neurotransmitters in the CNS of a subject are present at reduced levels in the sample from the subject as compared to control levels.
In some embodiments, the subject has a disease associated with a change in the levels of one or more neurotransmitters in the subject's CNS (e.g., as compared to control levels of the one or more neurotransmitters). In some embodiments, the subject has a disease or condition associated with a dopamine deficiency in the CNS. In some embodiments, the disease or condition associated with dopamine deficiency is PD, depression, schizophrenia, dystonia, or restless leg syndrome. In some embodiments, the disease or condition associated with dopamine deficiency is PD. In some embodiments, the levels of the one or more odorants, or the change in the levels of the one or more odorants is associated with the subject having a neurodegenerative disease, such as PD. In embodiments, the sample comprising the one or more odorants comprises sebum, saliva, blood, and/or urine of a subject.
In embodiments, the control levels for the one or more odorants are the levels of the one or more odorants in a sample from one or more individuals that do not suffer from a disease associated with a change in the levels of the one or more odorants. In embodiments, the control levels for the one or more odorants are the levels of the one or more odorants in a sample from one or more individuals that do not suffer from a disease associated with a change in the levels of the one or more neurotransmitters as described herein. In embodiments, the control levels for the one or more odorants are the levels of the one or more odorants in a sample from one or more individuals that do not suffer from PD, depression, schizophrenia, dystonia, and/or restless leg syndrome. In embodiments, the control levels for the one or more odorants are the levels of the one or more odorants in a sample obtained from the subject at a different time. In some embodiments, the control levels for the one or more odorants are obtained from the same subject at an earlier time. In some embodiments, the control levels for the one or more odorants are obtained from the same subject at a later time.
In embodiments, the control levels for the one or more neurotransmitters are the levels of the one or more neurotransmitters in the CNS in one or more individuals that do not suffer from a disease associated with a change in the levels of the one or more neurotransmitters. In some embodiments, the control levels for the one or more neurotransmitters are the levels of the neurotransmitter in the CNS of one or more individuals who do not suffer from a disease associated with a change in the level of dopamine (e.g., a decrease in dopamine) in the CNS. In some embodiments, the control level of a neurotransmitter is the level of that neurotransmitter in the CNS of one or more individuals who do not suffer from a disease associated with a change in the level of serotonin in the CNS. In some embodiments, the control level of a neurotransmitter is the level of that neurotransmitter in the CNS of one or more healthy individuals. In some embodiments, the control level of a neurotransmitter is the level of that neurotransmitter in the CNS of one or more individuals who do not suffer from PD, depression, schizophrenia, dystonia, and/or restless leg syndrome. In some embodiments, the control levels for the one or more neurotransmitters are the levels of the one or more neurotransmitters in the CNS of the subject obtained at a different time. In some embodiments, the control levels for the one or more neurotransmitters are obtained from the same subject at an earlier time. In some embodiments, the control levels for the one or more neurotransmitters are obtained from the same subject at a later time. In some embodiments, the neurotransmitter is a catecholamine. In some embodiments, the neurotransmitter is selected from the group consisting of dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and serotonin. In some embodiments, the neurotransmitter is dopamine or serotonin.
Provided herein are biosensors comprising one or more populations of olfactory neurons, or cilia derived therefrom, that preferentially express certain ORs; biosensors comprising a cell or a population of cells engineered to express certain ORs; biosensors comprising certain isolated ORs; transgenic animals and tissues derived therefrom that preferentially express certain ORs; isolated cells or populations of cells engineered to express certain ORs; expression constructs for the preferential expression of certain ORs; and methods of using the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein.
Provided herein are methods for detecting one or more odorants associated with the levels of one or more neurotransmitters, or the change in the level of one or more neurotransmitters, in the CNS of a subject. For example, dopamine deficiency in the brain can lead changes in bodily odors or odorant secretions. Accordingly, the present application provides biosensors and methods for using such biosensors for the detection of one or more odorants associated with a disease or condition characterized by altered levels, or change in the levels, of one or more neurotransmitters (including, but not limited to dopamine) in the CNS of a patient.
ORs are amongst the most sensitive and specific chemical detectors in nature, and the discriminatory power of the olfactory system rivals that of any other sensory system. Mammals detect odors using millions of ORs expressed by OSNs that line the nasal cavity. In mammals, olfactory perception of odorants occurs by the combinatorial activation of ORs. OR genes form a large multigene family with about 1,000 members in rodents and about 350 members in humans. The main olfactory epithelium (MOE) expresses ORs through a singular gene choice mechanism whereby only one OR gene allele is expressed in each OSN. Thus, the olfactory sheet is a broad chemical detector, in which each OR is equally distributed in the main olfactory epithelium and only expressed in a small percentage of OSNs—about 0.1% of all OSNs in rodents.
The present disclosure addresses the limitations of current methods for an early and reliable diagnosis of patients with changed levels of one or more neurotransmitters in the CNS by utilizing a cohort of ORs that show activation or lack of activation in presence of one or more odorants in a sample from a patient that exhibits a change in the levels of one or more neurotransmitters in the patient's CNS (e.g., as compared to the control levels for the one or more neurotransmitters).
Provided herein are ORs responsive to odorant that are associated with the levels of one or more neurotransmitters in the CNS of a subject. Provided herein are ORs responsive to odorants that are associated with a change in the levels of one or more neurotransmitters in the CNS of a subject. Provided herein are odorants responsive to odorants associated with a change in the levels of one or more neurotransmitters in the CNS of a subject (e.g., as compared to the control levels for the one or more neurotransmitters).
The ORs described herein are activated by one or more odorants present in a sample from a subject and/or present in a control sample. In embodiments, the odorant is present at elevated levels in the sample from a subject leading to increased activation of the responsive OR. In embodiments, other odorants are present at reduced levels in the sample from the subject and at elevated levels in control samples leading to reduced activation in the responsive OR to the sample from the subject. In embodiments, the ORs described herein are activated by one or more odorants the levels of which are associated with the levels of one or more neurotransmitters in the CNS of a subject or a control. The one or more neurotransmitters can include, for example, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and/or serotonin. In embodiments, the ORs described herein are activated by one or more odorants the levels of which are associated with a disease characterized by a change in the levels of one or more neurotransmitters in the CNS (e.g., as compared to control levels of the one or more neurotransmitters), including, e.g., a disease associated with a dopamine deficiency in the CNS such as PD, depression, schizophrenia, dystonia, or restless leg syndrome. In embodiments, the ORs described herein are activated by one or more odorants, the levels of which are associated with a neurodegenerative disease in the subject. In embodiments, the neurodegenerative disease is PD.
Provided herein is an OR, wherein the level of activation or lack of activation of the OR is associated with the levels of one or more neurotransmitters in the CNS of a subject. Provided herein is an OR, wherein the activation or lack of activation of the OR is associated with a change in the levels of one or more neurotransmitters in the CNS of a subject. In some embodiments, the OR shows activation, or increased activation, upon exposure to a sample from a subject or to an extract from the sample and shows lack of activation, or reduced activation, upon exposure to a sample from a control or to an extract from the sample. In some embodiments, the OR shows lack of activation, or reduced activation, upon exposure to a sample from a subject or to an extract from the sample and shows activation, or increased activation, upon exposure to a sample from a control or to an extract from the sample. In some embodiments, the OR is capable of being activated by an odorant molecule that is present in a sample from a subject and not present, or present at reduced levels, in the sample of a control. In some embodiments, the OR is capable of being activated by an odorant molecule that is present in a sample from a control and not present, or present at reduced levels, in the sample of a subject. In some embodiments, the OR binds to an odorant present in a sample from a subject and not present, or present at reduced levels, in a sample from a control. In some embodiments, the OR binds to an odorant present in a sample from a control and not present, or present at reduced levels, in a sample from a subject.
In some embodiments, the ORs show activation upon exposure to an odorant, wherein the odorant is present in a sample of a subject and wherein the odorant is present in a sample of a control, but wherein the odorant is present at a different level in the sample of the subject and in the sample of the control. In some embodiments, the level of the odorant in the sample of the subject is higher than the level of the odorant in the sample of the control. In some embodiments, the level of the odorant in the sample of the subject is lower than the level of the odorant in the sample of the control.
ORs useful for the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein and methods disclosed herein include, but are not limited to, rat ORs Olr836, Olr837, Olr838, Olr839, Olr840, Olr841 (and paralogs), Olr300, Olr1396, Olr292, Olr804, Olr661, Olr749, Olr1185, Olr1381, Olr1558, Olr607, Olr712, 041160. In embodiments, the OR used in the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein and methods disclosed herein comprises an amino acid sequence selected from SEQ ID NOs: 1-40. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to any one of SEQ ID NOs: 1-40. In embodiments, the OR used in the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein and methods disclosed herein comprises an amino acid sequence selected from SEQ ID NOs: 1 and 6-18. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to any one of SEQ ID NOs: 1 or 6-18. In embodiments, the OR used in the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein and methods disclosed herein comprises an amino acid sequence of an OR described in any of Tables 1-5. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described in any of Tables 1-5.
In embodiments, the OR comprises an amino acid sequence of an OR described in Table 1. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described in Table 1. In embodiments, the OR comprises an amino acid sequence of an OR described in Table 2. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described Table 2. In embodiments, the OR comprises an amino acid sequence of an OR described in Table 3. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described in Table 3. In embodiments, the OR comprises an amino acid sequence of an OR described in Table 4. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described in Table 4. In embodiments, the OR comprises an amino acid sequence of an OR described in Table 5. In embodiments, the OR comprises an amino acid sequence with greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity to an amino acid sequence of an OR described in Table 5.
In some embodiments, the OR is encoded by a patch gene. Patch genes are a group of genes encoding OR proteins expressed in a localized region, a “patch”, of the olfactory epithelium. The patch domain of expression for this group of ORs has been well described for mouse genes, but is also conserved amongst other terrestrial mammals. Patch OR genes tend to be linked and show high degree of sequence homology amongst paralogues in the same species and orthologues across species. A subset of patch OR genes, respond to sebum derived odors including long chain aldehydes such as C14-C18. In mice, trace amine-associated receptor (TAAR) chemosensory genes are expressed in a domain and respond to amines, whereas Class I OR genes are expressed in a different domain and respond to acids. As described herein, certain patch ORs preferentially bind to sebum related odors.
Also useful for the biosensors, transgenic animals, tissues, cells, population of cells, and expression constructs disclosed herein and methods disclosed herein are ORs that are homologs of any one of the ORs disclosed herein. In some embodiments, the OR is an orthologue or a paralogue of an OR disclosed herein. As used in this specification, a homolog of an OR is an OR that shares 85% or more homology (amino acid identity plus amino acid similarity) as compared to an OR. As used in this specification, an orthologue of an OR is an OR (i) that is encoded by a gene that is located at an orthologous position in the genome as compared to the OR gene or that is encoded by a gene that exhibits synteny with the OR gene and (ii) that exhibits greater than 85% protein homology (amino acid identity plus amino acid similarity) as compared to the OR. As used herein, a paralogue is a gene that shares a high degree of homology to another gene due to a gene duplication event.
Once an OR has been identified in, for example, in a rat or a mouse, a person of ordinary skill in the art can readily identify homologous ORs derived from other species and can verify that these homologous ORs serve the same or a very similar function. Methods for identifying homologous proteins are well known in the art (see for example Pearson W R, An introduction to sequence similarity (“homology”) searching. Curr Protoc Bioinformatics. 2013 June; Chapter 3: Unit 3.1, incorporated herein by reference). Thus, provided herein are for example, rat, mouse, or other mammalian ORs that are homologs or orthologs to the ORs identified herein. A non-exhaustive, non-limiting list of orthologues and paralogues for the ORs identified in this disclosure can be found in Tables 2-5.
As used herein, a “biosensor” is an analytical device or system which may be used to detect, quantitatively or qualitatively, the presence, absence, and/or concentration of a biological analyte (such as an odorant molecule) in a sample. In some embodiments, the biosensor converts a biological response into a signal that can be detected, for example an electrical signal or light signal. In some embodiments, the biosensor comprises a recognition element (e.g., an OR described herein), which can recognize or capture a specific analyte, and a transducer, which transmits the presence or absence of an analyte into a detectable signal. In some embodiments, the biosensor comprises a chip or is utilized as part of a biochemical assay. A schematic of a method for making a biosensor according to the present disclosure and detecting one or more odorants is shown in
In embodiments, the biosensors described herein are for use in detecting one or more odorants, or a change in the levels of one or more odorants (e.g., as compared to control levels of the one or more odorants), in a sample from the subject. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, is associated with the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters (e.g., as compared to control levels of the one or more neurotransmitters), in the CNS of a subject. Thus, in embodiments, the biosensors described herein are for use in detecting the levels of one or more neurotransmitters, or a change in the levels of one or more neurotransmitters in the CNS of a subject. The one or more neurotransmitters can include, for example, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), histamine, and/or serotonin.
In embodiments, the biosensors described herein are for use in detecting one or more odorants, or the change in the levels of the one or more odorants, associated with a disease characterized by a change in the levels of one or more neurotransmitters in the CNS (e.g., as compared to control levels of the one or more neurotransmitters), including, e.g., a disease associated with a dopamine deficiency in the CNS such as PD, depression, schizophrenia, dystonia, or restless leg syndrome. Thus, in embodiments, the biosensors described herein are for use in detecting a disease characterized by a change in the levels of one or more neurotransmitters in the CNS including, e.g., a disease associated with a dopamine deficiency in the CNS such as PD, depression, schizophrenia, dystonia, or restless leg syndrome. In embodiments, the one or more odorants, or the change in the levels of the one or more odorants, detected by the biosensors disclosed herein, are associated with a neurodegenerative disease in the subject. Thus, in embodiments, the biosensors described herein are for use in detecting a neurodegenerative disease in the subject. In embodiments, the neurodegenerative disease is PD.
In one aspect, provided is a biosensor comprising a cell or population of cells engineered to express an OR described herein. In such embodiments, the cell may be an isolated eukaryotic cell or the population of cells may be a population of isolated eukaryotic cells. In some embodiments, the biosensor comprises a eukaryotic cell or a population of eukaryotic cells genetically engineered to express an OR described herein. As used herein, the terms “genetically engineered,” “genetically modified,” or “engineered” are used interchangeably and refers to any human or human-caused manipulation intended to introduce a genetic change in a cell or organism. Such manipulation includes altering the genetic material (such as DNA or RNA) existing in a cell or in an organism or introducing exogenous genetic material into a cell or into an organism.
Genetic alterations include, for example, a gene deletion or some other functional disruption of the genetic material. Genetic alterations also include modifications that introduce expressible nucleic acids encoding polypeptides (including, but not limited to ORs). In some embodiments, the genetic alteration restores, corrects, or modifies expression of a gene. In some embodiments, genetic alteration includes the introduction of a regulatory element (including, but not limited to, an enhancer, silencer, promoter, or other transcriptional regulator) that affects the expression of a naturally present gene.
The nucleic acid introduced into the cell or into the organism can originate from any species. In some embodiments, the nucleic acid sequence introduced into the cell or into the organism is derived from the same species or a different species. Alternatively, the nucleic acid sequence introduced into the cell or into the organism might not occur anywhere in nature and may be created by the chemical synthesis of nucleic acid. Accordingly, “genetically engineered” may refer to a cell or an organism that contains one or more artificial or recombinant sequences of nucleotides which have been created through molecular cloning techniques to bring together genetic material that is not natively found together.
The genetic alteration of the cell or organism may be achieved by a variety of techniques, including, but not limited to, calcium-phosphate-mediated transfection, diethylaminoethyl (DEAE)-mediated transfection, microinjection, viral transformation, protoplast fusion, lipofection, and/or the use of meganucleases and zinc finger nucleases, transcription activator-like effector nucleases (TALENs) or a Cas9-guideRNA system (adapted from Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)).
The genetically altered cell or organism may contain and/or express the foreign nucleic acid (e.g., an OR coding sequence) in either a transient or long-term manner. In general, transient expression occurs when foreign DNA does not stably integrate into the chromosomal DNA of the transfected cell or organism. In contrast, long-term expression of foreign DNA occurs when the foreign DNA has been stably integrated into the chromosomal DNA of the transfected cell or organism.
Not all eukaryotic cells in a population that is genetically engineered to express a polypeptide (such as an OR) or that is genetically engineered to change the expression level of a polypeptide (such as an OR) will express the polypeptide (such as an OR) in a significant amount. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the eukaryotic cells genetically engineered to express a polypeptide (such as an OR) will express the polypeptide (such as an OR) at a given time.
In some embodiments, the biosensor comprises a eukaryotic cell or a population of eukaryotic cells, wherein the eukaryotic cell is an OSN and the population of eukaryotic cells is a population of OSNs. OSNs can be obtained from transgenic animals such as the transgenic animals described herein by methods known in the art.
In some embodiments, the biosensor comprises a eukaryotic cell or a population of eukaryotic cells, wherein the eukaryotic cell is yeast cell and the population of eukaryotic cells is a population of yeast cells, HEK cells, or CHO cells.
In some embodiments, the biosensor comprises a eukaryotic cell other than an OSN that expresses an OR disclosed herein. In some embodiments, the OR may be fused to a processing/transport segment that directs the processing and transport of the OR to the cell membrane of the host cell. In some embodiments, the biosensor comprises a eukaryotic cell other than an OSN that expresses the hypervariable segment of an OR, wherein such segment contains at least one odorant binding site of the OR. Methods for the expression of ORs and detection of OR activation in yeast have been described in U.S. Pat. No. 7,223,550 and Patent Application No. PCT/2017/019179, both of which are incorporated herein by reference.
In embodiments, the biosensor comprises one or more populations of eukaryotic cells wherein each population of eukaryotic cells preferentially expresses an OR described herein. In embodiments, the biosensor comprises one or more populations of OSNs, wherein each population preferentially expresses a different OR described herein. In some embodiments, the biosensor comprises at least two, at least three, at least four, or at least five populations of eukaryotic cells (e.g., OSNs), wherein each population preferentially expresses a different OR described herein. In embodiments, the biosensor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more cell populations, wherein each population preferentially expresses a different OR described herein. In embodiments, the preferentially expressed OR comprises (1) an amino acid sequence selected from SEQ ID NOs: 1-40; (2) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1-40; (3) an amino acid sequence selected from SEQ ID NOs: 1 and 6-18; (4) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1 or 6-18; (5) an amino acid sequence of an OR described in any of Tables 1-5; and/or (6) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to an amino acid sequence of an OR described in any of Tables 1-5.
In a non-limiting example, the biosensor comprises two populations of cells with each population preferentially expressing a different OR comprising (1) an amino acid sequence selected from SEQ ID NOs: 1-40; (2) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1-40; (3) an amino acid sequence selected from SEQ ID NOs: 1 and 6-18; (4) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1 or 6-18; (5) an amino acid sequence of an OR described in any of Tables 1-5; and/or (6) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to an amino acid sequence of an OR described in any of Tables 1-5. In another non-limiting example, the biosensor comprises three, four, five, six, or more populations of cells with each population preferentially expressing a different OR comprising (1) an amino acid sequence selected from SEQ ID NOs: 1-40; (2) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1-40; (3) an amino acid sequence selected from SEQ ID NOs: 1 or 6-18; (4) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1 or 6-18; (5) an amino acid sequence of an OR described in any of Tables 1-5; and/or (6) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to an amino acid sequence of an OR described in any of Tables 1-5.
As used herein, “preferential expression” or “preferentially express” refers to an increase in the number of cells in a population of cells that express a specific OR as compared to the wild type or unmodified population of cells. For example, in the case of Rattus norvegicus, the preferential expression of an OR is compared to the expression of other Rattus norvegicus ORs. In the case of a Mus musculus OR, expression of the OR is compared to the expression of other Mus musculus ORs. For example, in the mouse, a typical OR is expressed in about 10,000 OSNs out of approximately 10,000,000 OSNs. By cloning a suitable enhancer upstream of an OR gene, that OR can be preferentially expressed in an increased number of OSNs, for example 500,000 to 2 million neurons. In embodiments, the percentage of cells in a population of cells that expresses an OR described herein is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, or greater than 60%. In embodiments, the percentage of cells in a population of cells that expresses the OR is between 10 and 90%.
In one embodiment, the methods described in International Patent Publication WO2017/024028, said methods are hereby incorporated by reference, are used in conjunction with a sequence encoding an OR, including the disclosed OR coding sequences SEQ ID NOs: 41-80 or OR polynucleotide sequences encoding the disclosed amino acid sequences SEQ ID NOS: 1-40. WO2017/024028 describes OR expression constructs, vectors and methods for producing genetically modified non-human vertebrates that preferentially express a selected OR in the OSNs, said expression constructs and methods are incorporated herein by reference (see FIG. 1 of WO2017/024028).
Accordingly, in some embodiments, provided is a nucleic acid construct for the preferential expression of an OR described herein, wherein the nucleic acid construct comprises an enhancer operably linked to the sequence encoding the preferentially expressed OR. In embodiments, the enhancer is a singular gene choice enhancer. In some embodiments, the enhancer comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sequential repeats of a 21 base pair (bp) sequence wherein each 21 bp sequential repeat comprises the sequence AACTTTTTAATGA (SEQ ID NO: 81). In some embodiments, the enhancer comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten sequential repeats of ACATAACTTTTTAATGAGTCT (SEQ ID NO: 82). In embodiments, the enhancer comprises three, four, five, six, seven, eight, nine, ten, or more sequential repeats each containing YTTTTAATGAR where Y=C/T and R=A/G (SEQ ID NO: 83). In some embodiments, the enhancer comprises ten or fewer of the 21 bp sequential repeats. In embodiments, the enhancer comprises four to ten (e.g., four, five, six, seven, eight, nine, or ten) of the 21 bp sequential repeats described herein.
In one aspect, provided are compositions and methods for preferentially expressing an OR using a tetracycline-controlled transactivator protein in combination with a TetR DNA binding site (TetO sequence). Together, these regulatory DNA sequence control expression of an OR in response to the presence or absence of tetracycline (Tc) or Tc derivatives including, but not limited to, doxycycline (Dox).
In embodiments, the tetracycline-controlled transcriptional transactivator protein is tTA, which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the transactivating domain of VP16 from Herpes simplex virus. In the absence of Tc or Dox, tTA binds to TetO and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TetO, and expression from the target gene remains inactive. In one embodiment, one or more repeats of the TetO sequence are placed upstream of a minimal promoter. In one embodiment, the minimal promoter is a CMV promoter.
In another embodiment, a reverse tetracycline-controlled transcriptional transactivator called rtTA is used. rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the TetO sequence in the presence of the Dox effector. Thus, here, transcription of the target gene is stimulated by rtTA only in the presence of Tc or Dox.
In some embodiments, the rTA or rtTA comprise viral structural protein VP32, VP48, or VP64 instead of VP16 as the activator. In some embodiments, the rTA or rtTA comprise GAL4-VP16 as the activator. GAL4-VP16 comprises the DNA-binding fragment of the yeast activator GAL4 and part of VP16, in combination with a promoter containing upstream activating sequences (UAS). In some embodiments, the promoter region further comprises a CMV promoter or fragment thereof.
In embodiments, the biosensors disclosed herein comprise cilia isolated from one or more populations of OSNs that express one or more ORs described herein. In the olfactory system, millions of hair-like olfactory cilia protrude from the dendrites of the OSNs into the mucus of the MOE that lines the nasal cavity. ORs present in the membranes of these cilia signal their activation by odorants through a G protein-mediated signaling cascade in which binding of the odor activates type III adenylate cyclase (ACIII) and causes a rapid rise in levels of cAMP, which binds to cyclic-nucleotide gated channels causing influx of Ca2+. There is also evidence that olfactory receptors can signal via G-protein activation of phosphoinositidase C, with subsequent production of inositol 1,4,5-triphosphate and 1,2-diacylglycerol second messengers.
In certain embodiments, the biosensor comprises cilia isolated from a population of OSNs that preferentially expresses an OR disclosed herein. Olfactory cilia can be detached from the main olfactory epithelium thereby providing an ex vivo system amenable to monitor OR activation, as olfactory signal transduction events are exclusively initiated within these cilia. Cilia can be obtained from olfactory epithelial tissue by methods known in the art. For example, Kuhlmann et al., (Molecular & Cellular Proteomics (2014), 13:1828-1843) and Mayer et al., (Proteomics (2009), 9:322-334) provide protocols for isolation of olfactory cilia and those protocols are incorporated herein by reference. Sklar et al. (J. of Biological Chemistry (1986), 261:15538-15543), and Pfeuffer et al. (J. of Biological Chemistry (1989), 264:18803-18807) also provide protocols for isolation of olfactory cilia and those protocols are also incorporated herein by reference. Following isolation, cilia preparations may be stored at −80° C. for months without significant loss in activity.
For example, a portion of the OSN (i.e., olfactory cilia) is extracted as follows: Olfactory epithelia from 6-week old mice are dissected and briefly washed in cold buffer. The tissue is incubated in cold extraction buffer containing calcium for 20 minutes and subsequently spun down for 10 minutes, in which the supernatant is collected for the following steps. This extraction process is repeated once on the tissue. The combined supernatant is collected and spun down at high speed in a cooled ultracentrifuge for 30 minutes. The resulting pellet contains olfactory cilia and is reconstituted in buffer with glycerol and protease inhibitor, aliquoted and snap-frozen with liquid nitrogen, and stored at −80° C. until use.
The methods recited above can be used to isolate cilia from a non-human mammal, for example a rat or mouse, where the population of OSNs in the OE of the mammal preferentially express an OR disclosed herein. The OR can be an endogenous OR where the OR gene has been modified to drive the preferential expression of the OR in the population of OSNs. In other embodiments, the OR is an exogenous OR expressed from a nucleic acid, vector, or construct that drives the preferential expression of the OR in the population of OSNs.
In embodiments, the biosensor is a chip or otherwise involves attachment of populations of cells or cilia to a solid support. Accordingly, the biosensor may comprise (i) an array of individual populations of cells (each population preferentially expressing a different OR disclosed herein), or (ii) an array of individual populations of cilia, where each population of cilia is derived from a population of OSNs, where each population of OSNs preferentially expresses a different OR disclosed herein. Such an array can also be used when the biosensor comprises a multi-well format.
In some embodiments, the biosensor comprises populations of eukaryotic cells disposed on a solid support. In some embodiments, the biosensor comprises populations of OSNs or cilia derived therefrom that were extracted from a transgenic non-human mammal and subsequently disposed on a solid support. Examples of suitable solid supports include, but are not limited to, silicon, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. Preferably, the solid support allows optical detection and does not appreciably fluoresce. In one embodiment, the surface of the solid support is modified to contain microwells, i.e. depressions in the surface of the solid support. This may be done as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, pressing, casting, molding, microetching, electrolytic deposition, chemical or physical vapor deposition employing masks or templates, electrochemical machining, laser machining or ablation, electron beam machining or ablation, and conventional machining. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the solid support. In one embodiment, the interior surfaces of the microwells may be coated with a thin film or passivation layer of biologically compatible material. For example, materials known to support cell growth or adhesion may be used, including, but not limited to, agar, fibronectin, any number of known polymers including collagen, polylysine and other polyamino acids, polyethylene glycol and polystyrene, growth factors, hormones, cytokines, etc. In addition, coatings or films of metals such as a metal such as gold, platinum or palladium may be employed. In an alternative embodiment, an indicator compound, for example, a fluorophore, a chromophore or dye, may be attached to the microwell surface for detecting cellular responses to OR activation. In some embodiments, the biosensor further comprises one or more of an electromagnetic radiation source, a detection element, an optical filter, components to deliver or remove fluids, a collection chamber, a cover plate, an electrode, an integrated circuit, and a hydrogel.
In one aspect, provided is a biosensor, wherein the biosensor comprises an isolated OR described herein. In some embodiments, the biosensor comprises a lipid bilayer comprising the OR. In some embodiments, the OR is present in a nanovesicle, nanosome, nanodisc, or is suspended in a lipid bilayer. In some embodiments the biosensor further comprises a marker for detecting activation or lack of activation of the OR, wherein the activation or lack of activation occurs upon exposure of the one or more populations of OSNs to a sample from a subject exhibiting a change in the levels of one or more neurotransmitters in the CNS (e.g., as compared to the control levels the one or more neurotransmitters).
A person skilled in the art will appreciate that the activation or lack of activation of an OR can be measured in various ways. For instance, activation of an OR may be detected by monitoring a decrease in ATP levels or an increase in Ca2+, GDP, cAMP, inositol 1,4,5-triphosphate and/or 1,2-diacylglycerol levels using conventional methods. Conversely, lack of activation of an OR may be detected by observing a lack of decrease in ATP levels or a lack of increase in Ca2+, GDP, cAMP, inositol 1,4,5-triphosphate and/or 1,2-diacylglycerol levels using conventional methods.
In some embodiments, a marker may be provided to detect the activation (or lack thereof) of an OR upon exposure to a sample from a patient or to an extract from the sample. The use of markers permits the measurement of OR activation (or lack thereof) using conventional methods, including the measurement of fluorescence, luminescence, phosphorescence, visible light, radioactivity, colorimetry, X-ray diffraction or absorption, electricity, change in electric potential, or magnetism. In some embodiments, the marker may be a fluorescent dye. Examples of suitable dyes include calcium-sensitive dyes such as fura-2, fluo-3, fluo-4, fluo-5F, indo-1, and Oregon Green BAPTA. The marker may be integrated into the biosensor using, for example, the techniques described in International Patent Publication WO2017024028, incorporated herein by reference. Marker proteins may be co-expressed with the one or more preferentially expressed ORs. Examples of suitable marker proteins include GECO2.1, GCaMP6f, Flamindo, Flamindo2, and Pink Flamindo.
In some embodiments, the OR is further genetically or chemically modified to allow detection of OR activation by inter- or intra-molecular fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), or bimolecular fluorescence complementation (BiFC).
The responsiveness of a transgenic mammal that preferentially expresses one or more ORs described herein can be determined using behavioral analysis. For example, the control (e.g., wild type) animal and transgenic animal are compared in terms of reaction to (e.g., time spent near) to a sebum sample derived from a patient as compared to a control sample.
In some embodiments, the activation of the OR is determined in a biochemical assay. In some embodiments, populations of OSNs, or cilia derived therefrom, that express an OR are isolated and the activation of the OR is detected ex vivo. In one embodiment, the cilia of the OSNs are further isolated using a deciliation protocol and used for the detection of activation of the OR.
Provided herein are transgenic animals, tissues, and cells isolated from the transgenic animals, wherein the transgenic animals have been engineered to express one or more ORs described herein. In embodiments, the transgenic animal comprises an olfactory epithelium, wherein the neurons of the OE preferentially express an OR described herein. In some embodiments, the olfactory epithelium is the main olfactory epithelium.
In some embodiments, the transgenic animal is a non-human mammal. In some embodiments, the non-human mammal belongs to the family of Bovidae, Canidae, and Muridae. In some embodiments, the non-human mammal is a rat, mouse, dog, cat, goat, chicken, sheep, pig, or primate.
In one aspect, provided is a transgenic animal comprising an olfactory epithelium in which the OSNs preferentially express an OR disclosed herein. In embodiments, the transgenic animal comprises: (a) a transgene sequence encoding an OR comprising an amino acid sequence selected from the group consisting of (1) an amino acid sequence selected from SEQ ID NOs: 1-40; (2) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1-40; (3) an amino acid sequence selected from SEQ ID NOs: 1 and 6-18; (4) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to any one of SEQ ID NOs: 1 or 6-18; (5) an amino acid sequence of an OR described in any of Tables 1-5, and/or (6) an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 98, or at least 99% identity to an amino acid sequence of an OR described in any of Tables 1-5; and (b) an enhancer operably linked to the transgene sequence. In embodiments, the enhancer is a singular gene choice enhancer described herein.
Also provided herein is a tissue isolated from any of the transgenic animals described herein. In some embodiments, the isolated tissue is an olfactory epithelium. In some embodiments, the isolated tissue is a main olfactory epithelium.
Also provided herein is a cell or a population of cells isolated from any of the transgenic animals described herein. In some embodiments, the isolated cell or a population of cells is an olfactory epithelium cell or a population of olfactory epithelium cells.
A sample from a subject, as used herein, refers to any substance or material obtained from, or derived from, a subject, comprising an odorant that can be detected by one or more of the ORs disclosed herein. A sample, may be obtained from breath, sebum, saliva, blood, urine, sweat, or semen of a subject and materials derived therefrom including, for example plasma, lipids, proteins, and small molecules. A sebum sample, for example, may be obtained from a cotton pad, swab, gauze, bandage, sportive tape, fabric, tissue, adsorbent-coated fiber, absorbent paper, clothing, or other material placed in contact with a subject's skin.
Methods for the extraction of odorants from samples are known in the art. For example, the sample (e.g., a sebum sample) may be collected with a suitable matrix, for example, with a cotton pad, swab, gauze, bandage, sportive tape, fabric, tissue, adsorbent-coated fiber, absorbent paper or clothing worn by the patient. Tape specially designed for the collection of sebaceous lipids is available under the name Sebutape® Adhesive Patches (cat #S100) or Sebutape® Indicator Strips (cat #5232) (CuDerm Corporation, Dallas, TX).
In some embodiments, the one or more odorants are extracted from the sample using an extraction medium. An extraction medium may be composed of polar and/or non-polar organic solvents such as chloroform, methanol, ether, propanol, isopropanol, di-chloromethane, tri-methyl-pentene, hexane, or heptane or their combinations and may contain an aqueous phase with or without modifiers (such as acids or bases). In some embodiments, the odorant is collected using dynamic headspace adsorption onto various porous polymers (e.g., Tenax, Porapak Q). Such methods may be used for collecting airborne odorants. In some embodiments, the odorant is directly collected into an adsorbent trap. In some embodiments, the odorants are collected using solid-phase microextraction (SPME), solvent-assisted flavor evaporation (SAFE), or simultaneous distillation extraction (SDE).
In some embodiments, during the days before collection of the sample(s), the patient is asked to follow particular instructions related to diet and the use of fragrance soap/shampoo. In some embodiments, the patient is asked to avoid spicy food and garlic several days before sampling. In some embodiments, the patient is asked to use no deodorant, no perfume, and to use fragrance-free soaps the days before odor collection. In some embodiments, the patient is instructed to do some exercise so that the skin became sweaty.
Provided herein are methods of using the biosensors, transgenic animals, tissues, and cells (including OSNs and cilia derived therefrom) disclosed herein for detecting one or more odorants. In one aspect, provided is a method of detecting one or more odorant molecules, the method comprising: (a) obtaining a sample from a subject, wherein the sample comprises one or more odorant molecules; (b) exposing one or more populations of eukaryotic cells to the sample obtained from said subject, wherein each population of eukaryotic cells preferentially expresses a set of ORs comprising an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 1-40, (2) an orthologue or a paralogue of an OR represented by any one of SEQ ID NOs: 1-40, (2) an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1-40, (3) an amino acid sequence selected from SEQ ID NOs: 1 or 6-18, (4) an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1 or 6-18, (5) an amino acid sequence of an OR described in any of Tables 1-5, and/or (6) an amino acid sequence with greater than 85% identity to an amino acid sequence of an OR described in any of Tables 1-5; and (c) measuring in each of the one or more populations of eukaryotic cells the activation or lack of activation of the preferentially expressed set of ORs by the one or more odorant molecules in the sample obtained from said subject. In some embodiments, the one or more populations of eukaryotic cells are one or more populations of OSNs, or cilia derived therefrom. In embodiments, the odorant molecules in the sample are compared to the odorant molecules of a control sample.
The biosensors provided herein are useful for detecting odorants, wherein the odorants are associated with a change in the levels of one or more neurotransmitters in the CNS of a patient (e.g., as compared to the control levels for the one or more neurotransmitters). Accordingly, the biosensors disclosed herein can be used in a method for diagnosing a disease or condition associated with a change in the levels of one or more neurotransmitters in the CNS as compared to the controls levels for the one or more neurotransmitters. In some embodiments, the biosensors is used to make a diagnosis prior to the subject manifesting the clinical symptoms that are used in the diagnosis of the disease. In some embodiments, the biosensors can be used to confirm a previous diagnosis of a disease or condition associated with a change in the levels of one or more neurotransmitters in the CNS.
Additionally, the biosensors disclosed herein can be used in a method for monitoring the disease status of a patient over time, wherein the patient has a disease or condition associated with a change in the levels of one or more neurotransmitters in the CNS as compared to the controls levels for the one or more neurotransmitters, wherein the controls levels for the one or more neurotransmitters were obtained from one or more individuals that do not have the disease or condition.
The biosensors disclosed herein can also be used to identify patients at increased risk of and/or with a predisposition of developing a disease or condition associated with a change in the levels of one or more neurotransmitters in the CNS as compared to the controls levels for the one or more neurotransmitters, wherein the controls levels for the one or more neurotransmitters were obtained from one or more individuals that do not have the disease or condition.
In some embodiments, the patient has a neurotransmitter deficiency in the CNS. In some embodiments, the patient has a dopamine and/or a serotonin deficiency in the CNS. In some embodiments, the patient has PD, depression, schizophrenia, dystonia, and/or restless leg syndrome. In some embodiments, the patient has an increased risk of developing a neurotransmitter deficiency in the CNS. In some embodiments, the patient has an increased risk of developing a dopamine and/or a serotonin deficiency in the CNS. In some embodiments, the patient has an increased risk of developing PD, depression, schizophrenia, dystonia, and/or restless leg syndrome.
As used herein, “subject” or “patient” includes individuals that are exhibiting signs of a change in the levels of one or more neurotransmitters in the CNS as well as individuals that have not yet begun exhibiting symptoms of a change in the levels of one or more neurotransmitters in the CNS.
Accordingly, in one aspect, provided is a biosensor for diagnosing a disease or condition associated with the levels of one or more neurotransmitters or a change in the levels of one or more neurotransmitters in the CNS of a subject, or for identifying an individual with an increased risk of developing diagnosing a disease or condition associated with the levels or a change in the levels of one or more neurotransmitters in the CNS of a subject, wherein the biosensor comprises: one or more populations of OSNs, or cilia derived therefrom, wherein each population of OSNs preferentially expresses an OR. In embodiments, the OR is (1) an OR that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, (2) a orthologue or a paralogue of an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, (3) an OR comprising an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1-40, (4) an OR that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, (5) a orthologue or a paralogue of an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 or 6-18, (6) an OR comprising an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1 or 6-18, (7) an OR comprising an amino acid sequence of an OR described in any of Tables 1-5, and/or (8) an OR comprising an amino acid sequence with greater than 85% identity to an amino acid sequence of an OR described in any of Tables 1-5.
Thus, in embodiments, the biosensor comprises one or more populations of OSNs, or cilia derived therefrom, wherein the one or more populations of OSNs comprises at least a first population that preferentially expresses a first amino acid sequence and a second population that preferentially expresses a second amino acid sequence, wherein the first amino acid sequence and the second amino acid sequence are different and are independently selected from the OR sequences described herein, including for example, a group consisting of SEQ ID NOs: 1-40, an orthologue or a paralogue of an OR represented by any one of SEQ ID NOs: 1-40, an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1-40, an amino acid sequence described in any of Tables 1-5, and/or an amino acid sequence with greater than 85% identity to an amino acid sequence described in any of Tables 1-5.
Also provided is a method of (i) diagnosing a disease or condition associated with the levels of one or more neurotransmitters or a change in the levels of one or more neurotransmitters in the CNS of a subject and/or (ii) identifying an increased risk of developing a disease or condition associated with the levels of one or more neurotransmitters or a change in the levels of one or more neurotransmitters in the CNS, the method comprising: (a) exposing one or more populations of eukaryotic cells to a sample obtained from said subject, wherein each population of eukaryotic cells preferentially expresses an OR comprising an amino acid sequence selected from the group consisting of (1) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, (2) an orthologue or a paralogue of an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-40, (3) amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1-40, (4) amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, (5) an orthologue or a paralogue of an OR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6-18, (6) an amino acid sequence with greater than 85% identity to any one of SEQ ID NOs: 1 or 6-18, (7) an amino acid sequence of an OR described in any of Tables 1-5, and/or (8) an amino acid sequence with greater than 85% identity to an amino acid sequence of an OR described in any of Tables 1-5; (b) measuring in each of the one or more populations of eukaryotic cells the activation or lack of activation of the preferentially expressed OR by the one or more odorant molecules in the sample obtained from said subject; and (c) (1) diagnosing a disease or condition associated with the levels of one or more neurotransmitters or a change in the levels of one or more neurotransmitters in the CNS of a subject or (2) identifying an increased risk of developing a disease or condition associated with the levels of one or more neurotransmitters or a change in the levels of one or more neurotransmitters in the CNS when activation of one or more preferentially expressed ORs is detected in the one or more of the populations of eukaryotic cells. In some embodiments, the one or more populations of eukaryotic cells are one or more populations of OSNs, or cilia derived therefrom.
Provided herein are biosensors for detecting one or more odorants associated with a change in the levels of one or more neurotransmitters in the CNS of a subject as compared to the control levels for the one or more neurotransmitter. As used herein, a “control level” of a neurotransmitter in the CNS may refer to a level of that neurotransmitter in the CNS of one or more individuals who do not suffer from a disease associated with a change in the level of that neurotransmitter in the CNS. The control level may be measured on an individual-by-individual basis, or on an aggregate basis such as an average.
In some embodiments, a control level of a neurotransmitter in the CNS is the level is of that neurotransmitter in the CNS of one or more individuals who do not suffer from a disease associated with a change in the level of dopamine in the CNS. In some embodiments, a control level of a neurotransmitter in the CNS is the level is of that neurotransmitter in the CNS of one or more individuals who do not suffer from a disease associated with a change in the level of serotonin in the CNS. In some embodiments, a control level of a neurotransmitter in the CNS is the level is of that neurotransmitter in the CNS of one or more individuals who do not suffer from PD, depression, schizophrenia, dystonia, and/or restless leg syndrome.
In some embodiments, the one or more individuals are healthy individuals.
In some embodiments, the control level of a neurotransmitter in the CNS is the level of that neurotransmitter in the CNS in the same individual for whom a diagnosis is sought or whose condition is being monitored, but is obtained at a different time. A control level may refer to a level obtained from the same patient at an earlier time, e.g., weeks, months, or years earlier. A control level may refer to a level obtained from the same patient at a later time, e.g., weeks, months, or years later.
Likewise, a “control subject” refers to one or more individuals who do not suffer from a disease associated with a change in the level of that neurotransmitter in the CNS. In some embodiments, the control subject has a control level of a neurotransmitter in the CNS. In some embodiments, the control subject does not suffer from a disease associated with a change in the level of dopamine in the CNS. In some embodiments, the control subject does not suffer from a disease associated with a change in the level of serotonin in the CNS. In some embodiments, the control subject does not suffer from PD, depression, schizophrenia, dystonia, and/or restless leg syndrome. In one embodiment, the control subject is a healthy individual.
As used herein, a difference from a control level refers to a difference that is statistically significant, as determined by any statistical analysis method now or hereafter used by those in the art.
Once a subject is diagnosed with a disease or condition associated with the levels or a change in the levels of one or more neurotransmitters in the CNS of a subject, therapy can be initiated to treat, prevent, slow the onset of symptoms, or slow or halt the progression of the disease or condition associated with the levels or a change in the levels of one or more neurotransmitters in the CNS of a subject. The ability to detect diseases or conditions associated with a change in the levels of one or more neurotransmitters in the CNS of a subject early, may, for example, allow the early use of one or more therapies designed to slow or prevent onset of symptoms. Disease-modifying therapies are intended to prevent, slow or halt the progression of diseases and conditions associated with a change in the levels of one or more neurotransmitters in the CNS of a subject.
In some embodiments, the disease or condition associated with a change in the level of one or more neurotransmitters in the CNS of a subject is a neurodegenerative disease, including, e.g., PD. The ability to detect neurodegenerative diseases like PD early, before neurological symptoms appear, allows for the administration of therapeutic agents in order to prevent, or delay the onset or progression of, the neurodegeneration. Accordingly, in some embodiments, the subject is administered a neuroprotective agent. The term “neuroprotective” agent, as used herein, refers to agents intended to prevent neurodegeneration, including agents that slow down or stop the progression of neuronal degeneration. Neuroprotective agents include, but are not limited to, caffeine, nicotine, urate, UA, vitamin E, vitamin C, vitamin D, beta-carotene, riboflavin, coenzyme Q10, creatine, docosahexaenoic acid (DHA), uridine, uric acid, melatonin, glutathione (GSH), phytic acid (IP6), non-steroidal anti-inflammatory drugs (NSAID), isradipine, phenylbutyrate, exendin-4 (Ex-4), rasagiline, minocycline, EMD 1195686 (Safinamide), Zonisamide, GM1 ganglioside, and acetaminophen.
In some embodiments, a PD patient is administered levodopa-based preparations, designed to replace the dopamine in the depleted striatum. L-dihydroxyphenylalanine (levodopa or L-DOPA) is a dopamine precursor levodopa that is able to cross the BBB. After absorption and transit across the BBB, levodopa is converted into the neurotransmitter dopamine by DOPA decarboxylase. Administration of levodopa may cause side effects resulting from the conversion of levodopa to dopamine outside the CNS (peripheral conversion) by DOPA decarboxylase. Accordingly, in some embodiments, levodopa is administered in combination with peripheral inhibitors of DOPA decarboxylase, including, but not limited to, benserazide and carbidopa.
In some embodiments, the PD patient is administered a dopamine agonist which stimulates the activity of the dopamine system by binding to the dopaminergic receptors. In some embodiments, the dopamine agonist is administered during the initial therapy for PD.
In some embodiments, the dopamine agonist is an ergot-derived dopamine agonist including, but not limited to, bromocriptine (Parlodel), pergolide (Permax), cabergoline, or lisuride. In some embodiments, the dopamine agonist is a non-ergot-derived dopamine agonist including, but not limited to apomorphine (Apokyn), pramipexole (Mirapex), ropinirole (Requip), and rotigotine (NeuPro).
In some embodiments, the PD patient is administered an inhibitor of an enzyme involved in dopamine metabolism, wherein the inhibitor preserves the levels of endogenous dopamine.
In some embodiments, the inhibitor of inhibitor of an enzyme involved in dopamine metabolism is a MAO-B inhibitor, including, but not limited to, selegiline (Deprenyl, Eldepryl, Zelapar), rasagiline (Azilect), and safinamide (Xadago).
In some embodiments, the inhibitor of an enzyme involved in dopamine metabolism is an inhibitor of catechol-O-methyl transferase (COMT). Non-limiting examples of COMT inhibitors include entacapone (Comtan), tolcapone (Tasmar), and opicapone (Ongentys).
In some embodiments, the inhibitor of an enzyme involved in dopamine metabolism is used in combination with levodopa-based preparations and may allow for a reduction in the levodopa dose.
In some embodiments, the PD patient is administered an anticholinergic, which reduces the activity of the neurotransmitter acetylcholine by acting as an antagonist at cholinergic receptors. Non-limiting examples of anticholinergics include benztropine, orphenadrine, procyclidine, and trihexyphenidyl (Benzhexol).
In some embodiments, the PD patient is administered amantadine (Symmetrel) (which acts as a weak glutamate antagonist at the N-methyl-d-aspartate receptor (NMDAR)), Exenatide (Byetta), or Isradipine,
In some embodiments, the PD patient is administered gene therapy. Transgenes used in gene therapy for PD include, but are not limited to, genes encoding for DOPA decarboxylase, tyrosine hydroxylase (TH), and guanosine triphosphate cyclohydrolase-1 (GTPCH1). In some embodiments, the PD patient is administered ProSavin, a lentivirus vector comprising genes encoding DOPA decarboxylase, TH, and (GTPCH1). In some embodiments, the PD patient is administered a gene therapy targeting a gene linked to PD including, but not limited to, α-synuclein (AS) (SNCA), parkin (PARK2), UCH-L1 (PARKS), PINK1 (PARK6), DJ-1 (PARK7), leucine-rich repeat kinase-2 (LRRK2; PARKS), and ATP13A2 (PARKS). In some embodiments, the PD patient is administered gene therapy targeting a gene linked to an increased risk of developing PD including, but not limited to, GBA1, VPS35, EIF4G1, and PARK16.
In some embodiments, the PD patient is administered c-Abl tyrosine kinase inhibitor (including, but not limited to, nilotinib) or a glucagon-like peptide-1 receptor agonist (including, but not limited to, exenatide).
In some embodiments, the PD patient is administered a therapy that targets a protein or pathway known to play a role in PD, including antioxidants (glutathione, inosine) or Neurotrophic Factors (GDNF, CERE-120).
In some embodiments, the PD patient is administered a therapy that reduces α-synuclein production, inhibits α-synuclein aggregation, increases intracellular and extracellular degradation of α-synuclein aggregates, and/or reduces uptake of extracellular α-synuclein by neighboring cells. In some embodiments, the PD patient is administered affitope, NPT088, or NPT200-11.
In some embodiments, the PD patient is administered a cell-based therapy to replace nigrostriatal dopamine terminals lost by the disease process, with fetal or stem cell derived DA neurons placed directly into the caudate-putamen, and/or in substantia nigra. In some embodiments, induced pluripotent stem cells, embryonic stem cells, or universal donor cells are used for the cell-based therapy. In some embodiments, somatic cells are converted to dopamine neurons in vivo using virus technology.
In some embodiments, the PD patient receives deep brain stimulation (DBS).
To identify the ORs that are involved specifically in response to samples from patients with PD, a technique called “Deorphanization of Receptors based on Expression Alterations of mRNA levels” (DREAM) was used. This technique utilizes the generalized reduction in OR mRNA levels that occur after specific OR activation (von der Weid, B., Rossier, D., Lindup, M., Tuberosa, J., Widmer, A., Col, J. D., Kan, C., Carleton, A., and Rodriguez, I. (2015). Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs in vivo. Nat Neurosci 18, 1455-1463; see also US2017/0285009, both incorporated herein by reference).
Samples were obtained from two independent cohorts of PD patients (PD1 and PD2) along with age matched healthy volunteers (HV) (n=10 in cohort 1 and n=20 in cohort 2 in each group), see
Subsequently, the gauzes were cut up in pieces. Samples from PD patients and healthy volunteers respectively were pooled and placed in breather bags (similar to the bags used to train dogs to sniff-out explosives) (see
Differential gene expression (DGE) analysis was performed by sequencing the olfactory cDNA library for each animals and comparing the sequence results for the different groups. To generate the cDNA libraries, total RNA from each sample was quantified using a NanoDrop ND-1000 instrument. About 1 to 2 μg total RNA was used to prepare the sequencing library in the following steps: (1) Total RNA was enriched by oligo (dT) magnetic beads (rRNA removed); (2) RNA-seq library preparation using KAPA Stranded RNA-Seq Library Prep Kit (Illumina), which incorporates dUTP into the second cDNA strand and renders the RNA-seq library strand-specific. The completed libraries were qualified with Agilent 2100 Bioanalyzer and quantified by absolute quantification qPCR method.
Several rounds of different sequencing approaches were performed. A first round utilized a targeted capture approach amplifying the OR gene sets from rats. The second round of sequencing utilized a more shotgun sequencing approach using Illumina Novaseq to allow for a very deep sequencing averaging over 285 million reads per sample/animal with the PD2 cohort and over 100 million reads per sample/animal with the PD1 cohort.
Once fastq files were obtained from the different sequencing runs, sequence quality was examined using the FastQC software v11.9. The trimmed reads (trimmed 5′, 3′-adaptor bases using trimmomatic v0.39 and quality trimming) were aligned to reference genome Rnor6.0 (ensembl98) using STAR software v 2.7.3a along with the annotation gtf file for the reference genome. The transcript abundances for each sample was estimated with the “--quantMode GeneCounts” option within STAR, and the differential gene expression was analyzed with the DeSeq2 v1.26.0 package in R (version 3.6.3). An alternate sequencing analysis was performed in order to analyze the expression data under a secondary approach.
Eleven OR genes (Olr292, Olr836, Olr661, Olr841, Olr749, Olr1185, Olr1381, Olr1558, Olr607, Olr712, 041160) were identified as differentially expressed in both cohorts as compared to the control (highlighted with * in Table 1). See
In olfaction, it is assumed that every odor activates a subset of receptors, which is referred to as the odor combinatorial code. Here, a combinatorial code in rats was identified comprising 14 different rat ORs that are differentially activated by sebum from PD patients or from control sebum. The fact that the differentially activated ORs were identified in two different cohorts of PD patients indicates that this odor combinatorial code is characteristic for PD rather than patient-specific. The patients' disease unrelated, characteristic smell was controlled for by merging pieces of gauzes from separate individuals in order to minimize individual smells and amplify the PD-associated odorant signal to be captured. With samples from two independent cohorts, the calculated statistical power of this analysis was sufficient for the identification of ORs that are differentially activated by PD or control sebum samples.
$OR was identified in the PD1 cohort and was differentially expressed as compared to the control.
Once an OR has been identified, a person skilled in the art can identify homologous or orthologous proteins that fulfill the same function. A non-exhaustive list of orthologues and paralogues of the rat ORs in Table 1 can be found in Tables 3 and 4. A non-exhaustive list of orthologues and paralogues of patch genes can be found in Table 5. All sequences for the NCBI Gene IDs, as well as NCBI mRNA and protein accession numbers provided in Tables 1-4 are incorporated herein by reference.
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Canis
Lupus (dog)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Rattus
norvegicus (rat)
Mus
musculus (mouse)
Mus
musculus (mouse)
Mus
musculus (mouse)
Rattus
norvegicus (rat)
Genes encoding ORs were designed with Stu and AscI restriction sites flanking the two ends and synthesized as sequence-verified, double-stranded DNA fragments. These DNA fragments were digested with StuI and AscI, then ligated into the MouSensor vector (˜9 kB) (as described in D'Hulst C, Mina R B, Gershon Z, et al. MouSensor: A Versatile Genetic Platform to Create Super Sniffer Mice for Studying Human Odor Coding. Cell Rep. 2016; 16(4):1115-1125., incorporated herein by reference) digested with StuI and AscI. Ligated constructs were transfected into DH5alpha Escherichia coli cells, and positive clones were grown for plasmid purification. To create constructs expressing a different fluorescent reporter IRES-MP-Gcamp6f, the OR constructs were digested with PacI to isolate the OR fragment and ligated with PacI-digested reporter genes. The final constructs (˜10 kB) were digested with PmeI to linearize the DNA for pronuclear injection, in which the DNA randomly integrates into the mouse genome (
The resulting chimeric offspring are subsequently genotyped to verify incorporation of the transgene into the host genome. Molecular analysis of the founders (transgenic mice which have integrated the transgenic construct) was performed by utilizing an internal ribosomal entry site (IRES) in the OR expression vector that allows for bicistronic translation and simultaneous expression of the fluorophore (
Because the transgenic ORs that were expressed in mouse OSNs are not necessarily of mouse origin, the transgenic OR-RNA levels were not compared with the endogenous mouse-OR RNA levels. To estimate the level of preferential expression of the transgenic ORs, a total glomerular volume (TGV) analysis was performed as a surrogate measurement for the calculation of the transgenic OSN numbers, because it is known that a positive correlation is present between the TGV and the number of OSNs expressing the corresponding OR (Bressel, O. C., M. Khan, and P. Mombaerts, J Comp Neurol, 2016. 524(1): 199-209, incorporated herein by reference).
The olfactory epithelium from individual 6 to 8 week old, transgenic mice that preferentially express a PD-discriminating OR are dissected and washed briefly in cold buffer containing proteinase inhibitors. The buffer is replaced with solution containing calcium to “shock” the cilia off of the olfactory neurons (protocol adapted from Mayer et al. 2009; Kuhlmann et al. 2014, incorporated herein by reference). Tissue debris is removed by a brief centrifugation step. After two rounds (20 min shock and 10 min centrifugation) of the above shock procedure, the pooled supernatant is spun at high speed in an ultracentrifuge for 30 min at 4° C. The resulting cilia pellet is resuspended in buffer with 5% glycerol and proteinase inhibitors, aliquoted and flash-frozen in liquid nitrogen. Cilia aliquots are stored at −80° C.
Cilia are obtained from a transgenic mouse preferentially expressing an OR described herein (generated using the methods described above). One μg of cilia bioextracts are incubated with 5 μM Forskolin or sample extract for 15 minutes at 37° C. in a total volume of 8 μL induction buffer (1×PBS, 100 μM Ro 20-1724[4-(3-butoxy-4-methoxybenzyl) imidazolidone], 500 μM IBMX (3-isobutyl01-methylxanthine)) in a covered white 96-well half-area plate. The cAMP-Glo™ assay (Promega) is performed on these samples according to manufacturer's suggestions adapted for 384-well plates. Forskolin (FSK) is used as a positive control to show viability of the cilia extracts. FSK binds directly with ACIII, which converts ATP into cAMP. Each sample (including controls) measurement is an average of technical triplicates.
The assay employed to test activation of PD-discriminating ORs takes advantage of the fact that ORs are G-protein coupled receptors (GPCRs) that couple with adenylate cyclase III. Activated adenylate cyclase produces cyclic AMP (cAMP), which stimulates protein kinase A (PKA) activity, leading to a decrease in ATP levels. This decrease in ATP is measured using a luciferase reaction, using a commercially available assay, for example, the Promega cAMP-Glo™ Assay. In this assay, which can be adapted for a 384 well format, a lower level of ATP leads to decreased bioluminescence, indicating increased activity of the OR.
Sebum from a subject is collected on gauze, and extracted from the gauze using methanol. Extracted sebum samples can be stored at −80° C.
Freshly-thawed cilia (100-1,000 ng) isolated from either (i) mice that preferentially express an OR disclosed herein or (ii) wild type mice, are placed in triplicate wells and incubated with control (solvent alone) or sample for 15 minutes at 37° C. All subsequent steps are performed as per manufacturer's instructions for the Promega cAMP-Glo™ Assay. Analysis for cilia activation is performed by calculating the difference in the bioluminescent readout (DRLU) between PD-treated and control or untreated cilia for the cilia isolated from either (1) mice that preferentially express a PD-discriminating OR or (2) wild type mice.
For wild type cilia, neither a sample from a subject with PD nor the odor control causes activation of the ORs expressed in these cilia, and the ATP levels are about the same upon exposure of these cilia to either the odor control or the sample from a subject with PD. As such, the difference in DRLU observed for exposure to the odor control vs to the sample from a subject with PD is small.
For cilia isolated from mice that preferentially express a PD-discriminating OR, said OR is, for example, activated upon exposure to a sample from a subject with PD, leading to decreased ATP levels as compared to the same cilia exposed to the odor control. Therefore the difference in DRLU observed for exposure to the odor control vs to the sample from a subject with PD is significantly greater for these types of cilia.
Viability of the cilia is tested with Forskolin (5 μM). Forskolin (positive control) activates ACIII directly and increases the intracellular cAMP levels.
This application claims priority to U.S. Provisional Application No. 63/081,871, filed Sep. 22, 2020, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/051586 | 9/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63081871 | Sep 2020 | US |
