METHODS OF IDENTIFYING, ISOLATING AND USING ODORANT AND AROMA RECEPTORS

- Firmenich SA

Provided here are new methods to identify specific families of mammalian odorant receptors for odorants or aroma, particularly indole and skatole malodors and their use in assays that may be used to discover compounds that modulate (blocking, enhancing, masking or mimicking compounds) their activity. Orphan mouse odorant receptors are identified from olfactory sensory neurons that respond to target compounds. The resulting receptors as well as their human counterparts can be screened in assays against test compounds to confirm their identity as odorant or aroma receptors, particularly malodor receptors and subsequently discover for example modulators that inhibit the perception of the malodor in humans.

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Description
RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/318,641 filed Jun. 28, 2014, which claims the benefit of U.S. provisional application 61/841,282, filed on Jun. 29, 2013 and of U.S. provisional application 61/921,664, filed on Dec. 30, 2013. The entire contents of each of these applications are hereby incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 8950US_DIV_SequenceListing. The size of the text file is 175 KB, and the text file was created on Jan. 19, 2018.

FIELD

The technical field is directed to odorant and aroma receptors and assays that can be used to identify odorant and/or aroma compounds and more specifically inhibitors or counteractants of malodor compounds such as indole, or skatole.

BACKGROUND

Olfaction is one of the most complex and poorly understood of human sensory systems. From olfactory receptor (OR) activation to perception, there are many steps that still require further investigation. If we can understand how the OR code for individual odorants and mixtures translates into perception then we can exploit this knowledge to bring significant benefit in several areas. These areas include odor modulators like malodor counteractants that block the perception of unpleasant odors, new flavor and fragrance ingredients that replace non-biodegradable or toxic compounds, and odor enhancers that would limit our reliance on difficult to source compounds from natural sources. The ‘olfactory code’ combinatorial paradigm is centered on the observation that any single OR may be activated by multiple odorants, and conversely most odorants are capable of activating several ORs. In the mouse genome there are 1,200 distinct ORs. Humans, by contrast, have ˜400. In both cases, the repertoire of ORs is activated by many thousands of odorants in the world, and it is this combinatorial complexity that allows for the breadth of olfactory sensations we can perceive. However, odorants or ligands for only 95 mouse (˜8%) and 41 human ORs (˜10%) have been identified as of 2014 using traditional deorphanization methods. In addition, the physiological relevance of most ligands for the human ORs, essentially identified in vitro, has not been tested.

Different OR de-orphanization methods have been described in the literature [e.g. Touhara (2007) Deorphanizing vertebrate olfactory receptors: Recent advances in odorant-response assays Neurochem Int 51, 132-139, Saito et al (2009) Odor coding by a Mammalian receptor repertoire. Sci Signal 2, ra9, and Peterlin et al. (2014), The State of the Art of Odorant Receptor Deorphanization: a Report from the Orphanage, J Gen Physiol; 143(5): 527-42]. Many of these methods rely exclusively on cell-based assays where the OR is expressed in non-olfactory cells that are suitable for high-throughput screening. However, ORs are often retained in the endoplasmic reticulum of such heterologous cells. Failing to traffic to the cell surface, the ORs are thus unable to interact with the odorant [Min et al. (2004) Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression. BMC Cell Biol 5, 34]. Thus, a systematic approach where hundreds to thousands of different cell lines, where each cell line possess a unique OR protein that can be assessed for odorant activity, is not a suitable approach for comprehensive decoding of the combinatoral interactions between odorants and ORs since many or most of the receptors do not function properly in such cell lines. There is therefore a need for new methods that can rapidly and reliably identify the relatively small subset of ORs, within the entire repertoire of ORs that exist in an organism, that are specifically activated or inhibited by one or more odorants. There is a further need for a method to involve the identification of ORs from the olfactory neurons themselves, where the ORs are presumed to be fully functional, thus circumventing the well-known challenges of OR assays in non-olfactory cells.

Malodor compounds such as indole, skatole (3-methyl indole), and p-cresol generate unpleasant odors that arise for example from latrines and other “bathroom” sources that contain fecal matter. Hence, malodor counteractants that mask or reduce the perceived intensity or modify the perceived quality for example of human smell of the compounds are desirable. Odorant receptors and more particularly malodor receptors have a need to be identified. Receptors that bind to indole and skatole have also been identified and compounds that bind to those receptors have been discovered and reported as potential modulators of malodor. However, the rapid identification of the complete repertoire of receptors that bind to malodors, particularly indole or skatole receptors continues to be desirable due to the numerous ORs that exist in mammals. Assays that rely on new malodor receptors to identify new potent compounds that bind to these receptors are further desired.

SUMMARY

Provided herein is a method of identifying olfactory receptors that are activated by an odorant or aroma compound comprising:

    • a) dissociating an isolated olfactory epithelium containing native olfactory neurons into single cells from a non-human mammal species wherein each neuron expresses an olfactory receptor;
    • b) loading the olfactory cells with an indicator dye that allows for the measurement of odorant or aroma receptor binding activity of the olfactory receptors;
    • c) contacting the olfactory receptors with odorant or aroma compounds sequentially;
    • d) measuring changes of odorant or aroma-induced neuronal activity;
    • e) isolating one or more olfactory neurons that were activated by a odorant or aroma compound;
    • f) isolating or isolating and amplifying the mRNA of the isolated olfactory receptors;
    • g) sequencing at least a portion of the transcriptome of the mRNA by Next-Generation Sequencing; and
    • h) determining the identity of a group of olfactory receptors selected from the group consisting of odorant and aroma receptors by comparing the sequence of the transcriptome to a reference genome sequence of the same species and other vertebrate species.
      Further provided herein is a method of identifying olfactory receptors that are activated by a malodor compound comprising:
    • a) dissociating an isolated olfactory epithelium containing native olfactory neurons into single cells from a non-human mammal species wherein each neuron expresses an olfactory receptor;
    • b) loading the olfactory cells with an indicator dye that allows for the measurement of malodor receptor binding activity of the olfactory receptors;
    • c) contacting the olfactory receptors with malodor compounds sequentially;
    • d) measuring changes of odorant-induced neuronal activity;
    • e) isolating one or more olfactory neurons that were activated by a malodor compound;
    • f) isolating or isolating and amplifying the mRNA of the isolated olfactory receptors;
    • g) sequencing at least a portion of the transcriptome of the mRNA by Next-Generation Sequencing; and
    • h) determining the identity of a group of malodor olfactory receptors by comparing the sequence of the transcriptome to a reference genome sequence of the same species and other vertebrate species.

Further provided herein is an isolated nucleic acid sequence having at least 60% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85.

Also provided herein is an isolated nucleic acid sequence as describe above which encodes a polypeptide having at least 60% sequence identify with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

Further provided herein is an isolated polypeptide comprising an amino acid sequence having at least 60% sequence identify with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

Still yet further provided is a cell that is recombinantly modified to express a polypeptide, described above.

Further provided are assays for identifying compounds that bind to indole and/or skatole odorant receptors. In particular, provided herein is a method for identifying a compound that blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor that is activated by a compound selected from the group consisting of indole and skatole comprising

    • a) contacting the receptor, or a chimera or fragment with a compound;
    • b) assaying whether the compound has an effect on the activity of the receptor;
      • wherein the receptor is a polypeptide described above.

In one embodiment the activity of the compound is determined by comparing its binding to that of indole and/or skatole. In another embodiment, the receptor is contacted with a compound in the presence of skatole and/or indole under conditions that allow for the binding of the compound along with skatole and/or indole to the receptor.

In a further embodiment, provided herein, when a functional assay is used to measure binding activity, the step of measuring a signaling activity of receptors provided herein may comprise detecting a change in the level of a second messenger. In another embodiment, the measurement of a signalling activity is provided wherein the step of measuring a signaling activity comprises the measurement of guanine nucleotide binding/coupling or exchange, adenylate cyclase activity, cAMP, Protein Kinase C activity, Protein Kinase A activity phosphatidylinosotol breakdown, diacylglycerol, inositol triphosphate, intracellular calcium, calcium flux, arachidonic acid, MAP kinase activity, tyrosine kinase activity, melanophore assay, receptor initialization assay, FRET, BRET, or reporter gene expression. In a particular embodiment provided herein, the measuring of signaling activity comprises using a fluorescence or luminescence assay. Fluorescence and luminescence assays may comprise the use of Ca2+ sensitive fluorophores including Fluo-3, Fluo-4 or Fura dyes (Molecular Probes); Calcium 3 assay kit family (Molecular Devices) and aequorin.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a schematic overview of the malodor receptor de-orphanization process

FIG. 2 displays Ca2+ imaging traces that are shown for 6 independent olfactory sensory neurons specifically activated by both indole and skatole (50 μM each).

FIG. 3 shows malodor receptor genes identified using the procedures outlined herein.

FIGS. 4A and 4B display human and additional mouse malodor receptors within the Olfr740 (4A) and Olfr665 (4B) families identified using the procedures outlined herein

FIGS. 5A to 5E show the indole and skatole activity of mouse ORs Olfr743, Olfr746, and Olfr740 in HEK293T cells.

FIGS. 6A to 6J show the indole and skatole activity of human OR52N2, OR11G2, OR5AC2, OR4C15, OR8S1, OR11H6 and OR11H4 and mouse Olfr665 and Olfr740 in HEK293T cells.

FIG. 7 shows the inhibition of indole receptor Olfr743 activity in HEK293T cells by a test compound.

FIG. 8 shows the pairwise amino acid identities for the mouse Olfr740 family and their predicted human orthologs.

FIG. 9 shows the pairwise amino acid identities for the mouse Olfr665 family and their predicted human orthologs.”

DETAILED DESCRIPTION

For the descriptions herein and the appended claims, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of”

Definitions

The following terms have the meanings ascribed to them unless specified otherwise.

“OR” refers to one or more members of a family of G protein-coupled receptors that are expressed in olfactory cells. Olfactory receptor cells can also be identified on the basis of morphology or by the expression of proteins specifically expressed in olfactory cells. OR family members may have the ability to act as receptors for olfactory transduction.

“Indole” and/or “skatole OR” refers to a member of the family of G protein-coupled receptors that is expressed in an olfactory cell, which receptors bind and/or are activated by indole and/or skatole in a binding or activity assay for identifying ligands that bind and modulate GPCRs by activating, inhibiting or enhancing their activity. Such assays are described below. Indole and/or skatole receptors herein will include fragments, variants, including synthetic and naturally occurring, and chimeras that respond to or bind indole and/or skatole.

“OR” nucleic acids encode a family of GPCRs with seven transmembrane regions that have “G protein-coupled receptor activity,” e.g., they may bind to G proteins in response to extracellular stimuli and promote production of second messengers such as IP3, cAMP, cGMP, and Ca2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase.

“OR” polypeptides are considered as such if they pertain to the 7-transmembrane-domain G protein-coupled receptor superfamily encoded by a single ˜1 kb long exon and exhibit characteristic olfactory receptor-specific amino acid motifs. The predicted seven domains are called “transmembrane” or “TM” domains TM I to TM VII connected by three predicted “internal cellular loop” or “IC” domains IC I to IC III, and three predicted “external cellular loop” or “EC” domains EC I to EC III. The motifs are defined as, but not restricted to, the MAYDRYVAIC motif overlapping TM III and IC II, the FSTCSSH motif overlapping IC III and TM VI, the PMLNPFIY motif in TM VII as well as three conserved C residues in EC II, and the presence of highly conserved GN residues in TM I [Zhang and Firestein (2002), The Olfactory Receptor Gene Superfamily of the Mouse. Nature Neuroscience: 5(2):124-33; Malnic et al., The Human Olfactory Receptor Gene Family: PNAS: 101(8):2584-9].

The “N terminal domain” region starts at the N-terminus and extends to a region close to the start of the first predicted transmembrane region. “Transmembrane domain,” which comprises the seven predicted “transmembrane regions,” refers to the domain of OR polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. The seven transmembrane regions and extracellular and cytoplasmic loops can be identified using standard methods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32 (1982), or in Stryer. The general secondary and tertiary structure of transmembrane domains, in particular the seven transmembrane domains of G protein-coupled receptors such as olfactory receptors, are known in the art. Thus, primary structure sequence can be designed or predicted based on known transmembrane domain sequences, as described in detail below. These transmembrane domains are useful for in vitro ligand-binding assays, both soluble and solid phase.

The phrase “functional assay” in the context of assays for testing compounds that modulate OR family member mediated olfactory transduction includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signal transduction receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP IP3, or intracellular Ca2+), assays performed in vitro, in vivo, and ex vivo. It also includes other physiological effects such as increases or decreases of neurotransmitter or hormone release. Included herein are functional assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of an OR family member, e.g., functional, physical and chemical effects. Such functional assays or effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, luminescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte OR gene expression; tissue culture cell OR expression; transcriptional activation of OR genes; ligand-binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “blockers,” “activators,” “counteractants” and “modulators” of OR genes or proteins are used interchangeably to refer to inhibitory, activating, or modulating molecules identified using ex vivo, in vitro and in vivo assays for olfactory transduction, e.g., ligands, agonists, antagonists, inverse agonists and their homologs and mimics. Inhibitors and blockers are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate olfactory transduction, e.g., antagonists. Activators are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate olfactory transduction, e.g., agonists. Modulators include compounds that, e.g., alter the interaction of a receptor with: extracellular proteins that bind activators or inhibitors (e.g., odorant-binding proteins, lipocalin and other members of the hydrophobic carrier family); G proteins; kinases (e.g., homologs of rhodopsin kinase and beta adrenergic receptor kinases that are involved in deactivation and desensitization of a receptor); and arrestins, which also deactivate and desensitize receptors. Modulators can include genetically modified versions of OR family members, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., expressing OR family members in cells or cell membranes, applying putative modulator compounds, in the presence or absence of flavor or fragrance molecules, e.g., perfumery raw materials, perfume formulations, or malodors, and then determining the functional effects on olfactory transduction, as described above. Samples or assays comprising OR family members that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation. Control samples (untreated with modulators) are assigned a relative OR activity value of 100%. Inhibition of an OR is achieved when the OR activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of an OR is achieved when the OR activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.

The terms “purified,” “substantially purified,” and “isolated” as used herein refer to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the “purified,” “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample.

As used herein, the term “isolated,” when referring to a nucleic acid or polypeptide refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human, body. Any degree of purification or concentration greater than that which occurs naturally in the body, including (1) the purification from other naturally-occurring associated structures or compounds, or (2) the association with structures or compounds to which it is not normally associated in the body are within the meaning of “isolated” as used herein The nucleic acids or polypeptides described herein may be isolated or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processed known to those of skill in the art.

As used herein, the terms “amplifying” and “amplification” refer to the use of any suitable amplification methodology for generating or detecting recombinant of naturally expressed nucleic acid, as described in detail, below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo or in vitro.

The term “7-transmembrane receptor” means a polypeptide belonging to a superfamily of transmembrane proteins that have seven domains that span the plasma membrane seven times (thus, the seven domains are called “transmembrane” or “TM” domains TM I to TM VII). The families of olfactory and certain taste receptors each belong to this super-family. 7-transmembrane receptor polypeptides have similar and characteristic primary, secondary and tertiary structures, as discussed in further detail below.

The term “nucleic acid” or “nucleic acid sequence” refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions or single nucleotide polymorphisms) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant” means also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using a primer of the invention.

The term “expression vector” refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome (e.g. stable expression). The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression “cassettes” which contain only the minimum elements needed for transcription of the recombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, HEK-293, and the like, e.g., cultured cells, explants, and cells in vivo.

In one embodiment provided herein is an isolated nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85.

In one embodiment provided herein is an isolated nucleic acid sequence as described above which encodes a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

In a further embodiment provided herein is an isolated polypeptide comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

A method enables one skilled in the art to rapidly identify the relatively small subset of odorant receptors (ORs), within the total population of 1,200 ORs that exist in the rodent genome, whose human genetic counterparts likely encode a specific odor characteristic in humans. FIG. 1 is a schematic overview of the method. Intact mouse olfactory epithelium (OE) tissue is dissociated by mechanical and enzymatic treatment into single cells. The resulting individual olfactory sensory neurons (OSNs) are plated onto glass coverslips and loaded with a calcium indicator dye (e.g., Fura-2) to detect cell activity. The coverslips are then placed in a perfusion chamber and bathed in a physiological salt solution. Malodorous test odorants are diluted in physiological saline and perfused over the cells. OSN activity is then detected by monitoring the changes in intracellular calcium flux via a change in Fura-2 fluorescence and a fluorescent microscope. Importantly, in some instances, only one or two of ˜1200 possible OR genes are expressed in a given OSN. This means that the activity of a given OSN is driven exclusively by a single type, or two types, of OR protein(s) expressed on the cell surface. A semi-automated calcium imaging system with a finely controlled odorant injection system and a movable stage allows for monitoring of >1,500 OSNs in a single experiment. With ˜1,200 OR genes in mouse, this throughput ensures a representative sampling of the entire OR repertoire. One or more OSNs that respond to one or more test odorant are collected using a glass micropipette and displaced in a microtube for subsequent RNA extraction, amplification, and Next-Generation Sequencing (NGS) of OSN transcriptomes. NGS-based transcriptome sequencing from very small amounts of starting material (<100 cells) has been reported in the literature. However, it has not yet been applied for the specific purpose of G-Protein Coupled Receptor (GPCR) deorphanization to the best of our knowledge; in particular olfactory receptors. Using the methods described herein, multiple OSNs can be pooled in the same microtube if desired. Raw sequence reads are then mapped to a reference genome, visually inspected for proper alignment and annotation, and analyzed for false-positive mapping due to, for example, genome repeat sequences. The result is a list of candidate mouse ORs that are likely activated by the same malodorous test odorant(s) used to screen mouse OSNs. The candidate mouse OR protein sequences are then used to query (e.g., BLASTP) public mouse genome databases to obtain a full list of candidate mouse OR sequences. These candidates are related by sequence homology (i.e. paralogous) to the NGS-derived OR sequences originally identified from the neurons that were activated by indole and/or skatole. The full list of mouse paralogs (NGS and BLASTP-derived) are then used to query public human genome databases to obtain a list candidate human OR sequences that are related (i.e. orthologous) to the mouse OR sequences. The result is a complete list of candidate mouse and human ORs that are likely activated by the test odorants. To confirm the activity of the resulting candidate ORs genes, cDNAs of said genes can be cloned into available expression vectors and transfected into cultured cells that are amenable to high-throughput screening (e.g. HEK293T). The resulting cell lines, each containing a recombinantly expressed candidate OR cDNA, can then be screened in a cell-based activity assay with the test odorants used in calcium imaging and other structurally- or organoleptically-related odorants.

In a particular embodiment olfactory epithelium containing native olfactory neurons from a non-human mammal (e.g., mouse) are isolated and dissociated into single cells according to for example Araneda et al. (2004), A Pharmacological Profile of the Aldehyde Receptor Repertoire in Rat Olfactory Epithelium. Journal of Physiology: 555:743-756. In a particular embodiment, each neuron expresses only one or two receptors at a time. The olfactory tissue used to generate olfactory neurons is, in one embodiment, from a non-human mammal that has publically available data concerning the receptors and associated sequences. This includes mouse, rat and hamster tissue. The dissociation protocol should be optimized in order to ensure high rate of surviving recordable neurons. In a particular embodiment a minimum of approximately 1,500 distinct sensory neurons are recorded.

In a further embodiment, the indicator is selected from a fluorescent calcium indicator dye, a calcium indicator protein, a fluorescent cAMP indicator, a cAMP response element (CRE) mediated reporter protein, a biochemical cAMP HTRF assay, a beta-arrestin assay, or an electrophysiological recording. Particularly, a calcium indicator dye is selected that can be used to monitor the activity of olfactory receptors expressed on the membrane of the olfactory neurons (e.g., Fura-2 AM).

In a particular embodiment, compounds are screened sequentially and the odorant-dependant changes in calcium dye fluorescence are measured using a fluorescent microscope or fluorescent-activated cell sorter (FACS).

As an example, olfactory neurons are isolated after screening with one or more malodor compounds using either a glass microelectrode attached to a micromanipulator or a FACS machine. More particularly at least 1 neuron is isolated. Mouse olfactory sensory neurons are screened by Ca2+ imaging similar to procedures previously described (Malnic et al., 1999; Areneda et al., 2004). Particularly, a motorized movable microscope stage is used to increase the number of cells that can be screened to at least 1,500 per experiment. Since there are approximately 1,200 different olfactory receptors in the mouse and each olfactory sensory neuron expresses only 1 or 2 of 1,200 olfactory receptor genes, this screening capacity will cover virtually the entire mouse odorant receptor repertoire. In other words, the combination of calcium imaging for high-throughput olfactory sensory neuron screening leads to the identification of nearly all of the odorant receptors that respond to a particular profile of odorants. In a particular aspect, odorants that respond to both indole and skatole, two common latrine malodor compounds can be isolated.

For calcium imaging of olfactory neurons, the main olfactory epithelium may be dissected from a mouse before neuronal dissociation. Dissected olfactory epithelium may then be transferred to a dissociation buffer for mechanical and enzymatic dissociation. Dissociated neurons may then be seeded onto a coverslip allowing the screening of thousands of cells by fluorescence microscopy and the cells may be loaded with a calcium sensitive dye (Fura-2 AM) for example for about 30 minutes at 31° C. and transferred onto the microscope ready for screening. Cells are stimulated by perfusing diluted solutions of odorants (in physiological saline) over the dissociated olfactory neurons. The rare cells that respond to the malodor compound are identified by for example stimulating the receptors with 50 μm of the malodor compounds and then by monitoring the intracellular Ca2+ flux indicated by changes in Fura-2 fluorescence. After analysis, responding cells may be retrieved from a glass coverslip with a suction micropipette. Isolated cells are then pooled into one sample for subsequent identification of the odorant receptor genes expressed as mRNA in the responding cells.

In a particular embodiment, the mRNA of olfactory neurons are purified and amplified according to the method generally described in Marko, N. F., et al., (2005) A robust method for the amplification of RNA in the sense orientation. BMC genomics, 6, 27; doi:10.1186/1471-2164-6-27 (Eberwine method). At least a portion of the transcriptome (up to and including the entire transcriptome) is sequenced using Next-Generation Sequencing (NGS) or hybridized to known genes using Microarray technologies. NGS is generally discussed and described in Metzker, M. L. (2010). Sequencing technologies—the next generation. Nature reviews. Genetics, 11(1), 31-46; doi:10.1038/nrg262. In a particular embodiment, a minimum of 5 neurons presenting the same response profile are pooled and the mRNA are amplified by two consecutive rounds of in vitro transcription (IVT). The mRNA is released by cell lysis immediately after picking; no DNAse and no purification steps are carried out. The amplification may be done according to MesageAmpll aRNA kit (Ambion, AMA1751) with the following parameters: two rounds of consecutive 14 hour long IVT.

In a further embodiment, the identity of a group or gene family of malodor olfactory receptors is determined (e.g., up to as many as the number of neurons picked) by comparing the results of the NGS to a reference genome sequence of the same species. Particularly, the putative malodor receptors will be the most highly abundant mRNA in the olfactory neuron-derived NGS sample or present in more than one independent biological replicate. Because of the combinatorial nature of the olfactory code (one compound activates many ORs and one OR can be activated by many compounds), pooling several neurons activated by given compounds allows the retrieval of virtually all of the receptors responsible for the perception of these molecules in a single NGS experiment. Pooling functionally similar neurons thus greatly improves the deorphanization throughput and speed.

Standard bioinformatics tools are then used to identify the most closely related human odorant receptor(s) to other putative mammalian (non-human) malodor receptor(s) under the assumption that homologous receptor sequences retain similar function. One study demonstrated that ˜82% of orthologous OR gene pairs responded to a common ligand, suggesting that as many as ˜18% of orthologous gene pairs may respond to a different ligand; further, 33% of paralogous OR gene pairs responded to a common ligand, suggesting that at least ˜67% of paralogous pairs may respond to a different ligand [Adipietro et al. (2012) Functional Evolution of Mammalian Odorant Receptors. PLoS Genet 8(7): e1002821. doi:10.1371/journal.pgen. 1002821]. Default parameters of BLASTP and/or BLASTN algorithm may be used.

The human or non-human mammalian malodor receptor may be adapted to a functional assay that can be used to identify compounds that mimic, block, modulate, and/or enhance the activity of a malodor compound. In particular the assay may be a cell assay and the method for identifying compounds may be a high-throughput screening assay. More particularly, provided herein are receptor-based assays adaptable for high-throughput screening of receptors with compound libraries for the discovery of modulating compounds (e.g., blocking, enhancing, and masking).

In a particular embodiment, malodor receptor gene sequences are identified from indole and skatole-sensitive cells as follows: Pooled neurons are heated to 75° C. for 10 minutes to break the cell membrane and render their mRNA available for amplification. This amplification step is important when applying NGS technologies with limited amount of starting material, typically between 1 to 15 cells. A linear amplification according to the Eberwine method (IVT) ensures the maintenance of the relative transcription levels of expressed genes. Two consecutive overnight (14 h) rounds of in vitro transcription are used to yield sufficient amounts of cRNA; Amplified cRNA is then used to generate an Illumina HiSeq cDNA library. The resulting short sequences of typically 75-150 base pairs (commonly referred to as “reads”) are aligned against the reference genome of the mouse (such as UCSC version mm9 or mm10) in order to build the full transcriptome of these cells. Quantitative analysis of the transcriptome data yields a list of transcribed odorant receptor genes and their respective expression levels. Odorant receptor genes that show the most abundant levels of mRNA (most abundant “reads”) or are present in more than one replicate experiment are considered putative indole and skatole receptors.

The predicted mouse OR genes are then used to mine the latest versions of both the mouse and human genome databases in order to identify the most closely related receptors (i.e., highest sequence similarity) in mouse (paralogous genes) and in human (orthologous genes). This process may be performed using the BLAST search algorithm (publically available at the NCBI website), a sequence similarity search tool, where every putative gene sequence previously obtained from the initial transcriptome analysis is used as a query sequence. The newly identified genes identified from this data mining process are also considered as potential malodor receptors under the assumption that paralogous and orthologous genes are highly likely to possess similar activities.

In a particular embodiment, pairwise comparison of sequence homology is carried out to identify closely related receptors in mouse and humans using the following iterative scheme:

Step Query sequence BLASTN/BLASTP Result 1. Mouse candidate 1 → Mouse paralog 1 and human ortholog 1 2. Mouse paralog 1 → Human ortholog 2 3. Human ortholog 1 → Human paralog 1 4. Human ortholog 2 → Human paralog 2 Paralog = homolog in same species Ortholog = homolog in other species

Paralogous genes are then aligned using a multiple alignment tool in order to generate a phylogenetic tree. Functional in vitro data can be interpreted in the light of such a phylogenetic relationship between closely related but distinct receptors. This step is essential in the identification of complete OR gene families that respond, to varying degrees, to the test compounds, for example indole and skatole.

To complete the deorphanization process, the candidate OR genes are further expressed in vitro for confirmation of activity against the compounds used to isolate the olfactory sensory neurons and other structurally-related compounds of interest. In one embodiment, to complete the deorphanization process, the candidate OR genes are further expressed in vitro for confirmation of activity against the compounds initially used to isolate said olfactory sensory neurons. The same candidate OR genes expressed in vitro are further screened with other structurally-related compounds of interest to identify, e.g., activators, inhibitors, or modulators of the receptor.

Using the process and method described herein the following receptors have been identified directly by sequencing (NGS) the transcriptome of olfactory neurons responding to indole and skatole malodors (mouse ORs). Human indole and skatole receptors with the highest degree of amino acid identity to the mouse ORs were subsequently identified by searching available genome databases (Table 1). Mouse receptor Olfr740 and Olfr665 were initially identified through the NGS method described herein after isolating or “picking” responding cells after exposure to both indole and skatole. Corresponding human homologous receptors, OR11G2 and OR52N2 respectively, were further identified by amino acid sequence similarity comparisons. As demonstrated in examples 4 and 5, the candidate mouse receptors Olfr740 and Olfr665 and their human counterparts OR11G2 and OR52N2 were activated in vitro by indole and by skatole and are thus described as indole and/or skatole receptors. Similarly, the candidate mouse receptors Olfr 746, Olfr745, Olfr207, Olfr1211 and Olfr257 led to identification of the human receptors OR11H4, OR11H6, OR5AC2, OR4C15 and OR8S1, respectively, following the same steps. These human OR genes also responded to both indole and skatole in vitro. Taken together, seven out of seven tested putative human receptors identified by the method described herein were confirmed as indole and skatole receptors further supporting the method for efficient human indole and skatole receptor identification.

This approach to odorant receptor deorphanization has several major advantages over previously established single cell RT-PCR methods. First, by pooling multiple neurons sharing similar binding properties to odors and/or aromas, a unique mRNA sequencing experiment (NGS) identifies virtually all the receptors that are activated by the target malodor compounds. Therefore the throughput is higher than what was previously achieved. Second, because multiple cells are pooled into one sample, multiple ORs for a particular compound are revealed in a single experiment. In a particular embodiment, the selection of genes through a comprehensive comparison of replicate samples across experiments. Third, NGS does not require the use of PCR primers specific to an OR. NGS also does not require the use of degenerate primers specific to ORs, which are problematic and often lead to false positives due to non-linear or non-specific PCR amplification. In particular, since OR coding sequences lie within a single exon, sample contamination with genomic DNA can easily lead to an aspecific amplification of OR gene sequences. Fourth, RT-PCR analysis is difficult to perform on pooled samples because of the inherent false positive rate. Single cell mRNA hybridization experiments have been performed using of high-density DNA microarray chips. However, this approach is generally less sensitive than NGS and is further restricted to known genes for which corresponding DNA probes need to be synthesized. Hence, the use of NGS is significantly advantageous to rapidly identify OR and ultimately results in a more accurate selection of candidate receptors compared to the standard (e.g., RT-PCR and microarray) approaches.

In a further embodiment, mouse receptors identified from isolated olfactory neurons that respond to both indole and skatole are modified at their N-terminus with a short polypeptide sequence (e.g., bovine rhodopsin receptor—Rho, or Flag), transiently expressed in HEK 293T cells, and stimulated separately with indole and skatole to confirm their identity as bona fide indole/skatole receptors. Co-expression of the human G alpha subunit Gαolf (SEQ ID NO: 21) activates the Gs transduction pathway that leads to an internal cAMP increase upon binding to the appropriate ligand. The results confirm the identity of Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6), and Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) as indole-skatole receptors.

In another aspect, human receptor OR52N2 (SEQ ID NO: 11 and SEQ ID NO: 12) was identified because of its sequence similarity to mouse Olfr665 (SEQ ID NO: 9 and SEQ ID NO: 10), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole (Example 6). Human receptor OR11G2 (SEQ ID NO: 13 and SEQ ID NO: 14) was identified because of its sequence similarity to mouse Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole (Example 6). Human receptor OR5AC2 (SEQ ID NO: 75 and SEQ ID NO: 76) was identified because of its sequence similarity to mouse Olfr207 (SEQ ID NO: 77 and SEQ ID NO: 78), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole (Example 6). Human receptor OR4C15 (SEQ ID NO: 79 and SEQ ID NO: 80) was identified because of its sequence similarity to mouse Olfr1211 (SEQ ID NO: 81 and SEQ ID NO: 82), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole (Example 6). Human receptor OR8S1 (SEQ ID NO: 83 and SEQ ID NO: 84) was identified because of its sequence similarity to mouse Olfr257 (SEQ ID NO: 85 and SEQ ID NO: 86), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole (Example 6). Mouse receptor Olfr665 (SEQ ID NO: 9 and SEQ ID NO: 10) was identified directly from NGS data from isolated olfactory neurons responding to both indole and skatole (Example 6). Mouse receptor Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) was identified directly from NGS data from isolated olfactory neurons responding to both indole and skatole (Example 6). Mouse receptor Olfr736 (SEQ ID NO: 27 and SEQ ID NO: 28) was identified because of its sequence similarity to mouse Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), an indole/skatole receptor isolated from olfactory neurons and failed to respond to indole or skatole likely because of lack of cell surface expression. Mouse receptor Olfr747 (SEQ ID NO: 39 and SEQ ID NO: 40) and Olfr748 (SEQ ID NO: 41 and SEQ ID NO: 42) were also identified because of their sequence similarity to mouse Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) and exceptionally failed to respond to indole or skatole despite proper cell surface expression as evaluated by immunostaining assays. This underlines the diversifying nature of the olfactory receptors where highly similar sequences do not always share the same response profile.

The receptors are modified with the Rho sequence and stably expressed in HEK 293T cells. Co-expression of the human G alpha subunit Gα15 activates the Gq transduction pathway that leads to an internal Ca2+ increase upon binding to the appropriate ligand.

The above process and the results obtained so far serve to validate the process for rapid and reliable identification of mammalian odorant receptors for malodor compounds.

Still yet further provided is a cell that is recombinantly modified to express a polypeptide described above.

Further provided are assays for identifying compounds that bind to indole and/or skatole odorant receptors. In particular, provided herein is a method for identifying a compound that blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor that is activated by a compound selected from the group consisting of indole and skatole comprising

    • a) contacting the receptor, or a chimera or fragment thereof with a compound;
    • b) assaying whether the compound has an effect on the activity of the receptor;
      • wherein the receptor is a polypeptide described above.

In one embodiment the activity of the compound is determined by comparing its binding to that of indole and/or skatole. In another embodiment, the receptor or a chimera or fragment thereof is contacted with a compound in the presence of skatole and/or indole under conditions that allow for the binding of the compound along with skatole and/or indole to the receptor.

In a further embodiment, a compound is contacted to a receptor, or a chimera or fragment thereof that is activated by a compound selected from the group consisting of indole and skatole wherein the receptor, or a chimera or fragment thereof is expressed in a cell that is recombinantly modified to express the polypeptide.

The activity of the compound can be determined using in vivo, ex vivo, in vitro and synthetic screening systems, which typically allow for screening of large libraries of compounds, containing as many as 10,000s of compounds or mixtures of compounds.

In another embodiment, the contacting is performed with liposomes or virus-induced budding membranes containing the polypeptides described herein.

In another embodiment, the methods for identifying compounds that bind to the receptors that bind to skatole and/or indole, may be performed on a membrane fraction from cells expressing the polypeptides described here.

The following examples are illustrative only and are not meant to limit the scope of invention as set forth in the Summary, Description or in the Claims.

EXAMPLES Example 1

Screening of Mouse Olfactory Sensory Neurons to Identify Receptors that Bind Indole and/or Skatole

The olfactory epithelium containing native olfactory neurons from a mouse was isolated and dissociated into single cells and then transferred to a dissociation buffer for a gentle mechanical and enzymatic dissociation. In order to optimize the dissociation protocol, the general procedure for making fresh dissociation buffer was followed as set forth in Araneda et al. (2004), A Pharmacological Profile of the Aldehyde Receptor Repertoire in Rat Olfactory Epithelium. Journal of Physiology: 555:743-756 with the following modifications: DNAse I instead of DNAse II, Ca2+ concentration elevated to 5 mM and pH adjusted at 7.35 at 32°. Dissociated neurons were then seeded on a coverslip allowing the screening of >2,500 cells by fluorescence microscopy. The cells were then loaded with a calcium sensitive dye (Fura-2 AM) for 30 minutes at 31° C. and transferred onto a microscope ready for screening. Dissociated olfactory neurons were screened as previously described (Malnic et al., 1999; Araneda et al., 2004), but with a motorized movable microscope stage to increase the number of cells that can be screened. The cells were then stimulated by perfusing diluted solutions of odorants (in physiological saline) over the dissociated olfactory neurons.

Olfactory neurons that responded to indole and skatole were identified by stimulating receptors with 50 μm indole followed by 50 μm skatole and monitoring the intracellular Ca2+ flux indicated by changes in Fura-2 fluorescence. FIG. 2 displays Ca2+ imaging traces for 6 independent olfactory sensory neurons specifically activated by both indole and skatole (50 μM each). All cells were also stimulated with 20 μM forskolin (fork), a pharmacological activator of the enzyme adenylate cyclase ACIII, to confirm viability and neuronal identity of the cells. Timescale units on the X axis, 6 seconds frame. Average Fluorescence Intensity, relative fluorescent unit as a result of a ratiometric 340/380 nm recordings.

Example 2

Isolation and Amplification of mRNA from Indole and Skatole Sensitive Cells to Generate cDNA Sequences of the “Malodor Receptor” Gene for the Indole and Skatole Sensitive Receptors

The activated olfactory sensory neurons of Example 1 were isolated using a glass suction micropipette and then pooled into one sample for subsequent identification of the odorant receptor genes expressed as mRNA in the responding cells. Malodor receptor gene sequences were identified from indole and skatole-sensitive cells as follows: Four sets of more than five (5) olfactory neurons (6, 10, 15, and 15 cells each) activated by one or more malodor compounds were isolated using a glass microelectrode attached to a micromanipulator. Micropipettes were engineered from Borosilicate glass (Item # B150-86-10, Sutter Instruments) and pulled using a microelectrode puller (Model P-1000, Sutter Instruments) under the following conditions: Heat, ramp temp+20 0C; Pull=0; Vel=120; Pressure=500. Pulled pipette tips were broken manually and secured onto a Newport micromanipulator (model MW3R) for isolating olfactory sensory neurons activated by indole and skatole as determined in a calcium imaging assay. Activated OSNs were isolated by gentle suction applied to the back-end of the micropipette.

The mRNA of neurons was purified and amplified according to the Eberwine method (MessageAmp II aRNA kit—Ambion, AM1751). Pooled neurons were heated to 75° C. for 10 minutes in Resuspension Buffer (SuperScript III Cells Direct cDNA system, 46-6321-Invitrogen) to break the cell membrane and to render their mRNA available for amplification. A linear amplification according to the Eberwine method (in vitro transcription) ensures the maintenance of the relative transcription levels of expressed genes. First-strand cDNA was obtained according to the Ambion kit with a incubation for 2 h at 42° C. Second strand cDNA synthesis was performed using the same kit after a second incubation for 2 h at 16° C. Doubled stranded cDNA was used as a template to generate the corresponding cRNA (14 h incubation at 37° C.), which is then purified. These cRNA synthesis steps were repeated for a second IVT. The two consecutive overnight (14 h) rounds of in vitro transcription yielded sufficient amounts of cRNA; Amplified cRNA was then used to generate an Illumina HiSeq cDNA library. The sequence of the entire transcriptome was then obtained by Next-Generation Sequencing (NGS).

The identity of the mouse malodor, specificallyindole and/or skatole, olfactory receptors was determined by comparing the NGS results to a reference genome sequence of the same species. For this, the resulting short sequences of 150 base pairs (commonly referred to as reads) derived from NGS were aligned against the reference genome of the mouse (UCSC version mm9 or mm10) in order to build the full transcriptome of these cells. Quantitative analysis of the transcriptome data yielded a list of transcribed odorant receptor genes and their respective expression levels. Odorant receptor genes that showed abundant levels of mRNA (abundant reads) or were present in more than one replicate experiment were considered candidate indole and skatole receptors and are set forth in FIG. 3, which summarizes representatives NGS results from 4 independent skatole/indole cell picking experiments. The bars represent candidate indole/skatole OR genes with the most abundant mRNAs that were retained after visual inspection. The Y axis represents the mRNA abundance levels normalized to the level of OMP mRNA, accounting for the number of OSNs picked per sample. Star (*), receptors identified in more than one experiment. OMP (Olfactory Marker Protein) is a specific biomarker for mature olfactory neurons. By coupling calcium imaging and NGS as described above the following 5 receptor sequences were identified as putative mouse indole and skatole ORs: Olfr665 (SEQ ID NO: 9 and SEQ ID NO: 10), Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr741 (SEQ ID NO: 3 and SEQ ID NO: 4), Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6), and Olfr745 (SEQ ID NO: 7 and SEQ ID NO: 8.

Example 3 Identification of the Mouse and Human Malodor Receptors

The identification of a subset of 5 mouse indole and skatole ORs among ˜1,200 ORs in the mouse genome by coupling calcium imaging and NGS (Examples 1-2) enabled the rapid identification of additional mouse and new human indole and skatole ORs by in silico methods. Since ORs with similar overall amino acid identity are likely to display similar odorant response profiles [Adipietro et al. (2012)], a list of candidate indole and skatole ORs was expanded by querying public mouse and human genome databases with the mouse OR sequences derived from NGS analysis of indole/skatole sensitive olfactory sensory neurons isolated by calcium imaging. The identity of the mouse malodor olfactory receptors was determined by comparing the results of the NGS to a reference genome sequence of the same species. The putative malodor receptors will be the most highly abundant mRNA in the olfactory neuron-derived NGS sample or present in more than one independent biological replicate, but not in a control sample lacking olfactory neurons that respond to the malodor(s). Standard bioinformatics tools were used to identify the most closely related human odorant receptor(s) to the putative mammalian (non-human) malodor receptor(s) under the assumption that homologous sequence receptors retain similar function (Adipietro et al. (2012)). Default parameters of BLASTP and/or BLASTN algorithm were used. Table 1 lists the identified mouse odorant receptors (OR) and the predicted human orthologs.

TABLE 1 NGS Identified Mouse ORs Human Orthologs Amino Acid Identity Olfr665 OR52N2 77% (SEQ ID NO: 10) (SEQ ID NO: 12) Olfr740 OR11G2 77% (SEQ ID NO: 2) (SEQ ID NO: 14) Olfr741 OR11G2 73% (SEQ ID NO: 4) (SEQ ID NO: 14) Olfr743 OR11G2 75% (SEQ ID NO: 6) (SEQ ID NO: 14) Olfr745 OR11H6 82% (SEQ ID NO: 8) (SEQ ID NO: 16)

The related human protein sequences are within at least 60% sequence identity to the NGS identified mouse OR sequences listed in Table 1. Identity levels are given relative to the amino acid sequence pairwise comparison after BLASTP algorithm. Twenty six (26) new human and mouse ORs were identified through BLASTP queries of public genome databases (default parameters). Sixteen (16) were closely related to the NGS-derived genes Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), 741 (SEQ ID NO: 3 and SEQ ID NO: 4), 743 (SEQ ID NO: 5 and SEQ ID NO: 6), 745 (SEQ ID NO: 7 and SEQ ID NO: 8) and define the 740 family. Ten (10) were closely related to the NGS-derived receptor Olfr665 (SEQ ID NO: 9 and SEQ ID NO: 10) and define the Olfr665 family. FIGS. 4A and 4B depict the phylogenetic relationships of candidate mouse indole and skatole OR genes and their predicted human orthologs belonging to the Olfr740 (FIG. 4A) and Olfr665 (FIG. 4B) receptor families. The relationships were obtained by protein sequence-based Neighbor-Joining phylogenetic tree reconstruction. Corresponding human orthologs are indicated by arrows. Human OR gene names annotated as pseudogenes are marked with a ‘P’. Pseudogenes are likely non-functional. OR11H7P is a segregating pseudogene (SP) with known functional alleles. Diamonds (⋄) designate receptors initially identified by NGS. To determine the amino acid identities for additional candidate mouse and human indole and skatole ORs, the full length protein sequences were manually aligned based on highly conserved amino acid motifs across the entire OR repertoire (Bioedit sequence alignment tool, version 7.0.5.3). FIG. 8 displays the pairwise amino acid identities for the mouse Olfr740 family and their predicted human orthologs. FIG. 9 displays the pairwise amino acid identities for the mouse Olfr665 family and their predicted human orthologs.

Example 4 Indole and Skatole Activity of Mouse ORs Olfr743, Olfr746, and Olfr740 in HEK293T Cells.

The activity of candidate mouse receptors Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6), and Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) to both indole and skatole malodors were confirmed in cell-based cAMP assays. In FIGS. 5A and 5B, FLAG Rho-tagged (SEQ ID 18) Olfr743 (SEQ ID NO: 6) and Olfr740 (SEQ ID NO: 2) cDNA sequences were co-transfected with Gαolf. (SEQ ID NO: 21) and exposed to increasing concentrations of indole (left) or skatole (right). FIGS. 5C and 5D are repeat experiments showing similar activity of Olfr740 (SEQ ID NO: 2) with Olfr743 (SEQ ID NO: 6) with indole and skatole. In FIG. 5E, the FLAG Rho-tagged (SEQ ID 18) Olfr746 (SEQ ID NO: 38) cDNA sequence was co-transfected with Gαolf. (SEQ ID NO: 21) and exposed to increasing concentrations of indole or skatole. Odorant-induced activity was detected by measuring cAMP levels in the cytosol using the HTRF approach (CisBio kit). A dose-dependent increase of receptor activity is shown for all three receptors to both molecules. Co-transfection of HEK cells with Gαolf(SEQ ID NO: 21) alone (no receptor) were used as controls for non-specific activity (control). Activity is defined as the baseline corrected HTRF ratios normalized to the highest signal in the experiment. The results are displayed in FIGS. 5A to 5E. All three receptors show specific dose dependent activity to the malodor compounds and, as such, validate the process for identifying receptors for these compounds. Mouse receptors Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) and Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6) were identified from isolated olfactory neurons that responded to both indole and skatole. Mouse receptor Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) was identified in silico by database mining using Olfr740 (SEQ ID NO: 2) and Olfr743 (SEQ ID NO: 6) as query sequences. The receptors are modified at their N-terminus with the FLAG tag and the first 20 amino acids of the bovine rhodopsin receptor (SEQ ID 18), transiently expressed in HEK 293T cells, and stimulated separately with indole and skatole to confirm their identity as bona fide indole/skatole receptors. Co-expression of the human G alpha subunit Gαolf(SEQ ID NO: 21) activates the Gs transduction pathway that leads to an internal cAMP increase upon binding to the appropriate ligand. The results confirm the identity of Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6), and Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) as indole-skatole receptors.

Example 5 Indole and Skatole Activity of Human OR52N2, OR11G2, OR5AC2, OR4C15, OR8S1, OR11H6, and OR11H4 and Mouse Olfr665 and Olfr740 in HEK293T Cells.

The activity of candidate human indole and skatole malodors receptors OR52N2 (SEQ ID NO: 11 and SEQ ID NO: 12), OR11G2 (SEQ ID NO: 13 and SEQ ID NO: 14, OR5AC2 (SEQ ID NO: 75 and SEQ ID NO: 76), OR4C15 (SEQ ID NO: 79 and SEQ ID NO: 80), OR8S1 (SEQ ID NO: 83 and SEQ ID NO: 84), OR11H6 (SEQ ID NO: 15 and SEQ ID NO: 16) and OR11H4 (SEQ ID NO: 49 and SEQ ID NO: 50) and candidate mouse indole and skatole malodors receptors Olfr665 (SEQ ID NO: 9 and SEQ ID NO 10) and Olfr740 (SEQ ID NO: 1 and SEQ ID NO 2) were confirmed in cell-based calcium-flux assays (FIGS. 6A to 6J). Cells stably co-expressing Rho-tagged human OR52N2 (FIG. 6A, B) or OR11G2 (FIG. 6C) and Gα15 (SEQ ID NO: 25) were exposed to increasing concentrations of skatole or indole. FIG. 6B is a repeat experiment showing similar activity of OR52N2 with indole and skatole. Odorant-induced OR52N2 or OR11G2 activity was detected by measuring the maximum Calcium 5 dye (Molecular Devices) fluorescence change following odorant exposure. Activity is defined as the baseline corrected Relative Fluorescent Units (RFU) ratios normalized to the highest RFU in the experiment in FIG. 6A. Relative Fluorescent Units is used to measure the receptor activity in FIGS. 6B-J. A dose-dependent increase of receptor activity was recorded and a corresponding dose-response curve is shown for both compounds. A cell line lacking a receptor, for example hOR52N2, was used as a control for non-specific activity at high concentrations (‘Indole control’ and ‘Skatole control’). The human receptor OR52N2 (SEQ ID NO: 11 and SEQ ID NO: 12) was identified because of its sequence similarity to mouse Olfr665 (SEQ ID NO: 10), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole. The human receptor OR11G2 (SEQ ID NO: 13 and SEQ ID NO: 14) was identified because of its sequence similarity to mouse Olfr740 (SEQ ID NO: 2), an indole/skatole receptor isolated from olfactory neurons responding to both indole and skatole. The receptors were modified with the Rho sequence and stably expressed in HEK 293T cells. Further examples show indole and skatole dose-response curves for human receptors OR5AC2, OR4C15, OR8S1, OR11H6, OR11H4 and mouse receptors Olfr665 and Olfr740 (FIG. 6D-J). The human receptor OR5AC2 (SEQ ID NO: 75 and SEQ ID NO: 76) was identified because of its sequence similarity to mouse Olfr207 (SEQ ID NO: 78), a receptor isolated from olfactory neurons responding to both indole and skatole. The human receptor OR4C15 (SEQ ID NO: 79 and SEQ ID NO: 80) was identified because of its sequence similarity to mouse Olfr1211 (SEQ ID NO: 82), a receptor isolated from olfactory neurons responding to both indole and skatole. The human receptor OR8S1 (SEQ ID NO: 83 and SEQ ID NO: 84) was identified because of its sequence similarity to mouse Olfr257 (SEQ ID NO: 86), a receptor isolated from olfactory neurons responding to both indole and skatole. The human receptor OR11H6 (SEQ ID NO: 15 and SEQ ID NO: 16) was identified because of its sequence similarity to mouse Olfr745 (SEQ ID NO: 8), a receptor isolated from olfactory neurons responding to both indole and skatole. The human receptor OR11H4 (SEQ ID NO: 49 and SEQ ID NO: 50) was identified because of its sequence similarity to mouse Olfr746 (SEQ ID NO: 38), a receptor isolated from olfactory neurons responding to both indole and skatole. The mouse receptor Olfr665 (SEQ ID NO: 9 and SEQ ID NO: 10) was identified directly from NGS data from isolated olfactory neurons responding to both indole and skatole. The mouse receptor Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) was identified directly from NGS data from isolated olfactory neurons responding to both indole and skatole. Co-expression of the human G alpha subunit Gα15 activates the Gq transduction pathway that leads to an internal Ca2+ increase upon binding to the appropriate ligand. These results serve to validate the process disclosed here is useful for the rapid and reliable identification of mammalian odorant receptors for malodor compounds.

Example 6 Inhibition of Indole Receptor Olfr743 Activity in HEK293T Cells.

Once identified using the procedure outlined herein, the resulting malodor receptor cell lines can be used to screen chemical libraries for compounds that modulate their activity and possibly human perception. This experiment was performed under the same conditions as in Example 4. Cells transfected with Rho-tagged (SEQ ID NO: 18) Olfr743 (SEQ ID NO: 6) were exposed to increasing concentrations of indole in the presence or absence of 300 μM 2-actetonaphthone. 2-acetonaphthone inhibits the activity of Olfr743 (SEQ ID NO: 6) to indole. The results are presented in Table 2 and FIG. 7. In the presence of the antagonist compound, a rightward shift of in the dose-response curve is observed resulting in a 4 fold increase in the EC50 to indole. Activity is defined as the baseline corrected HTRF ratios normalized to the highest signal in the experiment. These results show that 2-acetonaphthone binds and inhibits the activity of the indole receptor.

TABLE 2 Indole + 2- Indole acetonaphthone 300 μM EC50 54 μM 206 μM

Claims

1. A method of identifying olfactory receptors that are activated by an odor and/or aroma compound comprising:

a) dissociating an isolated olfactory epithelium containing native olfactory neurons into a single cell from a non-human mammal species wherein each neuron expresses the olfactory receptor;
b) loading the olfactory cells with an indicator dye that allows for the measurement of malodor receptor binding activity of the olfactory receptors;
c) contacting the olfactory receptors with malodor compounds;
d) measuring changes of odorant-induced neuronal activity;
e) isolating one or more olfactory neurons that were activated by the malodor compound;
f) isolating or isolating and amplifying the mRNA of the isolated olfactory receptors;
g) sequencing at least a portion of the entire transcriptome of the mRNA by Next-Generation Sequencing; and
h) identifying a group of malodor olfactory receptors by comparing the sequence of the transcriptome to a reference genome sequence of the same species.

2. The method of claim 1, further comprising identifying a human receptor that is most closely related to a malodor olfactory receptor identified in claim 1.

3. The method of claim 1, further comprising modifying a cell line to express at least one malodor olfactory receptor identified in claim 1.

4. The method of claim 2, further comprising modifying a cell line to express at least one human receptor identified in claim 2.

5. The method of claim 3, further comprising contacting the malodor olfactory receptor with a test compound to determine if the test compound binds to the malodor olfactory receptor.

6. The method of claim 4, further comprising contacting the human receptor with a test compound to determine if the test compound binds to the receptor.

7. An expression vector comprising

a) a nucleic acid comprising a nucleotide sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 85; or
b) a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86.

8. A cell that is recombinantly modified to express an olfactory receptor polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86; or that comprises the expression vector of claim 7.

9. The cell of claim 7, wherein the cell is selected from the group consisting of HEK293, CHO, Xenopus oocytes, COS, yeast, bacteria and cells derived from the olfactory placode.

10. A method for identifying a compound that blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor selected from the group consisting of odorant or aroma receptors that is activated by a compound comprising indole and/or skatole comprising

a) culturing at least one cell of claim 8 under conditions conducive to expressing at least one olfactory receptor;
b) contacting a compound with the at least one receptor;
c) measuring the extent to which the compound blocks, inhibits, modulates, or enhances the activity of the receptor by measuring the response of the olfactory receptor in the presence and absence of the compound.

11. The method of claim 10, further comprising identifying a compound that blocks, inhibits, modulates, or enhances the response of the olfactory receptor on the basis of the response that was measured in the presence and absence of the compound; and selecting the identified compound as a compound that blocks, inhibits, modulates, or enhances the response of the olfactory receptor.

12. The method of claim 10, wherein the olfactory receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86.

Patent History
Publication number: 20180208637
Type: Application
Filed: Jan 22, 2018
Publication Date: Jul 26, 2018
Applicant: Firmenich SA (Geneva 8)
Inventors: Patrick PFISTER (Brooklyn, NY), Matthew ROGERS (Briarcliff Manor, NY), Khalid Jerod PARRIS (Brooklyn, NY)
Application Number: 15/877,215
Classifications
International Classification: C07K 14/705 (20060101); C12Q 1/6809 (20060101); G01N 33/50 (20060101);