(19)
(11)EP 3 726 218 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
21.10.2020 Bulletin 2020/43

(21)Application number: 20160003.8

(22)Date of filing:  09.06.2016
(51)International Patent Classification (IPC): 
G01N 33/566(2006.01)
C07K 14/72(2006.01)
C12N 9/22(2006.01)
C07K 14/435(2006.01)
C07K 14/47(2006.01)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 10.06.2015 US 201562173762 P
21.09.2015 US 201562221580 P

(62)Application number of the earlier application in accordance with Art. 76 EPC:
16731742.9 / 3308168

(71)Applicant: FIRMENICH SA
1242 Satigny (CH)

(72)Inventors:
  • JEONG, Hyo-Young
    Woodside, NY 11377 (US)
  • PFISTER, Patrick
    New York, NY 10009 (US)
  • ROGERS, Matthew
    New York, NY 10025 (US)

(74)Representative: Reitstötter Kinzebach 
Patentanwälte Sternwartstrasse 4
81679 München
81679 München (DE)

 
Remarks:
This application was filed on 28-02-2020 as a divisional application to the application mentioned under INID code 62.
 


(54)CELL LINES FOR SCREENING ODORANT AND AROMA RECEPTORS


(57) Provided herein is a cell line with improved odorant receptor function comprising an activated endogenous RTP1 gene, which further expresses an RTP1 protein. Further provided herein is a method for specifically activating an endogenous RTP1 gene in a eukaryotic cell using a CRISPR/Cas9 derived technique. Also provided herein is a method for identifying compounds with desired effects such as perfume or aroma modulators in said cell line.




Description

Field



[0001] The technical field is directed to odorant and aroma receptors and assays that can be used to identify odorant and/or aroma compounds or modulators of such. The assays are more specifically directed to engineered cell lines that exhibit improved odorant receptor activity.

Background



[0002] Odors are initially encoded in the peripheral olfactory system (i.e. the nose) through interactions between volatile flavor and fragrance compounds and odorant receptor (OR) proteins that reside on the membranes of olfactory receptor neurons of olfactory epithelia tissue. Such interactions occur in an odorant-specific combinatorial manner where any single OR may be activated by multiple odorants, and conversely most odorants are capable of activating several different ORs. For a given odorant/aroma compound, or mixture, these receptor interactions generate neurophysiological signals in the brain and ultimately give rise to conscious odor perception. Approximately ∼400 ORs genes in the human genome can be activated by thousands or more odorant stimuli and it is the inherent complexity of the combinatorial interactions between odorants and receptors that allows for the breadth of olfactory sensations we can perceive. Elucidating the these interactions can lead to the discovery of beneficial products including, but not limited to, 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.

[0003] There is a need for example, but not limited to, new methods that can functionally express ORs on the cell surface for reliable decoding of the OR codes. There is a further need for a method that functionally expresses the ORs in non-olfactory cells (for example, but not limited to, heterologous cell lines) that are amenable to high-throughput screens with libraries of volatile flavor and fragrance compounds for comprehensive characterization of OR activity. This could significantly expedite the discovery of highly desirable malodour counteractants, odor modulators, and new flavor or fragrance compounds.

[0004] Certain proteins derived from olfactory sensory neurons can improve cell surface localization of odorant receptors in non-olfactory cell lines. These proteins function by assisting in the trafficking of the odorant receptors from the endoplasmic reticulum to the Golgi apparatus and plasma membrane of the cell. Receptor-Transporting Protein 1 (RTP1), Receptor-Transporting Protein 2 (RTP2), and Receptor Expression Enhancing Protein 1 (REEP1) have been reported to improve odorant receptor plasma membrane localization and therefore function in non-olfactory cells. RTP1 has been reported to be the most effective odorant receptor chaperone. This protein has been shown to act, in part, by interacting with odorant receptors in the endoplasmic reticulum. A non-olfactory cell line that is amenable to high-throughput screening and that contains the RTP1 gene is therefore highly desirable for comprehensive decoding of the combinatorial interactions between odorants and odorant receptors.

[0005] Even more desirable is a cell line that consistently produces the RTP1 protein, including but not limited to RTP1S, by stably expressing the gene from the endogenous RTP1 gene locus. However, current techniques for developing cell lines that stably expresses the endogenous RTP1 gene involve inefficient and cumbersome molecular biology approaches for the insertion of DNA into cultured cells that otherwise do not express the gene. Hence it is desirable to use a technique that avoids such approaches to develop a stable cell line and that allows for the consistent expression of the endogenous RTP1 without the need to use recombinant methods.

[0006] CRISPR/Cas9 is a highly efficient genome editing tool used to generate precise genome modifications such as insertions and deletions. For example, a particular use of CRISPR/Cas9 enables the expression of a gene that may be otherwise silent in a cell line. By incorporating a transcriptional promoter upstream of the gene, a cell line may then express an endogenous gene that is otherwise inactive.

SUMMARY



[0007] A cell comprising an activated endogenous RTP1 gene within the cell wherein the cell further expresses a RTP1 protein.

[0008] Provided herein is a non-olfactory cell line with improved odorant receptor function comprising an activated endogenous RTP1 gene.

[0009] Provided herein is a non-olfactory cell line comprising an activated endogenous RTP1 gene within the cell which further expresses an RTP1 protein.

[0010] Further provided herein is a method for activating an endogenous RTP1 gene in a eukaryotic cell comprising:
  1. a. introducing a guide RNA complementary to a genomic target site upstream of the RTP1 gene;
  2. b. introducing a Cas nuclease protein to make a complex with the guide RNA; and
  3. c. using the guide RNA/Cas9 genome targeting complex to deliver the gene activating elements for the RTP1 gene specifically.


[0011] Also provided herein is a method for identifying a compound that activates, mimics, blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor in a non-olfactory cell wherein the cell comprises an activated endogenous RTP1 gene wherein the method further comprises:
  1. a. contacting the receptor, or a chimera or fragment thereof with a compound that activates, mimics, blocks, inhibit, modulates and/or enhances the receptor and
  2. b. determining whether the compound has an effect on the activity of the receptor.

DESCRIPTION OF THE DRAWINGS



[0012] 

Figure 1 shows a schematic of the endogenous RTP1 gene locus and the specific target site used for genome editing.

Figure 2 shows a schematic of the CMV promoter insertion process.

Figure 3 shows a schematic of the wild type and recombined alleles and the corresponding genotyping regions.

Figure 4 shows the characterization of the CMV promoter integration in the engineered cell line.

Figure 5 shows the characterization of RTP1 mRNA expression in the engineered cell line.

Figure 6 shows the characterization of RTP1 protein expression in the engineered cell line.

Figure 7 shows a mouse receptor Olfr741 dose-response curve in the presence of increasing concentrations of indole.

Figure 8 shows a mouse receptor Olfr742 dose-response curve in the presence of increasing concentrations of indole.

Figure 9 shows a mouse receptor Olfr96 dose-response curve in the presence of increasing concentrations of vulcanolide.

Figure 10 shows a human receptor OR11A1 dose-response curve in the presence of increasing concentrations of vulcanolide.

Figure 11 shows a mouse receptor Olfr740 dose-response curve in the presence of increasing concentrations of indole.

Figure 12 shows a human receptor OR1A1 dose-response curve in the presence of increasing concentrations of carvone-(-).


DETAILED DESCRIPTION



[0013] 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.

[0014] 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."

[0015] In one embodiment, a cell is provided comprising a nucleic acid encoding an odorant receptor.

[0016] In a further embodiment, the cell is provided comprising a nucleic acid encoding an odorant receptor selected from the group consisting of Olfr741, Olfr742, Olfr96, Olfr740, OR1A1, and OR11A1.

[0017] In yet a further embodiment, the cell is provided herein comprises a constitutive promoter upstream of an endogenous RTP1 gene locus such that the promoter drives the expression of the endogenous RTP1 gene.

[0018] In mouse olfactory sensory neurons, the RTP1 transcript contains two alternative translational start sites that can lead to two distinct forms of the RTP1 protein: a long version (RTP1L) and a short version (RTP1S). However, it is the RTP1S protein that is predominantly expressed in the mouse olfactory neurons. Also, non-olfactory cells (for example, but not limited to, HEK293T) heterologously expressing the full RTP1 coding sequence predominantly express RTP1L even though the RTP1S coding sequence is contained within RTP1L. However, RTP1S is preferred for odorant receptor screening in non-olfactory cells since it is known that RTP1S strongly outperforms RTP1L with respect to cell surface OR expression. We have surprisingly found that endogenous activation of the full RTP1 gene leads preferentially to the expression of RTP1S.

[0019] In a further embodiment, a cell is provided herein that comprises a constitutive promoter upstream of an endogenous RTP1 gene locus that drives the expression of the short version of RTP1 gene, RTP1S.

[0020] Provided herein is a non-olfactory cell line comprising an activated endogenous RTP1 gene within the cell which further expresses the short version of an RTP1 protein, RTP1S.

[0021] In yet a further embodiment, the constitutive promoter is selected from the group consisting of CMV, PGK, EF1a and SV40.

[0022] In one embodiment, the promoter is CMV, originating from the Cytomegalovirus.

[0023] In one embodiment, a cell provided herein comprises an inducible promoter upstream of an endogenous RTP1 gene locus such that the promoter drives the expression of the RTP1 gene when the corresponding operator is present.

[0024] In a further embodiment, the inducible promoter is a Tetracycline Response Element (TRE) promoter inducible by administration of tetracycline (or its analogue doxycycline).

[0025] In one embodiment, the Cas nuclease protein is a Cas9 protein.

[0026] In a further embodiment, the Cas9 protein is selected from the group consisting of Cas9, dCAs9 (deactivated Cas9) and Cas9 nickase.

[0027] In one embodiment, the guide RNA (gRNA) sequence may be designed based on state-of-the-art rules (Doench et al., Nat Biotech (2014)) and publicly available guide RNA design tools for efficient genomic targeting (e.g. wwws.blueheronbio.com/external/tools/gRNASrc.jsp). The homologous arms may be used that are 800bp long on each side of the specific Cas9 generated double strand DNA break. It is useful to carefully review the integration site of the CMV promoter to avoid unwanted translational start site before the endogenous RTP1 start site.

[0028] Preferably the original cell line used for CRISPR/Cas9 engineering should be from a mammalian origin and carry the RTP1 gene locus. Such cell lines include, but are not restricted to, HEK293, HEK293T, HeLa, CHO, OP6, HeLa-S3, HEKn, HEKa, PC-3, Calu1, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, BHK, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COS-7, HL-60, LNCap, MCF-7, MCF-10A, MDCK II, SkBr3, Vero cells, immortalized olfactory cells, immortalized taste cells, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (Manassus, Va.).

[0029] Hence, in some embodiments a stable cell line is selected from the group consisting of HEK293, HEK293T, HeLa, CHO, OP6, HeLa-S3, HEKn, HEKa, PC-3, , Calu1, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, BHK, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COS-7, HL-60, LNCap, MCF-7, MCF-10A, MDCK II, SkBr3, Vero cells, immortalized olfactory cells, immortalized taste cells, and transgenic varieties thereof.

[0030] In yet a further embodiment, the complex allows for the cleavage of the target nucleic acid sequence adjacent to the guide RNA sequence and Cas9 protein complex delivered to the cell and wherein the method further comprises introducing a donor DNA comprising a CMV promoter into the cell.

[0031] In one embodiment, the guide RNA sequence and Cas9 protein complex allows for the cleavage of the target nucleic acid sequence adjacent to the guide RNA sequence and Cas9 protein complex and wherein the method further comprises introducing a donor DNA comprising a CMV promoter through cellular homology directed repair mechanism at the cleavage site.

[0032] In a further embodiment is a cell line modified to stably express the endogenous RTP1 gene under the CMV promoter.

[0033] In a further embodiment a donor DNA may also comprise an antibiotic selection cassette (e.g., containing the puromycin resistance gene). Cultivating cells in antibiotic containing culture media after DNA delivery can be beneficial as it eliminates cells that did not undergo proper DNA integration and thus allows one to efficiently select recombined clonal cell populations that acquired a resistance marker hence the desired integration of the donor DNA (e.g. the constitutive promoter CMV). Such antibiotic resistance gene can subsequently be removed by engineering it with flanking "frt" sites that are specifically recognized by the Flippase enzyme. This cassette is then removed by delivering said enzyme to the cells.

[0034] In one embodiment the complex works by targeting specific locations in the genome and further recruiting transcription factors that activate downstream endogenous genes such as RTP1 without the need for cleaving the DNA. This is done by fusing the Cas9 protein to a transcription activation domain to form a complex wherein the complex is not capable of cleaving the target nucleic acid sequence. The method provides guide RNA directed DNA targeting (i.e. upstream of the RTP1 gene) of the Cas9 fused transcription activation domain. Instead of cleaving the DNA to allow for a promoter integration, it transiently binds and recruits transcription factors that activate the gene without the need for engineering or modifying the genome that is, the d. This may be done through the use of a deactivated version of Cas9 called dCas9, fused to specific transcription factor recruiting elements such as VP64, VPR and SAM, or through the use of a modified guide RNA (e.g. a truncated guide RNA). This fusion protein does not cleave the nucleic acid.

[0035] In one embodiment provided herein is a method comprising introducing a nucleic acid encoding an olfactory receptor into the cell.

[0036] In a further embodiment provided herein is a method for identifying a compound that activates, mimics, blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor in a non-olfactory cell wherein the cell comprises an activated endogenous RTP1 gene wherein the method further comprises:
  1. a. contacting the receptor, or a chimera or fragment thereof with a compound that activates, mimics, blocks, inhibit, modulates and/or enhances the receptor and
  2. b. determining whether the compound has an effect on the activity of the receptor.


[0037] In one embodiment, the olfactory receptor is from the group consisting of a musk and a malodor receptor.

[0038] In a particular embodiment, the malodor receptor is selected from a skatole or indole receptor.

[0039] In a particular embodiment the musk receptor is selected from a polycyclic musk and a nitromusk receptor.

[0040] In one embodiment nucleic acids encoding an odorant receptor is introduced in the substantial absence of a Golf protein.

[0041] In one embodiment, the follow steps are carried out:
  1. 1. editing a cell line genome using CRISPR/Cas9 technology, including: (1) designing a DNA encoding a 'guide RNA' specific to the desired genomic DNA integration site located near the endogenous RTP1 genomic locus; and (2) a 'donor DNA' to be integrated into the genomic locus that comprises a constitutively active transcriptional promoter;
  2. 2. introducing the DNAs engineered in step 1 into a mammalian cell line;
  3. 3. selecting a cell line that has integrated the donor DNA into the desired genomic locus and that produces RTP1 mRNA via activation of the endogenous RTP1 gene.
  4. 4. introducing an odorant receptor DNA sequence into the selected cell line.
  5. 5. contacting a receptor, or chimera or fragment with a compound and assay whether the compound has an effect on the activity of the odorant receptor.


[0042] The methods provided herein allow for the use of cell lines to discover ingredients such as odor enhancers and blockers for cosmetic and industrial use (e.g. perfumes, perfumer enhancers, flavour enhancers, home and body deodorants). New ingredients may provide more favourable fragrant, toxicity and biodegradation profiles and/or exhibit greater potency.

[0043] Accordingly, a compound or mixture of compounds that that activate, mimic, block, inhibit, modulate, and/or enhance the activity of an olfactory receptor in a non-odorant cell obtained by any one of the methods disclosed herein.

[0044] In one embodiment, enhanced functional odorant receptor expression in heterologous expression systems is provided using CRISPR/Cas9 to specifically and constitutively activate the RTP1 gene which is silent (inactive) in regular HEK293T cells.

DEFINITIONS



[0045] The following terms have the meanings ascribed to them unless specified otherwise.

[0046] "Endogenous gene" refers to a gene that originates from within an organism, tissue, or cell.

[0047] The phrase "functional effects" 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, but not limited to, 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+), in vitro, in vivo, and ex vivo and also includes other physiologic effects such increases or decreases of neurotransmitter or hormone release.

[0048] The phrase "determining whether the compound has an effect on the activity" in the context of assays is meant 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 effects can be measured by any means known to those skilled in the art, e.g., but not limited to, changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or 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.

[0049] 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, plan 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. 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.

[0050] 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.

[0051] "Inhibitors," "activators," "counteractants" and "modulators" of OR genes or proteins are used interchangeably to refer to inhibitory, activating, or modulating molecules identified using in vivo, in vitro and in vivo assays for olfactory transduction, e.g., ligands, agonists, antagonists, enhancers, and their homologs and mimetics. Inhibitors 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 inhibitor (e.g., odorant-binding proteins 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. Musks 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.

[0052] The "N terminal domain" region starts at the N-terminus and extends to a region close to the start of the first transmembrane region. "Transmembrane domain," which comprises the seven "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.

[0053] 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) 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.

[0054] Odorant Receptor or "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.

[0055] Odorant Receptor or "OR" nucleic acids encode a family of G-protein coupled receptors 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.

[0056] "OR" polypeptides are considered as such if they pertain to the 7-transmembrane-domain G protein-coupled receptor superfamily encoded by a single ∼1kb 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].

[0057] 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).

[0058] A "promoter" is defined as an array of nucleic acid sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

[0059] 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.

[0060] Nucleic acid and amino acid sequences identified and/or used herein are listed below:

Guide RNA target sequence (SEQ ID NO: 1 DNA)
SEQ ID NO: 1
ctgcaatctcagttcagggcc

Donor DNA FR homology directed repair (SEQ ID NO: 2 DNA)



Mouse Olfr741 (SEQ ID NO: 3 DNA; SEQ ID NO: 4 PROTEIN)





Mouse Olfr742 (SEQ ID NO: 5 DNA; SEQ ID NO: 6 PROTEIN)





Mouse Olfr96 (SEQ ID NO: 7 DNA; SEQ ID NO: 8 PROTEIN)



Human OR11a1 (SEQ ID NO: 9 DNA; SEQ ID NO: 10 PROTEIN)





Mouse Olfr740 (SEQ ID NO: 11 DNA; SEQ ID NO: 12 PROTEIN)



Human OR1a1 (SEQ ID NO: 13 DNA; SEQ ID NO: 14 PROTEIN)





Flag tag (SEQ ID NO: 15 DNA; SEQ ID NO: 16 PROTEIN)

SEQ ID NO: 15
gattacaaggacgacgacgataag

SEQ ID NO: 16
dykddddk

Rho tag (SEQ ID NO: 17 DNA; SEQ ID NO: 18 PROTEIN)

SEQ ID NO: 17
atgaacgggaccgagggcccaaacttctacgtgcctttctccaacaagacgggcgtggtg

SEQ ID NO: 18
mngtegpnfyvpfsnktgvv

Lucy tag (SEQ ID NO: 19 DNA; SEQ ID NO: 20 PROTEIN)

SEQ ID NO: 19
atgagaccccagatcctgctgctcctggccctgctgaccctaggcctggct

SEQ ID NO: 20
mrpqillllalltlgla


EXAMPLES



[0061] The below examples are illustrative only and are not meant to limit the claims or embodiments described herein.

Example 1


Genome editing strategy to induce constitutive activation of the endogenous RTP1 gene in HEK293T cells.



[0062] A strategy to develop enhanced functional odorant receptor expression in heterologous expression system is described. Taking advantage of a newly available genome editing technology called CRISPR/Cas9, an endogenous RTP1 gene which is silent (inactive) in regular HEK293T cells, is specifically and constitutively activated by introducing a constitutive promoter (CMV) upstream of its coding sequence. Figure 1) The RTP1 gene is located on chromosome 3 and the DNA sequence around its start site is shown. The Cas9 endonuclease is directed by a 20 base pair (bp) guide RNA (gRNA) homologous to the target. Upon delivery to the cells (GeneArt CRISPR Nuclease (CD4 enrichment) Vector Kit, cat# A21175) the guide RNA molecule and the Cas9 protein form an active complex that induces the desired double strand DNA break (DSB) upstream of the coding sequence. Boxes indicate the RTP1 gene on chromosome 3 (filled box, coding sequence (CDS); open box, untranslated region (UTR) in exon). The putative promoter region upstream of the RTP1 gene is inactive in HEK293T cells. The guide RNA target sequence (SEQ ID NO: 1) between position -150 and - 131 and a Protospacer Adjacent Motif (PAM) from -153 to -151 from the start codon (ATG) respectively are shown. Figure 1) DSB site for Cas9 nuclease 3bp away from the PAM motif, specified by a triangle, allows a donor DNA (SEQ ID NO: 2) to be inserted. Figure 2) A schematic of the CMV promoter insertion process is shown. The top configuration shows the RTP1 gene locus before modification and the bottom schematic shows the RTP1 locus after a donor DNA is targeted into the DSB site by Homology Directed Repair (HDR). The donor DNA is composed of a 5' homology arm, an FRT (Flippase Recognition Target) -flanked puromycin selection cassette, the CMV promoter and a 3' homology arm. The integration of the CMV promoter upstream of the RTP1 gene is then obtained by the cellular HDR mechanism inherent to eukaryotic cells. A donor plasmid containing two DNA stretches homologous to the sequences on either side of the desired entry point, flanking the puromycin resistance selection cassette (Puror) and the CMV DNA, is co-transfected into HEK293T cells. HDR results in the introduction of Puror and CMV upstream of the RTP1 coding sequence. Puromycin selection cassette can be subsequently removed by the use of the Flippase enzyme.

Example 2


Selection of a modified cell line endogenously expressing the RTP1 gene.



[0063] Several control steps help to characterize the modification of the cell line and its integrity. Figure 3) A schematic of the wild type and recombined alleles is shown. Grey lines indicate relative amplicon positions of PCR and RT-PCR experimental results for DNA genotyping and for RNA expression controls, respectively (not to scale). Figure 4) The genomic DNA from the Puromycin resistant cell line is extracted and a PCR is performed that discriminates between non-recombined wild type (WT) and modified cell lines (Mod.). PCR 1 amplifies a 2.0 kb band only in wild type HEK293T cells but not in a modified cell line. The modified line should yield a 4.0 kb band with PCR1 but did not likely because of the length and the complexity of the genomic structure. Genotyping results for the modified cell line failed to produce the 2.0 kb band indicating a homozygous integration of the CMV promoter. Proper integration of the donor DNA was further tested with PCR 2 and 3, as indicated. Figure 5) After mRNA extraction and cDNA synthesis, an RT-PCR experiment is performed to demonstrate that RTP1 mRNA is specifically expressed in the modified cell line but not in original HEK293T cells. This confirms that the CMV promoter that was integrated at the targeted genomic locus properly drives the expression of the RTP1 gene. The specificity of the RT-PCR bands was confirmed by direct sequencing of the amplified bands. Reverse-transcriptase negative (RT-) and GAPDH PCR conditions indicate the absence of contaminating genomic DNA and the presence of cDNA in all samples, respectively.

Example 3


Characterization of the RTP1 protein expression.



[0064] RTP1 protein expression in the selected modified cell line was determined by western blot analysis using an RTP1-specific antibody. A long (RTP1L) and a short (RTP1S) protein form can originate from the endogenous RTP1 gene. The genome modification strategy described herein involved the introduction of the CMV promoter upstream of the RTP1L start codon to avoid any modification of the endogenous coding sequence; hence RTP1L was expected to be expressed. However the results indicate that the modified cell line was heavily biased towards the expression of RTP1S, suggesting that the endogenous RTP1 gene preferentially expresses the short version without further genome editing. The latter is the preferred version as it is known to better promote odorant receptor cell surface expression. Figure 6) shows the western blots of RTP1 protein. Arrow heads indicate the expected protein sizes for RTP1S - 25 kDa, RTP1L - 28 kDa and the control protein β-actin - 42 kDa. The absence of RTP1 protein in a wild type HEK293T cell line (WT) and the presence of RTP1S in the modified cell line (Mod.) are shown. Surprisingly a much stronger band for RTP1S compared to RTP1L can be seen. Membrane protein extraction was prepared according to the Mem-Per Plus membrane protein extraction kit (Pierce, cat# 89842). Chameleon Duo Pre-stained used as size marker (LiCor, cat#92860000). Labelling was performed with the following primary antibodies: Rabbit anti-RTPl (Invitrogen, cat#PA5-24028) and mouse anti-β-actin (Pierce, cat#PIMA515739). Detection was performed with the following secondary antibodies: goat anti-rabbit (LiCor cat#925-32211) and goat anti-mouse (LiCor cat#925-68070). Imaging was performed on an Odyssey CLx (LiCor).

Example 4


Functional characterization of several odorant receptors in the modified cell line.



[0065] Functional dose-response experiments were performed in order to evaluate the level of functional enhancement of activity of odorant receptors in the modified cell line. Odorant receptors were modified at their N-terminus with short polypeptide sequences or tags [e.g. Flag (SEQ ID NO: 15), Rho (SEQ ID NO: 17; 20 first amino acids of the bovine rhodopsin receptor), or Lucy (SEQ ID NO: 19)], transiently expressed in WT or modified HEK293T cells, and stimulated with odorant compounds to determine the activity of the receptors. Figure 7 and 8) Using a cell-based odorant binding assay, the activity of Olfr741 (SEQ ID NO: 4) and Olfr742 (SEQ ID NO: 6) to indole was tested in the engineered RTP1 cell line and compared to HEK293T lacking RTP1 protein expression. Odorant receptors were transfected into both cell lines and exposed to increasing concentrations of indole. Odorant-induced activity was detected by measuring the level of cAMP increase in the cytosol using an HTRF based kit (CisBio, cAMP dynamic 2 kit, cat# 62AM4PEJ). Figure 9 and 10) Using the same cell-based odorant binding assay, the activity of Olfr96 (SEQ ID NO: 8) and OR11A1 (SEQ ID NO: 10) to vulcanolide was tested in the engineered RTP1 expressing cell line and compared to HEK293T lacking RTP1 protein expression. The activity of Olfr740 (SEQ ID NO: 12) to indole was also tested in both cellular backgrounds. A dose-dependent increase of receptor activity is recorded for all ORs in the modified RTP1 cell line and not in the unmodified control cell line lacking RTP1 expression. Furthermore, the activity of OR1A1 (SEQ ID NO: 14) to carvone-(-) was tested in both cellular backgrounds. Even though OR1A1 can be expressed in regular HEK293T, a more potent dose-dependent increase of receptor activity is recorded in the modified RTP1 cell line and compared to the unmodified control cell line lacking RTP1 expression.

[0066] Further embodiments:
  1. 1. A cell comprising an activated endogenous RTP1 gene within the cell wherein the cell further expresses an RTP1 protein.
  2. 2. The cell as recited in embodiment1 wherein the cell is a non-olfactory cell.
  3. 3. The cell as recited in embodiment2 wherein the cell is derived from a HEK293T cell line.
  4. 4. The cell as recited in any one of embodiments 1-3 further comprising a nucleic acid encoding an odorant receptor.
  5. 5. The cell as recited in embodiment4 wherein the odorant receptor is selected from the group consisting of indole, skatole, and musk odorant receptors.
  6. 6. The cell as recited in any one of embodiments 1-5 comprising a promoter up stream of an endogenous RTP1 gene locus and wherein the RTP1 gene is expressed in the cell.
  7. 7. The cell as recited in embodiment6 wherein the promoter is a CMV promoter.
  8. 8. The cell as recited in any one of embodiments 1-7 comprising a Cas protein.
  9. 9. The cell as recited in any one of embodiments 1-5 comprising a dCas protein fused to a transcriptional activation domain.
  10. 10. A method for activating an endogenous RTP1 gene in a eukaryotic cell comprising:
    1. a. introducing a guide RNA complimentary to a genomic target site upstream of the RTP1 gene; and
    2. b. Introducing a Cas nuclease protein to make a complex with the guide RNA to form a guide RNA-Cas protein complex.
  11. 11. The method as recited in embodiment10 further comprising introducing a donor DNA comprising a promoter into the genomic target site upstream of an endogenous RTP1 gene locus wherein the promoter drives the expression of the RTP1 gene.
  12. 12. The method as recited in embodiment10 wherein the complex further comprises a dCas9 protein fused to a transcriptional activation domain wherein the activation domain drives the expression of the RTP1 gene.
  13. 13. The method as recited in embodiment1 1 wherein the Cas protein is a Cas9 protein.
  14. 14. The method as recited in embodiment13 wherein the Cas9 protein is selected from the group consisting of Cas9 and Cas9 nickase.
  15. 15. The method as recited in embodiment 14 wherein the Cas protein is Cas9.
  16. 16. The method as recited in embodiment14 wherein the Cas protein is Cas9 nickase.
  17. 17. The method as recited in embodiment1 1 further comprising cleaving the target nucleic acid sequence adjacent to the guide RNA sequence with the guide RNA-Cas protein complex.
  18. 18. The method as recited in any one of embodiments 10-17 further comprising introducing a nucleic acid encoding an odorant receptor into the cell.
  19. 19. A method for identifying a compound or mixture of compounds that activates, mimics, blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor in a non-odorant cell wherein the cell comprises an activated endogenous RTP1 gene wherein the method further comprises:
    1. a. contacting the receptor, or a chimera or fragment thereof with a compound or mixture of compounds that activates, mimics, blocks, inhibit, modulates and/or enhances the receptor; and
    2. b. determining whether the compound has an effect on the activity of the receptor.
  20. 20. The method as recited in embodiment 19 wherein the olfactory receptor is from the group consisting of a musk and a malodor receptor.
  21. 21. The method as recited in embodiment 19 wherein the malodor receptor is selected from skatole or indole receptor.
  22. 22. The method as recited in embodiment19 wherein the musk receptor is selected from a polycyclic musk and a nitro musk receptor.
  23. 23. A compound or mixture of compounds that that activate, mimic, block, inhibit, modulate, and/or enhance the activity of an olfactory receptor in a non-odorant cell obtained by the method of any one of embodiments 19 to 22.



































Claims

1. A cell comprising an activated endogenous RTP1 gene within the cell wherein the cell further expresses an RTP1 protein.
 
2. The cell as recited in claim 1 wherein the cell is a non-olfactory cell.
 
3. The cell as recited in any one of claims 1 and 2 further comprising a nucleic acid encoding an odorant receptor, in particular an odorant receptor selected from the group consisting of indole, skatole, and musk odorant receptors
 
4. The cell as recited in any one of claims 1-3 comprising a promoter up stream of an endogenous RTP1 gene locus and wherein the RTP1 gene is expressed in the cell.
 
5. The cell as recited in claim 4 wherein the promoter is a CMV promoter.
 
6. The cell as recited in any one of claims 1-5 comprising a Cas protein.
 
7. The cell as recited in any one of claims 1-3 comprising a dCas protein fused to a transcriptional activation domain.
 
8. A method for activating an endogenous RTP1 gene in a eukaryotic cell comprising:

a. introducing a guide RNA complimentary to a genomic target site upstream of the RTP1 gene; and

b. Introducing a Cas nuclease protein to make a complex with the guide RNA to form a guide RNA-Cas protein complex.


 
9. The method as recited in claim 8 further comprising introducing a donor DNA comprising a promoter into the genomic target site upstream of an endogenous RTP1 gene locus wherein the promoter drives the expression of the RTP1 gene.
 
10. The method as recited in claim 8 wherein the complex further comprises a dCas9 protein fused to a transcriptional activation domain wherein the activation domain drives the expression of the RTP1 gene.
 
11. The method as recited in claim 10 wherein the Cas protein is a Cas9 protein.
 
12. The method as recited in claim 11 wherein the Cas9 protein is selected from the group consisting of Cas9 and Cas9 nickase.
 
13. The method as recited in claim 9 further comprising cleaving the target nucleic acid sequence adjacent to the guide RNA sequence with the guide RNA-Cas protein complex.
 
14. The method as recited in any one of claims 8-13 further comprising introducing a nucleic acid encoding an odorant receptor into the cell.
 
15. A method for identifying a compound or mixture of compounds that activates, mimics, blocks, inhibits, modulates, and/or enhances the activity of an olfactory receptor in a non-odorant cell wherein the cell comprises an activated endogenous RTP1 gene wherein the method further comprises:

a. contacting the receptor, or a chimera or fragment thereof with a compound or mixture of compounds that activates, mimics, blocks, inhibit, modulates and/or enhances the receptor; and

b. determining whether the compound has an effect on the activity of the receptor.


 
16. The method as recited in claim 15 wherein the olfactory receptor is from the group consisting of a musk and a malodor receptor and wherein in particular the malodor receptor is selected from skatole or indole receptor; and/or wherein in particular the musk receptor is selected from a polycyclic musk and a nitro musk receptor.
 
17. A compound or mixture of compounds that that activate, mimic, block, inhibit, modulate, and/or enhance the activity of an olfactory receptor in a non-odorant cell obtained by the method of any one of claims 15 and 16.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description