Genetically modified cells and methods for improving the expression and functional response of chemosensory receptors.

Abstract

Genetically modified cells (e.g., recombinant mammalian cells) and methods for improving the expression / functional response of olfactory receptors are described herein. Genetically modified cells and methods can generally modulate the levels of chaperone proteins that help target receptors to the cell membrane. Genetically modified cells and methods can also provide a platform for use in studying the function of a broader group of chemosensory receptors, including taste receptors and TRP channels.

Description

Technical Field

[0001] Reference to Electronic Sequence Listing The content of the electronic sequence listing (AROM_007_P01US_SeqList_ST26.xml; size: 35,936 bytes; creation date: June 12, 2023) is hereby incorporated by reference in its entirety.

[0002] Genetically engineered cells and methods for improving the expression and functional response of olfactory receptors are described herein. The genetically engineered cell lines and methods can regulate the level of chaperone proteins in culture, which, if unregulated, can interfere with receptor targeting to the cell membrane. The genetically engineered cells and methods can also provide a platform for use in studying the functions of a more diverse set of chemoreceptors.

Background Art

[0003] Odorant molecules can be detected by chemoreceptors such as olfactory receptors. Odor detection is mediated by olfactory receptors. Olfactory receptors are expressed in the olfactory epithelium and constitute the largest multigene family in the human genome. Approximately 400 different olfactory receptors are expressed in the human nose, and their combined function enables the detection and discrimination of thousands of different odors. Olfactory receptors and other chemoreceptors (e.g., taste receptors and TRP channels) generally follow a pathway where they are first synthesized in the cytoplasm, move to the endoplasmic reticulum (ER), and after undergoing folding and maturation in the ER and Golgi compartments, are trafficked to the plasma membrane of the cell by vesicles. This process is supported by a series of chaperones that assist in protein folding and enzymes that process oligosaccharide chains and covalent bonds.

[0004] While heterologous cell-based assays using chaperones and accessory factors have partially reproduced the combinations of olfactory receptors and their binding properties to diverse odor molecules, large-scale functional studies of olfactory receptors remain limited. The use of currently available experimental cell lines has a significant drawback: these cell lines do not effectively transport receptors, resulting in often insufficient localization of olfactory receptors on the plasma membrane, making functional assays of odor molecules extremely difficult.

[0005] However, it was discovered that a single chaperone protein called RTP1S (RTP Family Members Induce Functional Expression of Mammalian Odorant Receptors, Saito et al., Cell 2004 and Synergism in Accessory Factors in Functional Expression of Mammalian Odorant Receptors, Zhuang and Matsunami, J. Biological Chemistry, 2007) enables robust localization of several mouse olfactory receptors in HEK293 / HEK293T cells (hereinafter referred to as "HEK cells"). This discovery has enabled the use of HEK cells as an expression platform for olfactory receptors with the help of RTP1S, thus facilitating much research on both mouse and human olfactory receptors. Subsequent studies have revealed that RTP1S is expressed in almost all olfactory neurons in mice and humans, and that knocking out RTP1S from the mouse genome significantly impairs the expression and localization of olfactory receptors in vivo (Olfactory Receptor Accessory Proteins Play Crucial Roles in Receptor Function and Gene Choice, Sharma et al. eLife, 2017). However, further research has shown that the RTP1S chaperone protein enables localization in HEK cells only for a fraction of the 400 human olfactory receptors.

[0006] Therefore, having alternative approaches, cell lines, and methods to improve the surface expression and / or functional response of olfactory receptors and other chemosensory receptors would be beneficial. [Overview of the project]

[0007] Described herein are genetically modified cells and methods that can increase the trafficking and / or response (e.g., transport) of various chemosensory receptors to the cell membrane. Chemosensory receptors may be olfactory receptors, gustatory receptors, TRP channels, parts thereof, or combinations thereof. Genetically modified cells and methods can modulate (e.g., reduce) the expression of one or more chaperone proteins.

[0008] Generally, genetically modified cells may exhibit reduced expression of one or more chaperone proteins involved in the folding of chemosensory receptors that are trafficked to the cell membrane surface. By reducing (or inhibiting) the production of chaperone proteins, genetically modified cells may show increased transport of chemosensory receptors to the cell membrane surface compared to control cells.

[0009] The reduction in the expression of one or more chaperone proteins may be achieved by knocking down one or more genes encoding one or more chaperone proteins. Exemplary genes include, but are not limited to, CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1. In some variations, the CANX gene may be a useful gene to knock down.

[0010] Chaperone expression can be reduced using any suitable cell line, but mammalian cell lines are generally preferred. The mammalian cell line may be kidney cells or their hybrid cells. In one modification, the kidney cells may be HEK293 / HEK293T cells.

[0011] A system for increasing the trafficking of chemosensory receptors to the cell membrane surface is also described herein. The system may include a plurality of genetically modified cells and an imaging device or modality configured to detect the presence of chemosensory receptors on the cell membrane surface. In some modifications, the imaging device may be a microscope designed to detect fluorescence.

[0012] Methods for increasing the transport of chemosensory receptors to the cell membrane surface are also described herein. These methods may generally involve expressing chemosensory receptors in cells in which the production of one or more chaperone proteins has been reduced by knockdown of one or more genes encoding one or more chaperone proteins. The production of one or more chaperone proteins may be reduced by about 25% to about 75% (including all values ​​and subranges within the range). For example, the production of one or more chaperone proteins may be reduced by about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%. The reduction in the production of one or more chaperone proteins may be due to knockdown of one or more genes encoding one or more chaperone proteins.

[0013] One or more target chaperone proteins may be selected from the group consisting of CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1. An example of single knockdown targets CANX, and an example of combination knockdown targets CANX + CALR.

[0014] This method allows the use of various cell types. In some variations, the cells may be mammalian cells. Mammalian cells may be kidney cells or their hybrid cells. When kidney cells are used, they may be HEK293 / HEK293T cells.

[0015] Various chemosensory receptors may also be used in this method. For example, chemosensory receptors may be olfactory receptors, gustatory receptors, TRP channels, or parts thereof. Chemosensory receptors may be of mammalian origin and include human and non-human mammalian receptors.

[0016] In some variations of this method, HEK293 / HEK293T cells for use in reducing chaperone protein expression can be prepared by modifying HEK293 / HEK293T cells to knock down one or more genes encoding chaperone proteins. One or more genes may be CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, RPN1, or a combination thereof. One or more genes may be CANX. In some cases, one or more genes may be a combination of CALR and CANX. [Brief explanation of the drawing]

[0017] This patent or application file includes at least one drawing created in color. Copies of the color drawings in this patent or patent application publication will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees.

[0018] [Figure 1] Figure 1 is a graph showing adequate, moderate, or inadequate localization of olfactory receptors based on their expression on the cell membrane of HEK293 / HEK293T cells. The Y-axis represents the percentage of cells showing receptor localization, and each bar along the X-axis represents a different olfactory receptor. Light green represents olfactory receptors with adequate localization, light green represents receptors with moderate localization, and light gray represents receptors with inadequate localization. [Figure 2]Figures 2A and 2B show pie charts illustrating the relative distribution of nine different categories of genes that perform different cellular functions. The green areas represent the proportion of transcripts from protein folding and processing genes among the top 200 most expressed genes. Figure 2A shows the relative distribution of transcript numbers in HEK293 / HEK293T cells, where protein folding and processing categories account for approximately 11.8% of the top transcripts. Figure 2B shows the relative distribution of transcript numbers in human olfactory epithelial cells, where protein folding and processing categories account for approximately 1.3% of the transcripts in human olfactory epithelial cells. [Figure 3] Figures 3A to 3F show microscopic images of Rho-tagged human olfactory receptors expressed in HEK293 / HEK293T cells. Figures 3A to 3C show the localization of olfactory receptors in the presence of calnexin knockdown, while Figures 3D to 3F show the localization of olfactory receptors under conditions without knockdown. [Figure 4] Figures 4A to 4D and Figure 5 show the analysis of the images in Figures 3A to 3F using an image processing program. [Figure 5] Figures 4A to 4D and Figure 5 show the analysis of the images in Figures 3A to 3F using an image processing program. [Figure 6] Figure 6 shows data demonstrating that knockdown of a single or combined protein folding and processing gene improves the surface expression of multiple olfactory receptors. The Y-axis represents the control (no knockdown), a single knockdown target, or a combination of different knockdown targets, while the X-axis represents the human olfactory receptors tested for surface expression improvement under each condition. The scale bars represent the surface localization of olfactory receptors under each condition compared to their knockdown control. [Figure 7] Figures 7A and 7B show the increased functional response of human olfactory receptors following single or combined knockdown of chaperone proteins. [Modes for carrying out the invention]

[0019] Described herein are genetically modified cells that may increase trafficking of various chemosensory receptors by simultaneously regulating (e.g., reducing) the expression of one or more chaperone proteins. The genetically modified cells and methods may also provide a platform for use in studying the functions of a wider variety of chemosensory receptors. The genetically modified cells and methods may be useful for the expression of mammalian olfactory receptors and other mammalian chemosensory receptors. A system comprising multiple genetically modified cells and an imaging device for detecting the presence of chemosensory receptors on the cell membrane surface is also described herein.

[0020] While gene knockdown is described herein, it should be understood that any method or technique may be used to reduce (or inhibit) the expression of one or more chaperone proteins. For example, such methods may include, but are not limited to, siRNA targeting, targeted gene knockout, and knockdown or inhibition of transcription factors. In principle, any molecular biology, cell biology, or method of choice can be used to reduce (or inhibit) the expression levels of a single or combination of chaperone proteins.

[0021] When gene knockdown is used to regulate (e.g., reduce) the expression of chaperone genes compared to their expression in host cells, this regulation can be achieved using various techniques. For example, knockdown of a chaperone gene can be achieved by introducing into the cell a nucleic acid molecule that hybridizes to a portion of the gene's mRNA and causes its degradation (e.g., shRNA, RNAi, miRNA), or by modifying the gene sequence in a way that results in a decrease in transcription, a decrease in mRNA stability, or a decrease in mRNA translation. In one variant, a method of causing knockdown of a chaperone gene includes the step of introducing shRNA into the cell by using transient transfection via lipofectamine. The shRNA can target the mRNA of a human chaperone gene. For example, the shRNA can target the mRNA of human genes including, but not limited to, CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1. In another variant, a modified cell line can be generated in which one or a combination of the above genes can be targeted for knockdown.

[0022] Genetically modified cells Cells modified to reduce the expression or production of chaperone proteins can be any type of cell culture (mammalian or non-mammalian). In some variants, the eukaryotic cells are animal cells. The animal cells can be mammalian cells such as cells of cows, dogs, cats, hamsters, mice, pigs, rabbits, rats, or sheep. In other variants, the animal cells can be cells of primates including, but not limited to, monkeys, chimpanzees, gorillas, and humans.

[0023] More specifically, the cells to be recombined can be fibroblasts, epithelial cells (e.g., kidney, mammary gland, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, and hematopoietic cells. In some variations, the cells are adult cells (e.g., terminally differentiated cells, dividing cells or non-dividing cells) or stem cells. In some variations, the cells can be mammalian cells such as Chinese hamster cells, mouse NIH 3T3 fibroblasts, human kidney cells (e.g., HEK293 / HEK293T cells), or rodent myeloma cells or rodent hybridoma cells. In one variation, the genetically modified cells are HEK293 / HEK293T cells or mutants thereof (e.g., HEK293 / HEK293T cells). The genetically modified cells can be used to generate cell lines for odorant molecule binding assays (e.g., odorant molecule binding platforms), including reduction of the expression of one or more chaperone proteins, as further described in Example 6.

[0024] Chemosensory receptor The chemosensory receptors expressed by the genetically modified cells can include olfactory receptors, taste receptors, or fragments thereof. The trafficking and / or functional responses of these chemosensory receptors can be increased by reducing the production (e.g., expression) of one or more chaperone proteins, as described above. Most olfactory receptors are G protein-coupled receptors (GPCRs), which form a complex with G proteins for signal transduction after the receptor is activated by an odorant molecule. GPCRs generally have seven α-helix transmembrane domains as a conserved characteristic structure. Further, olfactory receptors are typically about 320 ± 25 amino acids long. The difference in length may be due to the diversity of the N-terminal region and the C-terminal region.

[0025] In one variation, the olfactory receptor described herein is a human olfactory receptor. In some variations, the olfactory receptor may be derived from other mammals such as mice, rats, cats, cows and cattle, horses, goats, pigs, dogs, and bears. In other variations, the olfactory receptor may be a hybrid olfactory receptor. Exemplary human olfactory receptors may be derived from one or more of the following 18 olfactory receptor (OR) families: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 51, 52, 55, and 56. Furthermore, one or more olfactory receptors from any one of the OR families can be expressed in the genetically modified cells described herein.

[0026] Family OR1 has 21 members: OR1A, OR1B, OR1C, OR1D, OR1E, OR1F, OR1G, OR1H, OR1I, OR1J, OR1K, OR1L, OR1M, OR1N, OR1P, OR1Q, OR1R, OR1S, OR1X, OR1AA, and OR1AB.

[0027] Family OR2 has 41 members: OR2A, OR2B, OR2C, OR2D, OR2E, OR2F, OR2G, OR2H, OR21, OR2J, OR2K, OR2L, OR2M, OR2N, OR2Q, OR2R, OR2S, OR2T, OR2U, OR2V, OR2W, OR2X, OR2Y, OR2Z, OR2AD, OR2AE, OR2AF, OR2AG, OR2AH, OR2AI, OR2AJ, OR2AK, OR2AL, OR2AM, OR2AO, OR2AP, OR2AS, and OR2AT.

[0028] Family OR3 has three members: OR3A, OR3B, and OR3D.

[0029] Family OR4 has 21 members: OR4A, OR4B, OR4C, OR4D, OR4E, OR4F, OR4G, OR4H, OR4K, OR4L, OR4M, OR4N, OR4P, OR4Q, OR4R, OR4S, OR4T, OR4U, OR4V, OR4W, and OR4X.

[0030] Family OR5 has 49 members: OR5A, OR5B, OR5C, OR5D, OR5E, OR5F, OR5G, OR5H, OR51, OR5J, OR5K, OR5L, OR5M, OR5P, OR5R, OR5S, OR5T, OR5V, OR5W, OR5AC, OR5AH, OR5AK, OR5AL, OR5AM, OR5AN, OR5AF, OR5AP, OR5AQ, OR5AR, OR5AS, OR5AU, OR5W, OR5X, OR5Y, OR5Z, OR5BA, OR5BB, OR5BC, OR5BD, OR5BE, OR5BH, OR5BJ, OR5BK, OR5BL, OR5BM, OR5BN, OR5BP, OR5BQ, OR5BR, OR5BS, and OR5BT.

[0031] Family OR6 has 21 members: OR6A, OR6B, OR6C, OR6D, OR6E, OR6F, OR6J, OR6K, OR6L, OR6M, OR6N, OR6P, OR6Q, OR6R, OR6S, OR6T, OR6U, OR6V, OR6W, OR6X, and OR6Y.

[0032] Family OR7 has nine members: OR7A, OR7C, OR7D, OR7E, OR7G, OR7H, OR7K, OR7L, and OR7M.

[0033] Family OR8 has 18 members: OR8A, OR8B, OR8C, OR8D, OR8F, OR8G, OR8H, OR81, OR8J, OR8K, OR8L, OR8Q, OR8R, ORBS, OR8T, OR8U, OR8V, and OR8X.

[0034] Family OR9 has 12 members: OR9A, OR9G, OR9H, OR9J, OR9K, OR9L, OR9M, OR9N, OR9P, OR9Q, OR9R, and OR9S.

[0035] Family OR10 has 29 members: OR10A, OR10B, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10N, OR10P, OR10Q, OR10R, OR10S, OR10T, OR10U, OR10V, OR10W, OR10X, OR10Y, OR10Z, OR10AA, OR10AB, OR10AC, OR10AD, OR10AE, OR10AF, OR10AG, OR10AH, and OR10AK.

[0036] Family OR11 has 11 members: OR11A, OR11G, OR11H, OR11I, OR11J, OR11K, OR11L, OR11M, OR11N, OR11P, OR11Q.

[0037] Family OR12 has one member: OR12D.

[0038] Family OR13 has 11 members: OR13A, OR13C, OR13D, OR13E, OR13F, OR13G, OR13H, OR131, OR13J, OR13K, and OR13Z.

[0039] Family OR14 has six members: OR14A, OR14C, OR141, OR14J, OR14K, and OR14L.

[0040] Family OR51 has 21 members: OR51A, OR51B, OR51C, OR51D, OR51E, OR51F, OR51G, OR51H, OR511, OR51J, OR51K, OR51L, OR51M, OR51N, OR51P, OR51Q, OR51R, OR51S, OR51T, OR51V, and OR51AB.

[0041] Family OR52 has 22 members: OR52A, OR52B, OR52D, OR52E, OR52H, OR521, OR52J, OR52K, OR52L, OR52M, OR52N, OR52P, OR52Q, OR52R, OR52S, OR52T, OR52U, OR52V, OR52W, OR52X, OR52Y, and OR52Z.

[0042] Family OR55 has one member: OR55B.

[0043] Family OR56 has two members: OR56A and OR56B.

[0044] Taste receptors that can be expressed by genetically modified cells can detect sweet, bitter, and umami (glutamate) tastes (e.g., G protein-coupled receptors). In some variations, the taste receptors can detect salty and sour tastes (e.g., ion channel receptors or ionotropic receptors).

[0045] Trigeminal nerve receptors (TRP channels) can be expressed by genetically modified cells and can detect TRP ligands.

[0046] chaperone proteins The expression of one or more chaperone proteins can be regulated to increase trafficking of chemosensory receptors to the cell membrane surface. As previously mentioned, chaperone gene expression can be regulated (e.g., reduced) compared to expression in host cells by knocking down one or more genes. Genes that can be knocked down include, but are not limited to, CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1. In some modifications, as described in Example 3, one or a combination of the following genes: CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1 can be knocked down. It is understood that other chaperone genes can also be knocked down to increase trafficking of chemosensory receptors to the cell membrane surface. [Examples]

[0047] The following embodiments are illustrative and should not be construed as limiting the disclosure in any way.

[0048] Example 1: Localization of human olfactory receptors (ORs) to RTPS1 As mentioned above, several studies have shown that the RTP1S chaperone protein enables localization in HEK cells only for a fraction of the 400 types of human olfactory receptors (ORs). The inventors co-transfected HEK293 / HEK293T cells with olfactory receptors and then performed quantitative analysis of immunostaining and fluorescence microscopy images. They found that only about one-third of the receptors were adequately localized, a small number were moderately localized, and the majority were inadequately localized (see Figure 1). In Figure 1, the Y-axis represents the percentage of cells showing receptor localization, and each bar along the X-axis represents a different human olfactory receptor. Bright green represents human OR with sufficient localization (e.g., OR where more than 40% of cells are localized to the surface), light green represents human OR with moderate localization (e.g., OR where approximately 10% to 30% of cells are localized to the surface), and light gray represents human OR with insufficient localization (e.g., OR where approximately 10% of cells are localized to the surface). These observations suggest that RTP1S may be necessary to support human OR trafficking during functional testing in cell culture, but RTP1S alone may not be sufficient.

[0049] Example 2: Comparison of chaperone protein and processing protein levels in HEK cells and olfactory epithelial cells As mentioned earlier, receptors expressed on the cell surface are first synthesized in the cytoplasm, then moved to the endoplasmic reticulum (ER), where they undergo folding and maturation in the ER and Golgi compartments before being transported to the cell's plasma membrane via a pathway involving vesicles. This process can be supported by one or more chaperone proteins that assist in protein folding and one or more enzymes that process oligosaccharides and covalent bonds. Olfactory neurons (OSNs) have a unique and poorly understood receptor trafficking mechanism compared to cell cultures. In this experiment, we compared transcripts of expressed genes in HEK293 / HEK293T cells and OSNs to quantify the differences in chaperone proteins and enzymes involved in protein folding and processing in the trafficking pathway, thereby enabling the regulation of chaperone protein and enzyme levels in HEK293 / HEK293T cells to match in vivo levels.

[0050] Transcriptome data from HEK293 / HEK293T cells and human olfactory epithelial cells were downloaded from publicly available literature (A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome, Sultan et al., Science 2008 and the Human Olfactory Transcriptome, Oleander et al., BMC Genomics 2016), and the relative gene expression levels of the top 200 most expressed transcripts in each dataset were compared. Transcripts from intergeneric regions and pseudogenes derived from human tissues were excluded. Instead, the analysis focused on protein-coding genes. Transcripts were classified based on the function of the proteins they encode (as listed in the Uniprot database).

[0051] Analysis revealed that across the two distinct datasets, the broadest transcript categories were related to the following functions: 1) gene expression (transcription, RNA processing, and translation), 2) ribosome function, 3) mitochondrial function, 4) structural / morphological function, 5) protein folding, 6) DNA replication and repair, 7) signal transduction, 8) enzyme function, and 9) molecular transport. Referring to Figure 2A, the protein folding and processing category accounted for approximately 11.8% of the top transcripts in HEK293 / HEK293T cells. In contrast, as shown in Figure 2B, this category accounted for only 1.3% of transcripts in human olfactory epithelial cells. As further tested in Example 3, the difference in receptor trafficking was thought to be due to an overzealous quality control system in HEK293 / HEK293T cells.

[0052] Example 3: Chaperone gene knockdown in HEK cells The candidate protein folding genes shown in Table 1 below were overexpressed in HEK cells (compared to olfactory epithelium) and were selected as targets for knockdown (in HEK cells). The hypothesis tested was that the difference in receptor trafficking between HEK293 / HEK293T cells and human olfactory epithelial cells observed in Example 2 was due to an excessive quality control system in HEK293 / HEK293T cells. Knockdown was performed using RNA interference technology. More specifically, double-stranded short hairpin RNA (shRNA) expression constructs targeting CANX, CALR, HASPA5, HSP90B1, PRKCSH, HYOU1, P4HB, and RPN1, shown as SEQ ID NOs. 1-40 in Table 1, were purchased from Sigma Aldrich (hereinafter referred to as "shRNA constructs"), and recombinant knockdown cells were created using these constructs. As further described below, co-expression of olfactory receptors was also performed to evaluate whether surface expression improved after knockdown of target genes. [Table 1]

[0053] In this experiment, HEK293 / HEK293T cells seeded on coverslips were transfected with human olfactory receptor (OR51D1), RTP1S, and green fluorescent protein (GFP) (as an indicator of transfection efficiency) along with a mixture of calnexin-targeting shRNA constructs (CANX KD: calnexin knockdown) as shown in Table 1. For comparison, a control was established using OR51D1, RTP1S, and GFP without the shRNA construct. OR51D1 was selected because it is not a well-expressed receptor. Staining was performed 48 hours after transfection, and Rho-tagged olfactory receptors were visualized using an anti-rhodopsin antibody (clone 4D2, EMD Millipore). Imaging was then performed approximately 24 hours after staining and mounting, and images were acquired under the same exposure conditions for all samples. Representative images of shRNA-transfected and untransfected olfactory receptors are shown in Figures 3A-3C and 3D-3F, respectively. Figures 3A-3C show the localization of OR51D1 in the presence of calnexin knockdown, while Figures 3D-3F show the localization of OR51D1 under conditions without knockdown. All images were acquired with a Keyence BZ-X810 microscope at an exposure time of 1 / 1.2 seconds.

[0054] Preliminary image analysis was performed using ImageJ (https: / / imagej.nih.gov / ij / ), a public domain Java image processing program. Regions containing cells were selected as shown in Figures 4A and 4B, and their grayscale intensities were measured as shown in Figures 4C and 4D, respectively. Referring to Figure 5, a comparison of the modal intensities of OR51D1 images in the presence and absence of calnexin knockdown revealed that calnexin depletion significantly increased the surface expression of OR51D1.

[0055] Example 4: Enhancement of olfactory receptor surface expression using knockdown We evaluated whether more underexpressed olfactory receptors could be enhanced by targeting one or a combination of the folding and / or processing genes of candidate proteins. In these experiments, HEK293 / HEK293T cells were transfected with human olfactory receptors (OR6P1, OR8D1, OR2J3, OR51D1, and OR9Q1) along with one or a combination of shRNA constructs (CANX+HYOU;CANX+HSP;CALR+HYOU;CALR+HSP;CALR+CANX; and CALR+CANX+HSP).

[0056] Forty-eight hours after transfection, staining and microscopic observation were performed as described in Example 3. Fluorescence image analysis was then performed using in-house developed software to quantify the percentage of cells showing human olfactory receptor expression under control and various knockdown conditions. Referring to Figure 6, it was found that increased surface expression of human olfactory receptors was achieved under most knockdown conditions (single or in combination). The degree of improvement in surface expression did not show a consistent pattern, but rather appeared to vary depending on the combination of olfactory receptor and knockdown target. However, the overall data indicated that knockdown of either single or combined protein folding and processing genes is likely to enhance the surface expression of human olfactory receptors. In Figure 6, the Y-axis represents control (no knockdown), single knockdown target, or combination of different knockdown targets, and the X-axis represents the human olfactory receptors tested for surface expression improvement under each condition. The scale bars represent the surface localization of olfactory receptors under each condition compared to their knockdown control, with localization indicated by the intensity of the green color. Areas with high localization are shown in dark green, areas with low localization are shown in light green, and areas with no localization are shown as colorless.

[0057] Example 5: Functional Assay The validation of chaperone knockdown approaches to increase olfactory receptor surface expression was completed by performing functional assays. It was expected that improved olfactory receptor surface expression, as described in Example 4, would increase the functional response to odor ligands. To test this, HEK293 / HEK293T cells were transfected with CALR or CANX+CALR shRNA constructs and controls (without shRNA). The cells were also co-transfected with OR2J3 and OR5L1, signaling elements, and a firefly reporter.

[0058] Forty-eight hours after transfection, cells were stimulated with either "odorless" or pentanol (odor stimulation of OR2J3 and OR5L1), and receptor activity was read at the end of the assay. OR2J3 was found to show increased signaling in CALR and CANX+CALR compared to control HEK293 / HEK293T cells (see Figure 7A). OR5L1 showed increased signaling under CANX+CALR knockdown (see Figure 7B). White bars represent assay readings without stimulation, and green bars represent assay readings with pentanol stimulation. Overall, the data suggest that knockdown of chaperones associated with protein folding and processing genes, either individually or in combination, is likely to increase olfactory receptor functional responses by increasing surface expression of olfactory receptors.

[0059] Example 6: Preparation of genetically modified cell lines We created stable knockdown cell lines to increase the surface expression of olfactory receptors using shRNA that specifically targets CALR (calreticulin). Briefly, we introduced a plasmid encoding short hairpin loop RNA (shRNA) into HEK293 / HEK293T cells via lipid transfection. Subsequently, we selected and propagated cell colonies that had incorporated the plasmid (polyclonal colonies). This was achieved by exposing the cells to a predetermined concentration of a selective antibiotic (puromycin) and selecting the viable colonies. A total of 10 polyclonal colonies were selected, their functional effectiveness was analyzed, and the knockdown of CALR mRNA expression levels was confirmed by quantitative PCR.

[0060] In the next step, 28 single clones were identified by limiting dilution and grown to form monoclonal colonies. The top five clones were evaluated for efficacy in our cell-based functional assay, and the top two monoclones were selected. These two colonies will be tested using various OR (odor) pairs to compare with commercially available HEK293 / HEK293T cells for potential positive effects on the sensitivity and efficacy of our assay.

Claims

1. Genetically modified cells in which the expression of at least one chaperone protein involved in the folding of chemosensory receptors that are trafficked to the membrane surface of the cell is reduced, and the trafficking of the chemosensory receptors to the membrane surface and / or functional response is increased compared to control cells.

2. The cell according to claim 1, wherein the reduction in the expression of the one or more chaperone proteins is achieved by knockdown of one or more genes encoding the one or more chaperone proteins.

3. The cell according to claim 2, wherein one or more of the genes are selected from the group consisting of CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1.

4. The cell according to claim 3, wherein one or more of the genes are CANX.

5. The cell according to claim 1, wherein the cell is a mammalian cell.

6. The cell according to claim 5, wherein the mammalian cell is a kidney cell or a hybrid thereof.

7. The cell according to claim 6, wherein the kidney cell is a HEK293 / HEK293T cell.

8. The cell according to claim 1, wherein the chemosensory receptor is an olfactory receptor.

9. The cell according to claim 1, wherein the chemosensory receptor is a taste receptor.

10. A system for increasing chemosensory receptor trafficking, comprising a plurality of genetically modified cells according to any one of claims 1 to 9.

11. The system according to claim 10, further comprising an imaging device configured to detect the expression of the chemosensory receptors on the membrane surface of the plurality of genetically modified cells.

12. A method for increasing the transport of chemosensory receptors to the cell membrane surface, comprising expressing the chemosensory receptors in cells in which the production of one or more chaperone proteins has been reduced by knockdown of one or more genes encoding one or more chaperone proteins.

13. The method according to claim 12, wherein the production of one or more chaperone proteins is reduced by about 25% to about 75%.

14. The method according to claim 12, wherein the reduction in the production of the one or more chaperone proteins is achieved by knockdown of one or more genes encoding the one or more chaperone proteins.

15. The method according to claim 14, wherein one or more of the genes are selected from the group consisting of CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1.

16. The method according to claim 15, wherein one or more of the genes are CANX.

17. The method according to claim 12, wherein the cells are mammalian cells.

18. The method according to claim 17, wherein the mammalian cell is a kidney cell or a hybrid thereof.

19. The method according to claim 17, wherein the kidney cells are HEK293 / HEK293T cells.

20. The method according to claim 12, wherein the chemosensory receptor is an olfactory receptor.

21. The method according to claim 12, wherein the chemosensory receptor is a taste receptor.

22. A method for preparing genetically modified cells, comprising modifying HEK293 / HEK293T cells to knock down one or more genes encoding CALR, HSPA5, HSP90B1, CANX, PRKCSH, HYOU1, P4HB, and RPN1.

23. The method according to claim 22, wherein one or more genes encode CANX.

24. The method according to claim 23, wherein one or more genes encode CALR and CANX.

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