Method for identifying target and use thereof

Abstract

The present invention relates to the field of biomedicine and particularly provides a method for identifying a target. The method specifically comprises determining the number of target-adjusting systems of a plurality of cells to be tested to determine the influence of adjusting a candidate target on cell function; and before and / or during the determination, adjusting the expression or activity of the candidate target in said plurality of cells by means of the plurality of target-adjusting systems, wherein each of the target-adjusting systems targets a different candidate target site, and during the determination, the number of said cells is at least 5 times the number of the target-adjusting systems. Further provided is a system for identifying a target.

Description

A method for identifying targets and its application

[0001] Cross-references to related applications

[0002] This application claims priority to PCT International Application No. PCT / CN2024 / 089092, filed on April 22, 2024, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention relates to the field of biomedicine, specifically to a method for identifying targets and its application. Background Technology

[0004] In the biomedical field, screening and identifying gene targets that can regulate cell function has significant application prospects. Existing target gene screening platforms typically use freshly obtained primary immune cells as test cells to identify target genes that affect cell function.

[0005] However, the number of primary cells is usually limited, and primary cell amplification is difficult. Furthermore, gene editing of primary cells is challenging (screening requires gene editing of cells), and the added difficulty of amplification after editing further complicates matters. Therefore, the number of primary cells is insufficient for screening large-capacity libraries. Using too few primary cells as test cells in a screening system increases the risk of unreliable screening results. Therefore, there is an urgent need in the field for a method to screen gene targets that can control the risk of inaccurate screening results when the source of test cells is limited. Summary of the Invention

[0006] Current immune cell target screening systems do not control the number of cells to be tested. This invention reveals that in target gene screening systems, if the number of cells used as test cells, such as NK cells and NKT cells, is too small, it may lead to inaccurate screening results. Specifically, a small number of harvested cells used for sequencing increases the risk of random gRNA loss, potentially resulting in the absence of the lost gRNA in the cell extraction sequencing results. This can lead to situations where sequencing results cannot be accurately interpreted; the absence of a particular gRNA in the sequencing can be interpreted as either a random loss of that gRNA or as the knockout of the corresponding target gene failing to improve the function of the test cells, thus resulting in their rejection by the screening system. This reduces the reliability of the screening results.

[0007] Therefore, this invention provides a novel method for identifying targets, which controls the number of cells to be tested during sequencing of screened cells to improve the reliability of the screening results.

[0008] On one hand, the present invention provides a method for identifying targets, the method comprising the following steps: (1) providing a plurality of test cells; (2) contacting the test cells with a plurality of target regulatory systems to regulate the expression or activity of candidate targets in the test cells, each target regulatory system targeting a different candidate target site; preferably, each test cell contains a target regulatory system; (3) contacting the test cells with an identification model, and then determining the presence and / or number of target regulatory systems in the plurality of test cells; and (4) determining the effect of regulating the candidate targets on the test cells by means of the readings obtained from the determination, thereby identifying the candidate targets.

[0009] On the other hand, the present invention provides a method for identifying targets, comprising determining the effect of regulating candidate targets on cell function by measuring the number of target regulatory systems in a plurality of test cells; regulating the expression or activity of candidate targets in a plurality of test cells by a plurality of target regulatory systems before and / or simultaneously with the measurement, each target regulatory system targeting a different candidate target site; and at the time of the measurement, the number of test cells is at least 5 times the number of target regulatory systems.

[0010] On the other hand, the present invention provides a method for identifying targets, the method comprising the following steps: (1) regulating the expression or activity of a candidate target in a plurality of test cells by a plurality of target regulation systems, each target regulation system targeting a different candidate target site; and (2) determining the effect of regulating the candidate target on cell function by measuring the number of target regulation systems in the plurality of test cells, wherein the number of test cells in step (2) is at least 5 times the number of target regulation systems.

[0011] In some embodiments, the number of cells to be tested in the assay is at least five times the number of the target regulation system.

[0012] In some embodiments, the cells to be tested comprise immune cells, preferably NK cells, NKT cells, macrophages, and / or γδT cells (such as Vδ1 subset, Vδ2 subset).

[0013] In some embodiments, the identification model contains tumor antigens before and / or simultaneously with the assay.

[0014] In some embodiments, the method of the present invention further includes introducing a receptor that recognizes a tumor antigen into the cells to be tested.

[0015] In some implementations, the antibodies that recognize tumor antigens are chimeric antigen receptors (CARs) or T-cell receptors (TCRs).

[0016] In some embodiments, the identification model is tumor cells, and the test cells are co-cultured with tumor cells before and / or simultaneously with the assay, the test cells expressing receptors that recognize tumor antigens expressed on the tumor cells.

[0017] In some implementations, the co-cultivation includes at least one stage of co-cultivation.

[0018] In some implementations, the co-cultivation described in each stage lasts for at least 12 hours.

[0019] In some implementations, after the co-culture is completed, the killing effect of the test cells on tumor cells is significantly reduced (e.g., to 50% or less of the initial killing ability).

[0020] In some implementations, the regulation step includes introducing components of a target regulation system into the test cells, wherein the target regulation system increases or decreases the expression or activity of the candidate target.

[0021] In some implementations, the regulation step includes providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a broad-spectrum nuclease system, and / or repressive RNA.

[0022] In some implementations, the regulation step includes introducing single-strand breaks, double-strand breaks, mutations, and / or expression regulation in the midstream, upstream, and / or downstream of the candidate nucleic acid sequence.

[0023] In some implementations, the regulatory steps include increasing or decreasing the expression or activity of the candidate target at the genomic level.

[0024] In some implementations, the regulation step includes introducing a target regulation system containing guiding nucleic acid molecules and nucleases into the cell to be tested.

[0025] In some implementations, the guiding nucleic acid molecule comprises a guide RNA (gRNA) that targets the target.

[0026] In some embodiments, the nuclease comprises Cas protein, Cas protein homologues, or functionally active fragments thereof.

[0027] In some embodiments, the nuclease comprises Cas 9 and / or Cas 12.

[0028] In some implementations, the proportion of cells expressing the candidate target in the cell population obtained by the conditioning step is reduced to less than about 95%.

[0029] On the other hand, the present invention provides a composition comprising a plurality of test cells, wherein the expression or activity of a candidate target in the plurality of test cells is regulated by a plurality of target regulation systems, each of which targets a different candidate target site; wherein the number of test cells is at least 5 times the number of target regulation systems.

[0030] On the other hand, the present invention provides a system for identifying targets, the system comprising: a measurement module for measuring the number of target regulatory systems in a plurality of test cells and determining the effect of regulating candidate targets on cell function; regulating the expression or activity of candidate targets in the plurality of test cells by a plurality of target regulatory systems before and / or simultaneously with the measurement, each target regulatory system targeting a different candidate target site; and at the time of the measurement, the number of test cells is at least 5 times the number of target regulatory systems.

[0031] On the other hand, the present invention provides a system for identifying targets, the system comprising:

[0032] Module (1) regulates the expression or activity of candidate targets in multiple test cells through multiple target regulation systems, with each target regulation system targeting different candidate target sites;

[0033] Module (2) determines the effect of regulating the candidate target on cell function by measuring the number of target regulatory systems in multiple test cells, wherein the number of test cells in module (2) is at least 5 times the number of target regulatory systems;

[0034] Module (1) and module (2) may be arranged in parallel, or module (1) may be arranged before module (2). Attached Figure Description

[0035] The features and advantages of the present invention can be better understood by referring to the accompanying drawings. A brief description of the drawings is as follows:

[0036] Figure 1 shows an exemplary screening process provided by the present invention.

[0037] Figure 2 shows the ratio of effector cells to target cells in co-culture of NK cells and tumor cells from two different donor sources.

[0038] Figure 3 shows the number of immune cells / gRNA library coverage for two different donor sources.

[0039] Figure 4 shows the results of gRNA detection loss percentage (vertical axis: uncovered fraction of sgRNAs) corresponding to different cell / gRNA coverage (horizontal axis: cell coverage).

[0040] Figure 5 shows the gene screening results obtained by the screening system of the present invention. Detailed Implementation

[0041] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0042] Terminology Definition

[0043] In this invention, the term "target" generally refers to a target. For example, a target gene is obtained through screening or identification from candidate genes. Depending on the results of the screening or identification, the protein encoded by the "candidate gene" is identified as a potential target suitable for regulating cells. In this invention, the term "gene" generally refers to a nucleic acid molecule that, when placed under the control of a suitable regulatory or control sequence, is transcribed into RNA or translated into a polypeptide in vitro or in vivo. In some embodiments, the target is a target gene. In some embodiments, the target is a key target and / or a novel target with cellular function regulatory effects. In some embodiments, the target is a key target and / or a novel target with the function of promoting cell tumor invasion. In some embodiments, the target is a key target and / or a novel target with the function of promoting cell exhaustion resistance.

[0044] In this invention, the term "target tissue" generally refers to a target, site, or biological tissue that is desired to be targeted by immune cells. For example, the target tissue of this invention may be a target or site for cell therapy. For example, the tissue of this invention may include in vitro, ex vivo, or in vivo biological tissue. For example, the tissue of this invention may include artificially induced biological tissue.

[0045] In this invention, the term "co-culture" generally refers to culturing two or more different populations of cells in contact with each other to a certain extent. This "contact" between the two or more different populations of cells can, in one embodiment, be direct contact, i.e., direct physical contact between cells from one population and cells from another population. Alternatively, in another embodiment, it can be indirect contact mediated by a shared culture medium. The shared culture medium may contain metabolites produced and released by at least one population of the co-cultured cells and is used to culture another population of cells.

[0046] In this invention, the term "identification model" generally refers to a system used for identifying targets (abbreviated as "identification system," synonymous with "identification model," and used interchangeably). For example, the identification model of this invention can be in vitro, ex vivo, or in vivo. For example, the identification model of this invention can include all or part of the functions of a circulatory system. For example, the identification model of this invention can simulate the process of test cells approaching target tissue. For example, the identification model of this invention can be a non-human animal, such as a non-human primate, like an ape or monkey, and livestock, such as a dog, rabbit, cow, pig, sheep, goat, horse, or donkey. For example, the identification model of this invention can be a cow, bull, bison, buffalo, pig, bighorn sheep, horse, mule, deer, elk, llama, or alpaca. For example, the identification model of this invention can be a rodent, such as a mouse or rat. In some embodiments, the identification model of this invention includes tumor antigens. In some embodiments, the identification model of this invention includes tumor cells. In some embodiments, the identification model of this invention is isolated tumor cells, tumor tissue, or in vitro cultured tumor cells or tumor tissue.

[0047] In this invention, the term "contact" generally refers to a process of proximity. For example, placing cells in close proximity to a substance so that the cells can interact with the substance and / or be affected by its presence. The contact can be temporary or transient. The contact can also last for a period of time or be permanent.

[0048] In this invention, the term "natural killer T cell (NKT)" generally refers to a T cell or population of T cells that exhibits both conventional T cell characteristics and natural killer (NK) cell characteristics. For example, in some embodiments, NKT cells are mature lymphocytes that possess both T cell receptors and NK cell receptors.

[0049] In this invention, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into cells.

[0050] In this invention, the term "expression vector" generally refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system.

[0051] In this invention, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. Unless specifically defined, the term covers nucleic acids containing known natural and / or non-natural nucleotide analogs that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions, such as conserved substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences.

[0052] In this invention, the terms "transfected," "transformed," or "transduced" generally refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary host cells and their progeny.

[0053] In this invention, the terms "about" and "approximately" generally refer to a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, and may include within 50%, preferably within 20%, more preferably within 10%, and most preferably within 5%. The permissible variations included in the terms "about" or "approximately" may depend on the specific system under investigation and will be readily understood by those skilled in the art.

[0054] In this invention, the terms "above", "below", "at most" and "at least" include the number itself.

[0055] In this invention, "comprising", "including", or "containing" are open-ended expressions, while "composed of" is a closed-ended expression. The former covers the latter, and the latter is a special form of the former.

[0056] Invention Details

[0057] This invention designs a target gene screening process. During sequencing of screened cells, the number of cells to be tested is controlled to improve the reliability of the screening results. This method is based on the following finding: in the screening process, system imperfections may lead to a high proportion of false positives or missed important targets. This invention also finds that increasing the number of harvested cells and reducing the risk of random gRNA loss are important methods to improve the reliability of the screening results.

[0058] On one hand, the present invention provides a method for identifying targets, the method comprising the following steps: (1) providing a plurality of test cells; (2) contacting the test cells with a plurality of target regulatory systems to regulate the expression or activity of candidate targets in the test cells, each target regulatory system targeting a different candidate target site; preferably, each test cell contains a target regulatory system; (3) contacting the test cells with an identification model, and then determining the presence and / or number of target regulatory systems in the plurality of test cells; and (4) determining the effect of regulating the candidate targets on the test cells by means of the readings obtained from the determination, thereby identifying the candidate targets.

[0059] In some embodiments, the number of cells to be tested in the assay is at least five times the number of the target regulation system. For example, when determining the number of target regulatory systems in multiple test cells, the number of test cells is at least 5-1,000,000,000 times, at least 5-1,000,000,000 times, at least 5-1,000,000, at least 5-1,000,000 times, at least 5-1,000,000 times, at least 5-1,000,000 times, at least 5-1,000, at least 5-100 times, at least 5-10 times, for example, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 1,000 times, at least 10,000 times, at least 100,000 times, at least 1,0 ... 000 times, at least 10,000,000 times, at least 100,000,000 times, or at least 1,000,000,000 times.

[0060] In some embodiments, the test cells comprise immune cells. Preferably, these are immune cells that are not readily available in the natural environment, such as NK cells, NKT cells, γδT cells (e.g., Vδ1 subset, Vδ2 subset, Vδ3 subset), macrophages, monocytes, dendritic cells (DCs), and / or B cells. In some embodiments, the immune cells are immune cells isolated from an individual (e.g., a cancer patient). In some embodiments, the immune cells are cultured cell lines.

[0061] This invention provides a target regulatory system library (e.g., a gRNA library) for modifying test cells to identify targets. In some embodiments, the method of this invention includes introducing multiple target regulatory systems into the test cells. For example, each target regulatory system targets a different candidate target site. In some embodiments, one target regulatory system is introduced into each test cell. For example, the target regulatory system targets different candidate target sites by containing different types of gRNAs. For example, the gRNA library capacity in the screening system is at least about 10 to 1,000,000,000, such as at least about 10, at least 20, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000, at least 1,000, at least 1,000,000, at least 10,000,000, at least 100,000,000, or at least 1,000,000,000. For example, the probability of a certain gRNA not being present in the sequencing is at most 0.1%-0.0001%, such as at most 0.1%, at most 0.01%, at most 0.001%, at most 0.0001%, or the probability of a certain gRNA not being present in the sequencing is approximately 0.

[0062] In some embodiments, the identification model comprises tumor antigens. In some embodiments, the identification model comprises tumor cells. In some embodiments, the method further includes introducing a receptor that recognizes the tumor antigen into the test cells to target and kill the tumor cells. In some embodiments, the antibody that recognizes the tumor antigen is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR). Any suitable tumor antigen recognition receptor can be used in the method. For example, the chimeric antigen receptor can be a CAR or TCR that specifically recognizes and binds to the CD19 antigen.

[0063] For example, the test cells are co-cultured with tumor antigens before and / or simultaneously with the assay. For example, the test cells are co-cultured with tumor cells (also referred to as "target cells" in this invention) before and / or simultaneously with the assay, the test cells expressing receptors that recognize tumor antigens, the tumor antigens being expressed on the tumor cells. For example, the co-culture comprises at least one phase of co-culture. For example, the co-culture comprises at least 1 to 10 phases, such as at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 phases of co-culture. For example, each phase of co-culture lasts at least 12 hours to 14 days. For example, the duration of each phase of co-culture is independent of each other and may optionally last at least about 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. For example, the ratio of the test cells to the tumor cells in the culture environment is at least 30:1 to 1:30, such as at least 30:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, or 1:30.

[0064] This invention screens test cells that survive and / or exhibit amplification capacity after multiple rounds of culture (e.g., cell killing) to identify targets related to cell killing ability, apoptosis, and anti-exhaustion status. For example, after the co-culture is completed (e.g., after multiple rounds of target cell killing), test cells that survive or show amplification and enrichment under conditions of low cell function and near-complete loss of cell killing ability are considered to possess enhanced function. The method of this invention identifies targets by measuring the presence and expression levels of targets in these cells (e.g., by measuring the content of target regulatory systems). For example, compared to test cells that have not been co-cultured with cells expressing tumor antigens, the co-cultured cells obtained by the method of this invention essentially do not kill tumor cells. For example, compared to functionally normal test cells, the co-cultured cells obtained by the method of this invention essentially do not kill all tumor cells. For example, compared to functionally normal test cells, the number of remaining undamaged tumor cells in the co-cultured cells obtained by the method of this invention when co-cultured with tumor cells is greater than 1 × 10⁻⁶. 5 -2×10 6 One, for example, greater than 1×10 5 1, greater than 2 × 10 5 1, greater than 3 × 10 51, greater than 4 × 10 5 Items, greater than 5 × 10 5 1×10 6 One, or more than 2 × 10 6 For example, the cytotoxicity of tumor cells is determined by flow cytometry. For example, when co-cultured with tumor cells, the number of remaining unkilled tumor cells in the cells obtained by the method of this invention is greater than 50%-99% of the number of tumor cells added at the beginning of co-culture, for example, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%. For example, the cytotoxicity of tumor cells is determined by flow cytometry.

[0065] For example, the state of "significantly reduced cell-killing ability of the test cells" in this invention can refer to the test cells' cell-killing ability remaining at only 50% or less of the initial killing ability; for example, after co-culture, the number of tumor cells that the test cells can kill after the initial killing (first round of co-culture with tumor cells) is only 50%-0.1% or less, such as 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, or 0.1% or less. For example, if the cell-killing ability of the test cells is significantly reduced after co-culture, and the number of tumor cells proliferates exceeds the number of cells killed by the test cells, then the cell-killing ability of the test cells can also be considered significantly reduced.

[0066] For example, the state of "the test cells have basically lost their cell-killing ability" in this invention can refer to the ratio of the number of remaining unkilled tumor cells to the number of tumor cells added at the beginning of the last round of co-culture of the test cells with tumor cells being greater than 50%-99%, such as greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%. For example, after co-culture, the cell-killing ability of the test cells is significantly reduced, the number of tumor cells proliferates exceeds the number of test cells killed, and the ratio of the number of remaining undamaged tumor cells to the number of tumor cells added at the beginning of this round of co-culture is approximately 1-10,000 times, such as greater than 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 10, 2, 50, 100, 1000, or 10,000 times; at this point, it can be considered that the test cells have essentially lost their cell-killing ability.

[0067] For example, the regulation step includes introducing components of a target regulation system into the test cells, wherein the target regulation system increases or decreases the expression or activity of the candidate target. For example, the regulation step in this invention includes providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a broad-spectrum nuclease system, and / or repressive RNA. For example, the regulation step in this invention includes targeting a specific nucleic acid sequence of the candidate target, introducing single-strand breaks, double-strand breaks, mutations, and / or expression regulation midstream, upstream, and / or downstream of the specific nucleic acid sequence. For example, the target regulation system may include elements that increase gene expression, such as the CRISPRa system. For example, the target regulation system may include elements that decrease gene expression, such as the CRISPRi system.

[0068] For example, the regulatory step described in this invention includes increasing or decreasing the expression or activity of the candidate target at the genomic level. For example, the regulatory step described in this invention includes introducing a target regulatory system containing a guiding nucleic acid molecule and a nuclease into the test cell. For example, the guiding nucleic acid molecule described in this invention includes a guide RNA (gRNA) targeting the target. For example, the nuclease described in this invention includes a Cas protein, a Cas protein homologue, or a functionally active fragment thereof. For example, the nuclease described in this invention includes Cas 9 and / or Cas 12.

[0069] For example, the regulation step described in this invention includes introducing a target regulation system into the test cells, the target regulation system increasing or decreasing the expression or activity of the candidate target. For example, the regulation step described in this invention includes introducing a target regulation system into the cells, the target regulation system increasing or decreasing the expression or activity of the candidate target. In some embodiments, a clustered regular interval short palindromic repeat (CRISPR) system is used to regulate the candidate target. Alternatively, a transcription activator-like effector nuclease (TALEN) system, a zinc finger nuclease system, or a wide range of nuclease systems can be used to regulate the candidate target. In some embodiments, an activating regulatory system regulates the candidate target. Alternatively, overexpression of the candidate target can be used to increase the expression or activity of the candidate target.

[0070] For example, the regulatory steps described in this invention include increasing or decreasing the expression or activity of the candidate target at the genomic level. Alternatively, the expression or activity of the candidate target may be increased or decreased transiently at the RNA level. Alternatively, the expression or activity of the candidate target may be increased or decreased at the transcriptional, translational, and / or post-translational modification levels. In some embodiments, antisense RNA, siRNA, shRNA, or short hairpin RNA is used to decrease the expression or activity of the candidate target. In some embodiments, activating RNA is used to increase the expression or activity of the candidate target. For example, the regulatory steps described in this invention include providing a substance that covalently binds and / or non-covalently binds the candidate target to increase or decrease the expression or activity of the candidate target.

[0071] For example, the method for editing candidate targets in cells according to the present invention can be carried out in vivo, in vitro, and / or in vitro. For example, the in vivo expression level of candidate targets can be reduced in cells by delivery and editing via an in vivo gene regulatory system. For example, in vivo editing of candidate targets can be performed by targeting immune cells or their precursor cells, such as bone marrow stem cells, by delivering LNPs containing gene regulatory systems or mRNAs encoding gene regulatory systems. The efficiency of in vivo target editing according to the present invention can be improved by adjusting the composition and / or ratio of LNP components or by introducing components with targeting capabilities.

[0072] For example, the regulatory step of the present invention includes targeting a specific nucleic acid sequence of the target, introducing single-strand breaks, double-strand breaks, and / or mutations in the midstream, upstream, and / or downstream of the nucleic acid sequence. For example, the regulatory step of the present invention includes providing a CRISPR system, a zinc finger nuclease system, a wide-range nuclease system, and / or antisense RNA, siRNA, shRNA, or short hairpin RNA.

[0073] For example, in this invention, knocking out or down (collectively referred to as "silencing") candidate target genes can be achieved using chemically synthesized or in vitro transcribed small interfering RNAs (siRNAs), as well as PCR- or DNA-vector-based short hairpin RNAs (shRNAs). Furthermore, the shRNA molecules provided herein can be operatively linked to T-cell-specific promoters to achieve T-cell-specific targeting of the shRNA molecules for silencing candidate target genes.

[0074] For example, the present invention may include a gene silencing method for candidate target genes by interfering with RNA. The method involves creating a construct encoding interfering (silencing) RNA using a promoter active in a specific cell type (e.g., T cells) to drive the expression of shRNA.

[0075] For example, the shRNA construct used in the method provided by this invention has a small nucleotide extension targeting the candidate target gene for regulating the expression of the nucleic acid molecule encoding the candidate target gene. Inhibition of the candidate target gene is achieved by providing an oligomeric compound that hybridizes with one or more target nucleic acid molecules encoding the candidate target gene.

[0076] For example, in this invention, candidate target genes can be silenced using a system based on transcription activator-like effectors and nucleases (TALENs). Transcription activator-like (TAL) effector sequences can be assembled to specifically bind to the target DNA sequence by assembling repeat variable-di-residue (RVD) sequences. The fusion protein of the TAL effector and TALEN can generate targeted double-strand breaks in the cellular DNA, which can be used to produce cell-specific modifications.

[0077] For example, in this invention, the TAL effector domain that binds to a specific nucleotide sequence within the target DNA may comprise 10 or more DNA-binding repeats, or 15 or more DNA-binding repeats. Each DNA-binding repeat may include an RVD that determines the recognition of a base pair in the target DNA sequence, wherein each DNA-binding repeat is responsible for recognizing one base pair in the target DNA sequence.

[0078] For example, in this invention, the substance binding to the target nucleic acid can be linked to an effector domain, including but not limited to transposases, integrases, recombinases, resolvases, invertases, proteases, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, nucleases, transcription repressors, transcription activators, transcription factor recruitment, protein nuclear localization signals, or cellular uptake signals. For example, the effector domains described in this invention include, but are not limited to, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, DNA methyltransferase activity, DNA demethylase activity, histone acetyltransferase activity, histone deacetylase activity, nuclease activity, nuclear localization signal activity, transcription repressor activity, transcription activator activity, transcription factor recruitment activity, or cellular uptake signal activity.

[0079] For example, in this invention, a zinc finger is a small protein structural motif stabilized by one or more zinc ions. The zinc finger may contain, for example, Cys2His2 and recognizes a sequence of approximately 3 bp. Various zinc fingers with known specificities can be combined to generate multi-zinc-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp. Zinc-finger nuclease systems can generate double-strand breaks in DNA, which, if not properly repaired, can lead to frameshift mutations, resulting in reduced expression of target genes in cells.

[0080] For example, vectors used for integrating the target sequence in this invention include, but are not limited to, retroviral vectors, such as lentiviral or retroviral vectors, adenovirus vectors, and baculovirus vectors. For example, expression vectors can be used for the stable or transient expression of polypeptides encoded by the nucleic acid sequence to be expressed; the vector can be a self-replicating extrachromosomal vector or a vector integrated into the host genome. In one embodiment, the vector is a genome integration vector, or "integrating vector," which can be converted into chromosomal DNA or RNA integrated into a host cell, cellular system, or non-cellular system. In some embodiments, nonviral methods include the use of transposons (also called transposition elements). In some embodiments, a transposon is a DNA strand that can insert itself into one location in the genome, for example, a DNA strand capable of self-replication and inserting a copy into the genome, or a DNA strand that can be spliced ​​from a longer nucleic acid and inserted into another location in the genome. For example, a transposon comprises a DNA sequence consisting of inverted repeat sequences flanked by genes for transposition.

[0081] For example, the regulatory step of this invention includes introducing a target regulatory system containing a guide nucleic acid molecule and a nuclease into the cell. For example, the components of the target regulatory system can be introduced into the cell sequentially. For example, a viral or non-viral vector containing a guide nucleic acid molecule can be introduced into the cell first, followed by the introduction of a nucleic acid containing a nuclease or encoding a nuclease into the cell. For example, a guide nucleic acid molecule library for screening can be packaged with lentivirus or retrovirus and introduced into the cell first, followed by the introduction of the nuclease into the cell via electroporation. For example, a nucleic acid containing a nuclease or encoding a nuclease can be introduced into the cell first, followed by the introduction of a viral or non-viral vector containing a guide nucleic acid molecule into the cell. For example, the nuclease can be introduced into the cell first via electroporation, and a guide nucleic acid molecule library for screening can be packaged with lentivirus or retrovirus and then introduced into the cell. For example, the guide nucleic acid molecule in this invention contains guide RNA (gRNA). For example, the nuclease in this invention contains Cas protein, Cas protein homologues, or functionally active fragments thereof. For example, the nuclease in this invention contains Cas 9 and / or Cas 12. For example, the gRNA can be used to bind to the sequence of the candidate target. For example, the binding of the gRNA to the sequence of the candidate target can be fully complementary, partially complementary, or under moderately or tightly hybridized conditions. For example, the binding of the gRNA to the sequence of the candidate target can cause the CRISPR system of the gRNA to specifically cleave the candidate target.

[0082] For example, when the gene editing system includes CRISPR / Cas9, the downstream region of the region targeted by the guiding nucleic acid molecule of the present invention may have a prototypical spacer adjacent motif (PAM), which may be AGG, TGG, GGG, or CGG. For example, once the PAM region of a candidate target is identified, those skilled in the art can readily determine the target sequence consisting of approximately 15 to 25 nucleotides (e.g., approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) upstream of the 5' end of the candidate target PAM, and can design a suitable gRNA for this target sequence. For example, the guiding nucleic acid molecule can bind to a sequence consisting of approximately 15 to 25 nucleotides upstream of the 5' end of a prototypical spacer adjacent motif (PAM) selected from the group consisting of AGG, TGG, GGG, and CGG.

[0083] For example, when the gene editing system includes CRISPR / Cas12, the upstream region of the region guiding the target nucleic acid molecule of the present invention may have a prototypical spacer adjacent motif (PAM). The prototypical spacer adjacent motif (PAM) may be NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, where N is A, T, C, or G; Y is T or C; V is A, C, or G; and R is A or G. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of approximately 15 to 25 nucleotides (e.g., approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence. For example, the guiding nucleic acid molecule can bind to a target sequence consisting of about 15 to about 25 nucleotides downstream of the 3' end of the prototypical spacer sequence adjacent to the motif (PAM) selected from the group shown: NTTN, TTYN, VTTV, TRTV, TTTV, TATV, TYCV, TNN, or NTN, where N is A, T, C, or G, Y is T or C, V is A, C, or G, and R is A or G.

[0084] For example, when the gene editing system of the present invention contains wild-type Cas12a (also known as Cpf1, such as AsCas12a, FnCas12a, LbCas12a, BbCas12a, CMaCas12a, and OsCas12a), the upstream region of the region guiding the nucleic acid molecule targeting of the present invention may have a PAM sequence selected from the following: NTTN, where N can be A, T, C, or G. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of about 17 to about 25 nucleotides (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence.

[0085] For example, when the gene editing system of the present invention contains mutant Cas12a, such as enAsCas12a (mutation sites E174R, S542R, and K548R), the upstream region of the region guiding the nucleic acid molecule targeting of the present invention may have a PAM sequence selected from the following: TTYN (TTTN / TTCN), VTTV (ATTV / CTTV / GTTV), or TRTV (TATV / TGTV), where N can be A, T, C, or G, Y can be T or C, V can be A, C, or G, and R can be A or G. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of about 17 to about 25 nucleotides (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence.

[0086] For example, when the gene editing system of the present invention contains mutant Cas12a, such as opAsCas12a (mutation sites: E174R and S542R), the upstream region of the region guiding the nucleic acid molecule targeting of the present invention may have a PAM sequence selected from the following: TTTV (TTTA, TTTC, or TTTG), where V can be A, C, or G. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of about 17 to about 25 nucleotides (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence.

[0087] For example, when the gene editing system of the present invention contains mutant Cas12a, such as AsCas12aUltra (mutation sites: M537R and F870L), the upstream region of the region guiding the nucleic acid molecule targeting of the present invention may have a PAM sequence selected from the following: TTTV, TATV, or TYCV, where V can be A, C, or G, and Y can be T or C. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of about 17 to about 25 nucleotides (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence.

[0088] For example, when the gene editing system of the present invention includes mutant Cas12a, such as hfCas12Max (mutation site: N243R / E336R / D892R) and Cas12Max (mutation site: N243R), the upstream region of the region guiding the nucleic acid molecule targeting of the present invention may have a PAM sequence selected from the following: TNN, or NTN, where N can be A, T, C, or G. For example, once the PAM region of a candidate target is determined, those skilled in the art can easily determine the target sequence consisting of about 17 to about 25 nucleotides (e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25) downstream of the 3' end of the candidate target PAM, and can design a suitable gRNA for this target sequence.

[0089] For example, the regulation step includes introducing single-strand breaks, double-strand breaks, and / or mutations into the midstream, upstream, and / or downstream of the target nucleic acid sequence. For example, the regulation step includes increasing or decreasing the expression or activity of the candidate target at the genomic level. For example, the regulation step includes introducing a target regulation system containing a guide nucleic acid molecule and a nuclease into the test cell. For example, the guide nucleic acid molecule includes a guide RNA (gRNA) targeting the target. For example, the nuclease includes a Cas protein, a Cas protein homologue, or a functionally active fragment thereof. For example, the nuclease includes Cas 9 and / or Cas 12.

[0090] For example, in the cell population obtained by the regulation step of the present invention, the proportion of cells expressing the candidate target is reduced to less than about 95%. For example, it can be reduced to about 95-1%, such as about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, or about 1%.

[0091] For example, in the identification method of the present invention, in order to screen for effective targets, the test cells are regulated to target the candidate targets. For example, the expression or activity of the candidate targets is regulated in the test cells. For example, in the test cells, the expression or activity of the candidate target is regulated to be reduced to approximately 99-0.0001% of its original level, e.g., approximately 99%, approximately 98%, approximately 97%, approximately 96%, approximately 95%, approximately 94%, approximately 93%, approximately 92%, approximately 91%, approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, approximately 15%, approximately 14%, approximately 13%, approximately 12%, approximately 11%, approximately 10%, approximately 9%, approximately 8%, approximately 7%, approximately 6%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, approximately 1%, approximately 0.5%, approximately 0.4%, approximately 0.3%. %, approximately 0.2%, approximately 0.1%, approximately 0.09%, approximately 0.08%, approximately 0.07%, approximately 0.06%, approximately 0.05%, approximately 0.04%, approximately 0.03%, approximately 0.02%, approximately 0.01%, approximately 0.009%, approximately 0.008%, approximately 0.007%, approximately 0.006%, approximately 0.005%, approximately 0.004%, approximately 0.003%, approximately 0.002%, approximately 0.001%, approximately 0.0009%, approximately 0.0008%, approximately 0.0007%, approximately 0.0006%, approximately 0.0005%, approximately 0.0004%, approximately 0.0003%, approximately or 0.0002%, or approximately 0.0001%. For example, in the test cells, the expression or activity of the candidate target is modulated to increase by approximately 0.1% to approximately 10,000-fold, such as approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately... 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 20 times, approximately 30 times, approximately 40 times, approximately 50 times, approximately 60 times, approximately 70 times, approximately 80 times, approximately 90 times, approximately 100 times, approximately 1000 times, or approximately 10000 times. For example, the test cells are divided into two groups, and the expression or activity of the candidate target is regulated to decrease or increase in the regulated test cell group compared to the unregulated test cell group. For example, the expression or activity of the candidate target is regulated to decrease or increase in the regulated test cells compared to the unregulated test cells.

[0092] For example, statistical analysis can be performed on the enriched guideRNAs in the selected enriched cells. For instance, based on the quantity of enriched guideRNAs, the corresponding candidate targets can be ranked, and the top 1 to top 1000 candidate targets can be identified, such as the top 1, top 2, top 3, top 4, top 5, top 6, top 7, top 8, top 9, top 10, top 15, top 20, top 25, top 30, top 35, top 40, top 45, top 50, top 60, top 70, top 80, top 90, top 100, top 200, top 300, top 400, top 500, top 600, top 700, top 800, top 900, or top 1000, as targets that can influence cells. For example, based on the amount of guideRNA enrichment, the corresponding candidate targets are ranked, and the candidate targets ranked from the bottom 1 to the top 1000 are identified, such as the bottom 1, top 2, top 3, top 4, top 5, top 6, top 7, top 8, top 9, top 10, top 15, top 20, top 25, top 30, top 35, top 40, top 45, top 50, top 60, top 70, top 80, top 90, top 100, top 200, top 300, top 400, top 500, top 600, top 700, top 800, top 900, or top 1000, as targets that can enhance cell killing function after cell knockout.

[0093] For example, the test cells in this invention include immune cells, preferably cells that are not readily available in large quantities. For example, the test cells in this invention comprise approximately 20% to 0.001% or less of the total number of human peripheral blood lymphocytes, such as 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less, or 0.001% or less. For example, the immune cells in this invention include phagocytes, lymphocytes, neutrophils, eosinophils, and / or basophils. For example, the immune cells in this invention include monocytes, macrophages, and / or dendritic cells. For example, the immune cells in this invention are derived from stem cell-differentiated immune cells. For example, the stem cells in this invention include induced pluripotent stem cells (iPSCs), embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, and / or peripheral blood stem cells. For example, the immune cells described in this invention include B cells, T cells, regulatory T cells, natural killer cells, and / or natural killer-like T cells (NKT). For example, the immune cells described in this invention include αβ T cells and / or γδ T cells. For example, the immune cells described in this invention include tumor-infiltrating lymphocytes (TILs). For example, the TILs described in this invention are TILs derived from tumor tissue, tumor-associated lymph nodes with or without tumor metastasis, metastatic lesions, fragments of adjacent normal tissue, pleural effusion, and / or ascites, and / or TILs derived from cryopreservation followed by thawing. Prior to amplification and genetic modification, the source of the cells (e.g., immune effector cells (e.g., T cells or NK cells)) is obtained from the subject. The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0094] On one hand, the present invention provides a composition comprising a plurality of test cells, wherein the expression or activity of a candidate target in the plurality of test cells is regulated by a plurality of target regulation systems, each of which targets a different candidate target site; wherein the number of test cells is at least 5 times the number of target regulation systems.

[0095] On one hand, the present invention provides a system for identifying targets, the system comprising: a measurement module for measuring the number of target regulatory systems in a plurality of test cells and determining the effect of regulating candidate targets on cell function; prior to and / or simultaneously with the measurement, regulating the expression or activity of candidate targets in the plurality of test cells by a plurality of target regulatory systems, each target regulatory system targeting a different candidate target site; and at the time of the measurement, the number of test cells is at least 5 times the number of target regulatory systems.

[0096] On one hand, the present invention provides a system for identifying targets, the system comprising:

[0097] Module (1) regulates the expression or activity of candidate targets in multiple test cells through multiple target regulation systems, with each target regulation system targeting different candidate target sites;

[0098] Module (2) determines the effect of regulating the candidate target on cell function by measuring the number of target regulatory systems in multiple test cells, wherein the number of test cells in module (2) is at least 5 times the number of target regulatory systems;

[0099] Module (1) and module (2) may be arranged in parallel, or module (1) may be arranged before module (2).

[0100] On one hand, the present invention provides a system for identifying targets, the system comprising the following modules for implementing the method provided by the present invention:

[0101] Module (1) is used to simultaneously or sequentially introduce components of multiple target regulatory systems selected from CRISPR system, zinc finger nuclease system, TALEN system, large-scale nuclease system and / or repressive RNA into the cells to be tested. The target regulatory system increases or decreases the expression or activity of the candidate target, and each target regulatory system targets different candidate target sites.

[0102] Module (1-a) is used to enable the test cells to express exogenous cell receptors or functional fragments thereof;

[0103] Module (1-b) is used to co-culture test cells (cells that have been or are about to be regulated to candidate targets) with the specific antigen of the exogenous cell receptor or its functional fragment or cells expressing the specific antigen, so that the test cells are essentially rendered incapable of killing.

[0104] Module (2) determines the effect of regulating the candidate target on cell function by measuring the number of target regulatory systems in multiple test cells, wherein the number of test cells in module (2) is at least 5 times the number of target regulatory systems;

[0105] There is no order restriction between modules (1), (1-a), and (1-b), and modules (1-b) can be arranged in parallel arbitrarily.

[0106] Exemplary Implementation

[0107] 1. A method for identifying a target, comprising determining the effect of regulating candidate targets on cell function by measuring the number of target regulatory systems in a plurality of test cells; regulating the expression or activity of candidate targets in a plurality of test cells by a plurality of target regulatory systems, each target regulatory system targeting a different candidate target site, prior to and / or simultaneously with the measurement; and at the time of the measurement, the number of test cells is at least 5 times the number of target regulatory systems.

[0108] 2. A method for identifying a target, the method comprising the following steps: (1) regulating the expression or activity of a candidate target in a plurality of test cells by a plurality of target regulation systems, each target regulation system targeting a different candidate target site; and (2) determining the effect of regulating the candidate target on cell function by measuring the number of target regulation systems in the plurality of test cells, wherein the number of test cells in step (2) is at least 5 times the number of target regulation systems.

[0109] 3. The method according to any one of embodiments 1-2, wherein the test cells comprise immune cells, preferably NK cells, NKT cells, macrophages, and / or γδT cells (such as Vδ1 subset, Vδ2 subset).

[0110] 4. The method as described in any one of embodiments 1-3, wherein the test cells are co-cultured with tumor antigens before and / or simultaneously with the assay.

[0111] 5. The method according to any one of embodiments 1-4, wherein the test cells are co-cultured with tumor cells before and / or simultaneously with the assay, the test cells expressing receptors that recognize tumor antigens, the tumor antigens being expressed on the tumor cells.

[0112] 6. The method as described in any one of embodiments 4-5, wherein the co-cultivation comprises at least one stage of co-cultivation.

[0113] 7. The method as described in Implementation Scheme 6, wherein the co-cultivation in each stage lasts for at least 12 hours.

[0114] 8. The method according to any one of embodiments 4-7, wherein after the co-culture is completed, the cell-killing ability of the test cells is significantly reduced (e.g., to 50% or less of the initial killing ability) and / or essentially loses its cell-killing ability.

[0115] 9. The method of any one of embodiments 1-8, wherein the regulation step comprises introducing components of a target regulation system into the test cells, wherein the target regulation system increases or decreases the expression or activity of the candidate target.

[0116] 10. The method of any one of embodiments 1-9, wherein the conditioning step comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a wide range of nuclease systems, and / or repressive RNA.

[0117] 11. The method of any one of embodiments 1-10, wherein the regulation step comprises introducing a specific nucleic acid sequence targeting the candidate target, introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation in the middle, upstream and / or downstream of the specific nucleic acid sequence.

[0118] 12. The method of any one of embodiments 1-11, wherein the regulation step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

[0119] 13. The method of any one of embodiments 1-12, wherein the regulation step comprises introducing a target regulation system containing a guide nucleic acid molecule and a nuclease into the cell to be tested.

[0120] 14. The method of embodiment 13, wherein the guiding nucleic acid molecule comprises a guide RNA (gRNA) targeting the target.

[0121] 15. The method of any one of embodiments 13-14, wherein the nuclease comprises Cas protein, Cas protein homologue, or a functionally active fragment thereof.

[0122] 16. The method of any one of embodiments 13-15, wherein the nuclease comprises Cas 9 and / or Cas 12.

[0123] 17. The method of any one of embodiments 1-16, wherein in the test cell population obtained by the conditioning step, the proportion of cells expressing the candidate target is reduced to less than about 95%.

[0124] 18. A composition comprising a plurality of test cells, wherein the expression or activity of a candidate target in the plurality of test cells is regulated by a plurality of target regulation systems, each of which targets a different candidate target site; wherein the number of test cells is at least 5 times the number of the target regulation systems.

[0125] 19. A system for identifying a target, the system comprising: a assay module for measuring the number of target regulatory systems in a plurality of test cells and determining the effect of regulating candidate targets on cell function; prior to and / or simultaneously with the assay, regulating the expression or activity of candidate targets in the plurality of test cells by a plurality of target regulatory systems, each target regulatory system targeting a different candidate target site; and at the time of the assay, the number of test cells is at least 5 times the number of target regulatory systems.

[0126] 20. A system for identifying a target, the system comprising:

[0127] Module (1) regulates the expression or activity of candidate targets in multiple test cells through multiple target regulation systems, with each target regulation system targeting different candidate target sites;

[0128] Module (2) determines the effect of regulating the candidate target on cell function by measuring the number of target regulatory systems in multiple test cells, wherein the number of test cells in module (2) is at least 5 times the number of target regulatory systems;

[0129] Module (1) and module (2) may be arranged in parallel, or module (1) may be arranged before module (2).

[0130] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the methods and uses of the present invention and are not intended to limit the scope of the invention.

[0131] Example

[0132] Example 1: Screening Platform Screening Process

[0133] This invention designs a target gene screening process, which employs an optimized culture method to obtain test cells with sustained tumor cell killing ability, used to identify target genes that can affect cell function in practical application environments. The screening process provided by this invention is shown in Figure 1.

[0134] Exemplary cell culture methods

[0135] NK cells

[0136] PBMCs (peripheral blood mononuclear cells) are derived from blood samples from healthy donors. PBMCs frozen in liquid nitrogen are resuscitated and cultured using EasySep. TM Human NK Cell Isolation Kit (Stemcell) for NK cell isolation, CTS culture medium. TM NK-Xpander TM(Thermo) medium + 10% AB Serum (Gemini) centrifuged and resuspended to 2E6 / ml (2×10⁻⁶). 6 (each cell / ml) overnight culture, using The NK cell expansion kit P01 (Excell) was used to coat 12-well plates (Corning) according to the instructions. The next day, the sorted and cultured NK cells were added to the pre-coated plates, and cytokines (Excell) were added to activate the NK cells. The cells were cultured for about 72 hours.

[0137] NKT cells

[0138] NKT cells can be derived from PBMCs, for example, by collecting human PBMCs from healthy donors and isolating and expanding them to obtain iNKT cells (PB-iNKT). NKT cells can be further expanded using PBMCs / α-GalCer as feeder cells.

[0139] γδT cells

[0140] PBMCs frozen in liquid nitrogen were revived and cultured. The concentration of PBMCs was adjusted to 2.5E6 / ml using T-cell culture medium (RPMI-1640 + 10% FBS + 100 IU / ml IL-2) and cultured overnight. After overnight culture, zoledronic acid was added to a final concentration of 5 μM for activation for 3 days. After 3 days, γδT cells were isolated and purified using the TCRγ / δ+T Cell Isolation Kit, human (Miltenyi). After isolation, γδT cells were adjusted to 1.5E6 / ml using T-cell culture medium and then expanded.

[0141] High-throughput screening library construction

[0142] (1) Target gene screening library plasmid construction

[0143] The design of the in vitro target gene screening library was based on the Brunello library reference by JG Doench et al. (DOI:10.1038 / nbt.3437). For example, it can contain 126 gRNAs, including 106 gRNAs that target 53 genes (each gene has 2 gRNA target sites) and 20 control gRNAs that are not targeted or target non-functional regions, as a quality control parameter.

[0144] First, the oligo sequence of the screening library was synthesized, with 25 nt homologous arms at both ends. A single round of PCR amplification based on the homologous arm sequences was performed to obtain the dsDNA fragment of the screening library. Then, the dsDNA fragment of the library was assembled into a pre-designed retroviral expression vector using a Gibson kit. This vector also carries an easily stained surface marker gene, which can be used to indicate the transduction and expression of the library in NK and NKT cells, and can also be used for enrichment of transduction-positive cells. The assembled library plasmid was electroporated into competent Endura cells and cultured overnight to amplify the library plasmid, and then the plasmid was extracted to prepare the library plasmid.

[0145] High-throughput NGS sequencing was used to perform quality control on the prepared library plasmids, and the coverage and distribution of gRNA were detected to confirm that the library used in the screening platform of this invention meets the library construction standards. Specifically, the sequencing data alignment rate of the plasmid library is greater than 70%, the gRNA coverage is greater than 99%, the gene coverage is 100%, and the gini coefficient of gRNA is less than 0.3.

[0146] (2) Construction of target genome knockout retroviral library

[0147] Retroviruses with RD114 or GALV envelopes containing the target genome selection library were packaged in HEK293T cells and concentrated 20-fold. The viral titer of the selection library was tested on Jurkat cells, with a biological titer of at least 2E7 / ml. The retroviruses carrying the selection library and retroviruses expressing CD19CAR (FMC63) were transduced into activated human NK and NKT cells according to the exemplary method of this invention. The transduction efficiency of the selection library was controlled at 30-40%, and the probability of two retroviruses carrying different gRNAs entering the same cell was controlled at around 10% to reduce the impact on subsequent results analysis.

[0148] Cell-transduced CAR and target genome knockout retroviral libraries

[0149] An exemplary pretreatment method for viral transduction used in this invention. One day before viral transduction, 24-well or 6-well suspension culture plates are coated with recombinant human fibrin fragment (Retronectin, Takara) at a final concentration of 15 μg / mL, using 250 μL per well of a 24-well plate and 1250 μL per well of a 6-well plate. The plates are protected from light and incubated overnight at 4°C. The coated plates are then removed, the coating solution is discarded, and blocking buffer containing 2% BSA is added for blocking at room temperature for 30 minutes. The blocking buffer is discarded, and the plates are washed twice with washing buffer containing 2.5% HEPES, and the washing buffer is discarded.

[0150] The exemplary chimeric antigen receptor transduction method used in this invention. In the experimental group, viral transduction was performed using an appropriate amount of viral dilution, and the retroviral vector contained a sequence encoding a CD19-targeting CAR. For example, the CD19 CAR of this invention has a CD19-targeting scFv, a hinge region derived from CD8, a transmembrane region derived from CD28, an intracellular co-stimulatory domain derived from CD28, and an intracellular signaling domain derived from CD3z.

[0151] The viral vector can contain guide RNA (which can be used as a screening library) for the CRISPR system. Centrifuge at 32°C, 2000g for 2 hours. Discard the supernatant in the plate and add an appropriate volume of CD19CAR-transduced NK or NKT cells to each well, with a cell concentration of approximately 1 × 10⁻⁶ cells. 6 Cells / mL. Centrifuge at 30-32℃, 1000g, for 10 minutes. After centrifugation, place the culture plate in a 37℃, 5% CO2 incubator to obtain transduced cells.

[0152] Six hours after transduction until the next day, the experimental group was transduced with an appropriate amount of viral dilution using the aforementioned method. The retroviral vector contained a target genome screening library, and transduced cells were obtained.

[0153] After transduction, the cells were cultured for about 3 days to enrich the virus-transduced positive cell population.

[0154] By using marker proteins from the screening library and magnetic beads carrying corresponding antibodies, T cells that are positive for viral transduction from the screening library are enriched. Then, the Lonza electroporation system is used to electroporate Cas9 protein for targeted gene editing.

[0155] Cellular introduction of CRISPR-Cas system

[0156] The method for generating Cas9 cells used in embodiments of the present invention involves electroporating Cas9 (Kactus Biosystem: Cas9 Nuclease) with cells using a Lonza electroporator. After Cas9 protein electroporation, appropriate amounts of NK and NKT cell culture medium are added according to the cell count density and viability, along with cytokines, to adjust the initial cell density to 1 × 10⁻⁶ cells / year. 6 The cells were cultured at a rate of 10 cells / ml. After approximately 7–10 days, it was confirmed that the NK / NKT cells had recovered their normal proliferation status.

[0157] The co-culture of CAR-NK or NKT with tumor cells of the present invention

[0158] This invention employs an optimized culture method to obtain test cells with sustained target cell killing ability. Specifically, Nalm-6 cells expressing natural CD19 antigen are co-incubated with edited CAR-NK and NKT cells, and the test cell population is obtained through multiple rounds of target cell addition.

[0159] At the start of co-culture, edited CAR-NK / NKT cells were co-cultured with Nalm-6 cells at an effector cell (test immune cells, E) to target cell (Nalm-6 cells, T) ratio of 4:1 to 1:1. For example, co-cultured for approximately 2 days at an E:T ratio of approximately 4:1 to 1:1, such as a 4:1 ratio of live NK cells to live Nalm-6 cells.

[0160] Two days later, the same amount of Nalm-6 cells as in the first round of co-culture were added to the co-culture system, and co-cultured for about 2 to 4 days.

[0161] Two days later, add half the amount of Nalm-6 cells added in the first round of co-culture to the above co-culture system and co-culture for about 2 to 4 days.

[0162] Through the optimized culture method described above, CAR-NK / NKT cells with sustained target cell killing ability were obtained after screening.

[0163] Functional Filtering Model Construction and Filtering Process

[0164] Functional screening models can screen out CAR-NK / NKT cells with sustained target cell killing ability, thereby discovering target genes that can significantly enhance the sustained target cell killing function of NK / NKT cells.

[0165] This invention tests CAR-NK / NKT cells to obtain a CAR-NK / NKT cell population with corresponding sustained target cell killing ability after 2-3 rounds of target cell stimulation. For example, after 2 rounds of target cell stimulation, CAR-NK / NKT cells with sustained target cell killing ability are collected. Finally, the cell genome is extracted, and enriched gRNA sequences are captured by high-throughput sequencing and bioinformatics analysis, which can then screen for potential genes that can enhance the sustained target cell killing ability of NK / NKT cells.

[0166] Example 2: Cell co-culture in the screening system of the present invention

[0167] CAR-mediated CD19 targeting and screening libraries were expressed in NK or NKT cells from two different donor PBMCs to obtain effector cells; Nalm-6 cells expressing the CD19 antigen were used as target cells. Co-incubation began (Day 0) with edited CAR-NK / NKT cells and Nalm-6 cells co-cultured at a ratio of effector cells (test immune cells, E):target cells (Nalm-6 cells, T) of 4:1. Each experimental group was co-cultured for approximately 2 days at an E:T ratio of 4:1. After 2 days, the same amount of Nalm-6 cells as in the first round of co-culture was added to the co-culture system, and co-cultured for approximately 2–4 days.

[0168] Figure 2 shows the effector cell (test immune cells, E) : target cell (Nalm-6 cells, T) ratio at 4:1, with target cells added after 2 days. The effector cell ratio relative to target cells was measured at days 0, 2, 4, and 6 of co-culture. Further analysis of cell numbers showed that after 6 days of co-culture, the number of tumor cells proliferated exceeded the number of test cells killed. Compared to the first round of co-culture from day 0 to day 2, which essentially completely eliminated tumor cells, the cytotoxic ability of the test cells was significantly reduced on day 6. The number of remaining undamaged tumor cells on day 6 was 259% of the number of tumor cells added at the beginning of this round of co-culture on day 2, indicating that the test cells had essentially lost their cytotoxic ability by day 6. The remaining effector cells were essentially in a state of functional decline, which is beneficial for screening and identifying genes related to antagonistic exhaustion states.

[0169] Figure 3 shows the effector cell (tested immune cells, E) : target cell (Nalm-6 cells, T) ratio at 4:1, with target cells added after 2 days. The number of immune cells / gRNA library coverage was measured at 0, 2, 4, and 6 days of co-culture. The results showed that after 6 days of co-culture, the final cell number / gRNA library coverage ratio was greater than 1000, with sufficient cells remaining for subsequent sequencing validation.

[0170] Example 3: Number of effector cells in the screening system of the present invention

[0171] In target gene screening systems, insufficient numbers of NK or NKT cells used as test cells can lead to inaccurate screening results. Specifically, a low harvest number increases the risk of random gRNA loss, which in turn results in the absence of the selected gRNA in the genome extraction and sequencing results. This can lead to inaccurate interpretation of sequencing results: the absence of the gRNA in the sequencing could be interpreted as either random gRNA loss or as the inability of the target gene knocked out by the gRNA to improve the function of the test cells, resulting in their rejection by the screening system.

[0172] Therefore, to avoid the problem of a certain gRNA being missing in the sequencing results, the screening system of the present invention provides a method to ensure that the risk of random gRNA loss is controllable by determining the minimum amount of harvested cells.

[0173] Figure 4 shows the results of gRNA detection loss percentage (vertical axis) corresponding to different cell / gRNA coverage (horizontal axis).

[0174] The vertical axis represents the percentage of undetected gRNAs; the horizontal axis represents the cell coverage ratio (number of cells harvested relative to the number of gRNAs in the screening library). After fitting the data to real data, cell / gRNA coverage was set from 1 to 200X to predict the corresponding percentage of undetected gRNAs (uncovered fraction of sgRNAs). The prediction results showed that when the number of cells harvested was 5 times or more than the number of gRNAs in the screening library, the probability of a gRNA sequencing count of 0 could be reduced to below 10%.

[0175] Example 4: Targets obtained in the screening system of the present invention

[0176] Figure 5 shows the gene screening results obtained by the screening system of the present invention. Specifically, the screening system of the present invention identified CISH as a target affecting NK / NKT cell function. This target is also a recognized target in the art for enhancing the function of NK or NKT immune cells. Existing literature reports that CISH knockout NK cells exhibit improved proliferative capacity and enhanced cytotoxic activity against various tumor cells; CISH knockout cells have shown significantly improved in vivo survival and inhibitory effects on tumor progression in a leukemia xenograft model. Furthermore, the screening system of the present invention identified AAVS1 as a target that does not affect NK / NKT cell function. Existing literature reports that AAVS1 is a safe harbor target for NK cells, meaning that knocking out AAVS1 in NK cells has no effect on NK cell function.

[0177] Therefore, the screening system of the present invention can effectively screen for targets that enhance the function of immune cells.

[0178] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments described herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. A method for identifying a target, the method comprising the following steps: (1) Provide multiple cells to be tested; (2) The test cells are contacted with multiple target regulatory systems to regulate the expression or activity of candidate targets in the test cells, each target regulatory system targeting different candidate target sites; preferably, each test cell contains one target regulatory system; (3) Contact the test cells with the identification model, and then determine the presence and / or number of target regulatory systems in multiple test cells; as well as (4) Based on the reading results obtained from the measurement, determine the effect of regulating the candidate target on the test cells, thereby identifying the candidate target.

2. The method of claim 1, wherein in the assay, the number of cells to be tested is at least 5 times the number of the target regulation system.

3. The method according to any one of claims 1-2, wherein the test cells comprise immune cells, preferably NK cells, NKT cells, macrophages, and / or γδT cells (such as Vδ1 subset, Vδ2 subset).

4. The method of any one of claims 1-3, wherein the identification model comprises a tumor antigen before and / or simultaneously with the assay.

5. The method of any one of claims 1-4, further comprising introducing a receptor that recognizes a tumor antigen into the cells to be tested.

6. The method of claim 5, wherein the antibody that recognizes the tumor antigen is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

7. The method of any one of claims 1-6, wherein the identification model is tumor cells, and the test cells are co-cultured with tumor cells before and / or simultaneously with the assay, wherein the test cells express receptors that recognize tumor antigens, and the tumor antigens are expressed on the tumor cells.

8. The method of claim 7, wherein the co-cultivation comprises at least one stage of co-cultivation.

9. The method of claim 8, wherein the co-cultivation in each stage lasts for at least 12 hours.

10. The method of any one of claims 7-9, wherein after the co-culture is completed, the killing effect of the test cells on tumor cells is significantly reduced (e.g., to 50% or less of the initial killing ability).

11. The method of any one of claims 1-10, wherein the regulation step comprises introducing components of a target regulation system into the test cells, wherein the target regulation system increases or decreases the expression or activity of the candidate target.

12. The method of any one of claims 1-11, wherein the conditioning step comprises providing a CRISPR system, a zinc finger nuclease system, a TALEN system, a wide range of nuclease systems, and / or repressive RNA.

13. The method of any one of claims 1-12, wherein the regulation step comprises introducing a specific nucleic acid sequence targeting the candidate target, and introducing single-strand breaks, double-strand breaks, mutations and / or expression regulation in the middle, upstream and / or downstream of the specific nucleic acid sequence.

14. The method of any one of claims 1-13, wherein the regulation step comprises increasing or decreasing the expression or activity of the candidate target at the genomic level.

15. The method of any one of claims 1-14, wherein the regulation step comprises introducing a target regulation system containing guiding nucleic acid molecules and nucleases into the cell to be tested.

16. The method of claim 15, wherein the guiding nucleic acid molecule comprises a guide RNA (gRNA) targeting the target.

17. The method of any one of claims 15-16, wherein the nuclease comprises Cas protein, Cas protein homologue, or a functionally active fragment thereof.

18. The method of any one of claims 15-17, wherein the nuclease comprises Cas 9 and / or Cas 12.

19. The method of any one of claims 1-18, wherein in the cell population to be tested obtained by the conditioning step, the proportion of cells expressing the candidate target is reduced to less than about 95%.

20. A composition comprising a plurality of test cells, wherein the expression or activity of a candidate target in the plurality of test cells is regulated by a plurality of target regulatory systems, each target regulatory system targeting a different candidate target site; wherein, The number of cells to be tested is at least 5 times the number of the target regulation system.

21. A system for identifying a target, the system comprising: a assay module for measuring the number of target regulatory systems in a plurality of test cells and determining the effect of regulating candidate targets on cell function; prior to and / or simultaneously with the assay, regulating the expression or activity of candidate targets in the plurality of test cells by a plurality of target regulatory systems, each target regulatory system targeting a different candidate target site; and at the time of the assay, the number of test cells is at least 5 times the number of target regulatory systems.

22. A system for identifying a target, the system comprising: Module (1) regulates the expression or activity of candidate targets in multiple test cells through multiple target regulation systems, with each target regulation system targeting different candidate target sites; Module (2) determines the effect of regulating the candidate target on cell function by measuring the number of target regulatory systems in multiple test cells, wherein the number of test cells in module (2) is at least 5 times the number of target regulatory systems; Module (1) and module (2) may be arranged in parallel, or module (1) may be arranged before module (2).

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