Compositions and methods for modulating genetic driving factors

Abstract

In various embodiments, the disclosure provides compositions, such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors, or host cells, comprising and / or modulating the expression or activity of an immunomodulatory-related protein, such as a cytokine. In various embodiments, the disclosure also provides for the treatment of a disease or condition (e.g., a disease or disorder) using an agent comprising and / or modulating the expression or activity of an immunomodulatory-related protein. The present invention relates to methods for identifying such agents (e.g., diseases or conditions associated with whole genome association studies (GWAS), cancer genome maps (TCGA), whole genome sequencing, whole phenotype group association studies (PheWAS), expression quantitative trait locus (eQTL) studies, or combinations thereof), and methods for identifying such agents.

Description

Materials Incorporated by Reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 345,787, filed May 25, 2022. The entire teachings of the above application are incorporated herein by reference. Incorporation of XML Materials by Reference

[0002] This application incorporates by reference the sequence listing contained in the following eXtensible Markup Language (XML) file filed simultaneously herewith: a) File name: 57081058002.xml; created on May 24, 2023, size: 8,059,812 bytes. Background Art

[0003] The lack of effective therapeutic agents and methods for diagnosis, prevention and / or treatment has led to unmet medical needs for many diseases and conditions. Many diseases and conditions have genetic components that may affect or directly cause dysfunction. The genetic tendency to suffer from a disease or condition may be caused by changes at a single genetic locus, or may be the result of changes in many genetic loci. The identification of novel genetic drivers of diseases and conditions provides opportunities for developing and / or improving the diagnosis, prevention and / or treatment options of various diseases and conditions. Summary of the invention

[0004] The present disclosure provided herein is based, in part, on the identification of non-canonical protein targets (eg, proteins encoded by non-canonical open reading frames (ORFs)) that are altered (eg, mutated) in various diseases, disorders, or pre-disease phases.

[0005] On the one hand, the disclosure relates to a medicament comprising a target protein identified herein (e.g., a target protein in a sequence table, Table A, or a variant thereof) and / or regulating (e.g., increasing or decreasing) the expression and / or activity of the target protein. In some embodiments, the medicament comprises a target protein identified herein (e.g., a target protein in a sequence table, Table A, or a variant thereof). In certain embodiments, the medicament regulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., a target protein in a sequence table, Table A, or a variant thereof). In some embodiments, the medicament comprises, is essentially composed of, or is composed of a polypeptide, a polynucleotide, a gene editing system, a small molecule, or a cell (e.g., a cell therapy). The medicament may be an inhibitor or activator of a target protein identified herein. In some embodiments, the medicament regulates the expression of a target protein identified herein. In some embodiments, the medicament regulates the activity of a target protein identified herein.

[0006] In another aspect, the present disclosure provides a pharmaceutical composition comprising a target protein identified herein and a pharmaceutically acceptable carrier.

[0007] In another aspect, the present disclosure provides a pharmaceutical composition comprising an agent that modulates the expression or activity of a target protein identified herein and a pharmaceutically acceptable carrier.

[0008] In other aspects, the disclosure relates to a polynucleotide encoding a polypeptide described herein, an expression vector comprising a polynucleotide encoding a polypeptide described herein, and a host cell comprising a polynucleotide encoding a polypeptide described herein.

[0009] In another aspect, the present disclosure provides a method for detecting a disease or condition in a subject or determining the likelihood that a subject suffers from the disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood that the subject suffers from the disease or condition. In certain embodiments, the disease or disorder is associated with genome-wide association studies (GWAS, see, e.g., www.genome.gov / about-genomics / fact-sheets / Genome-Wide-Association-Studies-Fact-Sheet), The Cancer Genome Atlas (TCGA, see, e.g., www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga), whole genome sequencing, phenome-wide association studies (PheWAS, see, e.g., https: / / phewascatalog.org / ), expression quantitative trait loci (eQTL) studies (see, e.g., Nica and Dermitzakis, Expression quantitative trait loci: present and future, Philos Trans R Soc Lond B Biol Sci. [Philosophical Transactions of the Royal Society of London B: Biological Sciences] 368(1620):20120362(2013) and www.ebi.ac.uk / eqtl / ) or a combination thereof (e.g., the diseases listed in paragraphs

[0219] and

[0220] ).

[0010] In another aspect, the present disclosure provides a method of preparing a sample that can be used to determine the likelihood that a subject has a disease or condition, the method comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide or a combination thereof to the sample to prepare a sample that can be used to detect the possibility of suffering from cancer; and c) quantifying the expression or activity of the target protein in the sample prepared in step b).

[0011] In some embodiments, the method further comprises treating a subject predicted to have a likelihood of developing the disease or condition, the treatment comprising administering to the subject an effective amount of an agent or a pharmaceutical composition comprising the agent, the agent comprising the target protein identified herein and / or modulating the expression or activity of the target protein.

[0012] In another aspect, the disclosure provides a method of treating a disease or condition in a subject in need thereof (e.g., a human subject suffering from cancer), the method comprising administering to the subject an effective amount of an agent or a pharmaceutical composition comprising the agent, the agent comprising a target protein identified herein and / or modulating the expression or activity of the target protein.

[0013] In another aspect, the present disclosure provides a method for selecting a subject suitable for treating a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, and selecting the subject suitable for treating the disease or condition based on the expression or activity level of the target protein in the sample.

[0014] On the other hand, the present disclosure provides a method for modulating the expression or activity of a target protein identified in the sequence listing, Table A, or a variant thereof in a cell (e.g., a cancer cell, such as a cancer cell of a subject), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent or a pharmaceutical composition comprising the agent, the agent comprising the target protein identified herein and / or modulating the expression or activity of the target protein.

[0015] In another aspect, the disclosure provides a method of identifying an agent that modulates the expression or activity of a target protein identified herein, the method comprising: a) contacting the target protein with an agent; and b) determining whether the agent modulates the expression or activity of the target protein, wherein a difference in the expression or activity of the target protein that has been contacted with the agent compared to a reference for the expression or activity of the target protein indicates that the agent modulates the expression or activity of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments.

[0017] Figure 1 The effect of SEQ ID NO:5391 on the cell proliferation of colon cancer cell line (HCT-116) is shown. WST-1 assay was performed on HCT-116 to check its cell viability and growth when transiently overexpressing SEQ ID NO:5391 using Lipofectamine 3000. After plasmid transfection, WST-1 assay was used to measure cell proliferation 48 hours later. Data are presented as mean ± SD of three independent experiments performed in triplicate.

[0018] Figure 2A-2C The analysis of cell viability of lung cancer (A549), liver cancer (HepG2) and colon cancer (HCT-116) using WST-1 assay is shown. Cells were transfected with SEQ ID NO:4538, SEQ ID NO:5392 and SEQ ID NO:4335 individually. Labeled pcDNA3.1-Myc was used as a negative control. After plasmid transfection, cell proliferation was measured using WST-1 assay 48 hours later. Data are presented as mean ± SD of three independent experiments performed in triplicate. Figure 2A ) Results of WST-1 in HCT-116 cells. Upper panel: low serum, lower panel: high serum. Figure 2B ) Results of WST-1 in HepG2 cells. Upper panel: low serum, lower panel: high serum. Figure 2C ) Results of WST-1 in A549 cells. Upper panel: low serum, lower panel: high serum.

[0019] Figure 3 Shown are the relative secretion levels of 4 different forms of SEQ ID NO: 4371 that differ by one amino acid at the human GWAS position for type 2 diabetes.

[0020] Figure 4 The effect of SEQ ID NO:5404 on the cell proliferation of breast cancer cell line (MCF-1) is shown. WST-1 assay is performed on MCF-7 to check its cell viability and growth when stably overexpressed. Cell proliferation is measured using WST-1 assay. Data are presented as mean ± SD of three independent experiments performed in triplicate. DETAILED DESCRIPTION

[0021] Following is a description of an exemplary embodiment. Target protein

[0022] On the one hand, the present disclosure provides a target protein identified herein. As used herein, the expression "target protein identified herein" and "target protein disclosed herein" include polypeptides disclosed in the sequence table (e.g., comprising a target protein selected from SEQ ID NO: 5391, SEQ ID NO: 5392, SEQ ID NO: 4335, SEQ ID NO: 4538, SEQ ID NO: 4371, SEQ ID NO: 5404, the amino acid sequence of SEQ ID NO in Table C herein), variants thereof (e.g., comprising a target protein selected from SEQ ID NO: 4335_64NS, SEQ ID NO: 4335_P43A, SEQ ID NO: 5391_P31L, SEQ ID NO: 4371rs221797V to A, V to G or V to D) and peptides disclosed in Table A herein. The target protein can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0023] In some embodiments, the target protein is an intracellular protein. In some embodiments, the target protein is an extracellular protein (e.g., a secreted protein). In certain embodiments, the target protein is a transmembrane protein. In specific embodiments, the target protein is membrane-bound and extracellular, but not transmembrane. In more specific embodiments, the target protein is embedded in a membrane, but is not transmembrane.

[0024] In various embodiments, the target protein is a protein comprising an amino acid sequence as shown in the sequence listing or Table A. In some embodiments, the target protein consists of an amino acid sequence as shown in the sequence listing or Table A. In some embodiments, the target protein comprises an amino acid sequence having an amino acid substitution relative to the amino acid sequence as shown in the sequence listing or Table A, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence listing or Table A with a methionine (Met) residue. In some embodiments, the target protein consists of an amino acid sequence having an amino acid substitution relative to the amino acid sequence as shown in the sequence listing or Table A, wherein the substitution is to replace the N-terminal residue in the amino acid sequence in the sequence listing or Table A with a methionine (Met) residue. In some embodiments, the target protein comprises an amino acid sequence as shown in the sequence listing or Table A, and further comprises a methionine (Met) residue at its N-terminus. In some embodiments, the target protein consists of an amino acid sequence as shown in the sequence listing or Table A and a methionine (Met) residue at its N-terminus.

[0025] Table A. Target peptides of the present disclosure SEQ ID NO: sequence 5139 MP 5203 VAV

[0026] Without wishing to be bound by theory, it is believed that mutations in certain target proteins disclosed herein are associated with, contribute to or cause a disease, disorder, and / or pre-disease, wherein the mutation results in a loss or gain of a biological function that contributes to or mitigates the disease, disorder, and / or pre-disease. In some embodiments, mutations and / or target proteins are used as biomarkers or surrogates for a disease, disorder, and / or pre-disease.

[0027] Certain target proteins in the sequence listing and Table A have been identified as differentially expressed (e.g., upregulated or downregulated) in diseases, disorders and / or pre-disease states (e.g., the diseases listed in paragraphs

[0219] and

[0220] ) associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, compared to a reference state (e.g., a normal state), such that modulation of the level and / or activity of the target protein serves to treat, alleviate the disease or disorder, and / or prevent the onset of the disease or disorder.

[0028] As used herein, the term "differential expression" refers to at least one identifiable difference in protein expression. It can be a quantitatively measurable, semi-quantitatively estimable or qualitatively detectable difference in protein expression. Therefore, a differentially expressed protein or "DEP" can have a higher expression level in a reference state (e.g., a normal state) than in a disease state where the DEP has a lower expression level or is not expressed at all. Conversely, a DEP can have a higher expression level in a disease state than in a reference state (e.g., a normal state) where the DEP has a lower expression level or is not expressed at all. In addition, if a DEP is identifiably changed (e.g., mutated) between the two states under comparison, the expression can be considered to be differential. Identifiable changes can include amino acid substitutions, insertions and / or deletions (including N-terminal and C-terminal truncations) and modifications (e.g., post-translational modifications).

[0029] As used herein, the term "reference" refers to a standard for one or more comparison purposes. Those skilled in the art can select an appropriate reference for one or more specific comparison purposes. Thus, for example, a reference for a disease state can be a normal healthy state; a reference for a mutated protein can be a protein that has not been mutated; a reference for a disease treatment can be no treatment or can be a standard of care treatment. In some embodiments, particularly in embodiments involving methods for identifying agents that regulate the expression and / or activity of a target protein, the reference is the activity and / or expression of the target protein in the absence of an agent. In some embodiments, the reference is based on a predetermined level, such as based on functional expression or empirical determination. In some embodiments, the reference is obtained from a cell, sample, or subject (e.g., a cell or sample from a healthy subject, a subject not suffering from a specific disease; a healthy subject, a subject not suffering from a specific disease). In some embodiments, the reference is obtained from more than one cell (e.g., a cell population), sample, or subject (e.g., a cell or sample from a healthy subject, a subject not suffering from a specific disease; a healthy subject, a subject not suffering from a specific disease), such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more or a statistically significant number of cells, samples, or healthy subjects. References obtained from more than one cell, sample, or subject can be expressed as a statistic (eg, a mean or median).

[0030] In some embodiments, the expression level of the target protein in a disease or disorder (e.g., a disease or disorder associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof) is at least about 0.5-fold higher, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) compared to the expression level of the target protein in a reference (e.g., a sample of cells or tissue from a subject not suffering from the disease or disorder).

[0031] In some embodiments, the expression level of the target protein in a disease or disorder (e.g., a disease or disorder associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ) is at least about 0.5-fold lower, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) compared to the expression level of the target protein in a reference (e.g., a sample of cells or tissue from a subject not suffering from the disease or disorder). In some embodiments, the target protein is not expressed or is expressed at an undetectable level (e.g., as determined from a sample comprising or obtained from cells or tissue of a subject suffering from the disease or disorder) in a disease or disorder (e.g., a disease or disorder associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ).

[0032] In some embodiments, the transcript level of the target protein in a disease or disorder (e.g., a disease or disorder associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ) is at least about 0.5 times higher, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10 times higher (e.g., 50 times higher, 100 times higher) than the transcript level of the target protein in a reference (e.g., a sample of cells or tissues from a subject not suffering from the disease or disorder). In specific embodiments, an increase in the transcript level of the target protein contributes to (e.g., causes) a disease or disorder described herein.

[0033] In some embodiments, the transcript level of the target protein in a disease or disorder (e.g., a disease or disorder associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ) is at least about 0.5-fold lower, e.g., at least about: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) compared to the transcript level of the target protein in a reference (e.g., a sample of cells or tissue from a subject not suffering from the disease or disorder). In some embodiments, transcripts for the target protein are not expressed or are expressed at undetectable levels in the disease or disorder (e.g., as determined from a sample comprising or obtained from cells or tissues of a subject suffering from the disease or disorder). In specific embodiments, a reduction in transcript levels for the target protein contributes to (e.g., causes) a disease or disorder described herein.

[0034] In certain embodiments, the gene encoding the target protein in the diseases described herein comprises at least one mutation (eg, fusion, deletion, insertion, point mutation, and / or expansion of amino acid repeats).

[0035] In some embodiments, mutations and / or target proteins are used as markers for a disease or condition (e.g., a disease or condition associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ).

[0036] In certain embodiments, the target protein has a higher expression level in cells and / or tissues related to diseases or disorders described herein. In certain embodiments, the target protein expression level in cells and / or tissues is at least about 0.5 times higher than the target protein expression level in a reference (e.g., different cell / tissue types), for example, at least about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 times (e.g., 50 times higher, 100 times higher).

[0037] In certain embodiments, the target protein has a lower expression level in cells and / or tissues related to diseases or disorders described herein. In certain embodiments, the expression level of the target protein in cells and / or tissues is at least about 0.5 times lower than the expression level of the target protein in a reference (e.g., different cell / tissue types), for example, at least about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 times (e.g., 50 times lower, 100 times lower).

[0038] Non-limiting examples of (biological) samples include blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., biopsy or microbiopsy), pancreatic juice, chorionic villus samples, cells, and the like isolated from a subject.

[0039] In some embodiments, the target protein is translated from non-coding RNA. In some embodiments, the non-coding RNA is a long intergenic non-coding RNA (lincRNA). In certain embodiments, the non-coding RNA is a long non-coding RNA (lncRNA). In some embodiments, the non-coding RNA is a microRNA (miRNA or miR).

[0040] In some embodiments, the target protein is translated from non-exon elements in unprocessed precursor mRNA (pre-mRNA). In some embodiments, the non-exon element is an intron in the pre-mRNA. In some embodiments, the non-exon element is a 5' untranslated region (5'UTR) in the pre-mRNA. In some embodiments, the non-exon element is a 3' untranslated region (3'UTR) in the pre-mRNA.

[0041] In some embodiments, the target protein is 2,000 or less amino acids in length, such as 1000 or less amino acids, 750 or less amino acids, 500 or less amino acids, 250 or less amino acids, 150 or less amino acids, or 100 or less amino acids. In some embodiments, the target protein is 7 or more amino acids in length, such as 8, 9, 10, 15, 18, 25, 50, 75 or 100 or more amino acids. In certain embodiments, the target protein is from about 50 to about 200 amino acids in length, such as from about 100 to about 150 amino acids. In a specific embodiment, the target protein is 7 or more amino acids in length. In a more specific embodiment, the target protein is about 18 amino acids in length.

[0042] In some embodiments, the target protein of the present disclosure is a modulator of one or more GPCRs. In some embodiments, the target protein is an agonist of one or more GPCRs. In some embodiments, the target protein is an antagonist of one or more GPCRs. In some embodiments, the target protein is a direct modulator of one or more GPCRs, such as a ligand of one or more GPCRs. In some embodiments, the target protein is an indirect modulator of one or more GPCRs.

[0043] The expression and / or activity of various GPCRs have been linked to different diseases / disorders, conditions and indications, including those shown in Table B (see, e.g., Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71:267-315, April 2019; Harmar, AJ et al., IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels, Nucleic Acids Research, 2009, Vol. 37; and Davenport AP, Scully CCG, deGraaf C, Brown AJH and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev. 55:1-13. DrugDiscov. [Nature Review: Drug Discovery] June 19, 2020 (6): 389-413; the contents of each are incorporated herein by reference in their entirety). Therefore, in some embodiments, the target proteins disclosed herein (which are modulators of GPCRs) can be used to treat and / or diagnose one or more diseases / disorders, conditions and / or indications, such as cancer or precancerous conditions, or any of the diseases / disorders, conditions and indications listed in Table B that are known to be associated with GPCR expression and / or activity.

[0044] Table B. Disease-related GPCRs Agents that modulate target proteins

[0045] Provided herein are agents that regulate the expression of a target protein disclosed herein (such as a target protein in a sequence listing or Table A) or a variant of the foregoing or a fragment of the foregoing (e.g., a biologically active fragment of a target protein). The expression of a target protein or a variant or fragment thereof can be regulated directly or indirectly by a wide range of processes, resulting in an increase or decrease in the level of the target protein. Non-limiting examples include changes in: the number of copies of the gene encoding the target protein, transcription initiation, extension or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modification of a protein, protein stability, protein degradation (e.g., cleavage, such as protease cleavage), or a combination of the foregoing.

[0046] In some embodiments, the agent regulates (e.g., increases or decreases) the expression of a gene or gene transcript encoding a target protein. In some embodiments, the agent regulates the expression or activity of a target protein. In some embodiments, the agent reduces (e.g., inhibits, reduces or neutralizes) the activity of a target protein. In some embodiments, the agent increases (e.g., activates) the activity of a target protein. In some embodiments, the agent reduces (e.g., inhibits or downregulates) the expression of a target protein. In other embodiments, the agent increases (e.g., activates or upregulates) the expression of a target protein.

[0047] As used herein, the term "increasing" or "increase" refers to a regulation that results in a higher level of expression, activity, function, or a combination thereof or a measure (e.g., cancer cell death or DNA methylation at a target site) of a target protein relative to a reference (e.g., a level before or in the absence of an agent). In some embodiments, the agent increases the expression or activity or measure of the target protein by at least about 5% relative to a reference, such as by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to a reference.

[0048] As used herein, the term "decreasing" or "decrease" refers to a regulation that results in a lower level of expression, activity, function, or a combination thereof or a measure (e.g., cancer cell death or DNA methylation at a target site) of a target protein relative to a reference (e.g., a level before or in the absence of an agent). In some embodiments, the agent reduces the expression or activity or measure of the target protein by at least about 5% relative to a reference, such as by at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to a reference.

[0049] Non-limiting examples of metrics include energy production or energy conversion in the liver (e.g., regulation of ATP synthesis, B-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial thermogenesis, hormone signaling (e.g., mitochondrial estrogen receptor (mtER) signaling), redox maintenance (e.g., NADH and / or FADH2), cell cycle regulation, cell migration, cell morphology, apoptosis, necrosis, membrane potential, ion (e.g., calcium or zinc) storage, ion (e.g., calcium or zinc) homeostasis, metabolite synthesis (e.g., heme biosynthesis or steroid biosynthesis), nutrient sensing, unfolded protein stress response pathways, signaling processes (e.g., calcium signaling).

[0050] In some embodiments, the level of expression, activity, function, or a combination or metric thereof of the target protein is measured, e.g., after a treatment regimen has begun, after the agent is contacted with (e.g., a cell) or administered (e.g., to a subject) for at least about 1 day, e.g., at least about: 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0051] In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, a polynucleotide, a gene editing system, a small molecule, or a cell (e.g., a cell therapy).

[0052] In some embodiments, the target protein activates immune cells, and the agent regulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of the target protein. In some embodiments, the target protein inhibits immune cells (e.g., inhibits activation of immune cells, induces immune cell death (e.g., apoptosis), or a combination thereof), and the agent regulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of the target protein.

[0053] In certain embodiments, the agent modulates (eg, increases or decreases) the level of expression, activity, function, or a combination thereof of a target protein in cells and / or tissues implicated in a disease or disorder described herein, or a combination of the foregoing.

[0054] In some embodiments, the agent modulates (eg, increases or decreases) protein function and / or signaling pathways involved in a disease or disorder described herein, or a combination of the foregoing.

[0055] In some embodiments, the agent induces downregulation of the target protein (e.g., increases degradation of the target protein); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); regulates (e.g., excites, antagonizes, or destroys) the known function of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); induces antibody-dependent cell killing, phagocytosis, and / or opsonization of cells expressing the target protein; or a combination of the foregoing. In certain embodiments, the agent lacks agonistic activity against the target protein. In certain embodiments, the agent has agonistic activity against the target protein. In some embodiments, the agent lacks antagonistic activity against the target protein. In some embodiments, the agent has antagonistic activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein, and the at least one residue is involved in binding to the binding partner. In some embodiments, the agent binds to one or more binding sites and / or domains of the target protein, and the one or more binding sites and / or domains are involved in binding of the target protein to the binding partner.

[0056] The binding partner of the target protein of the present disclosure can be any known protein in the genome of a mammal (eg, human).

[0057] In some embodiments, the agent induces downregulation of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); prevents multimerization (e.g., dimerization) of the binding partner of the target protein; sequesters the binding partner of the target protein (e.g., secreted binding partner); induces antibody-dependent cellular killing, phagocytosis and / or opsonization of cells expressing the binding partner of the target protein; regulates (e.g., excites, antagonizes or destroys) the known functions of the binding partner of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); or a combination of the foregoing. In certain embodiments, the agent lacks agonistic activity for the binding partner of the target protein. In certain embodiments, the agent has agonistic activity for the binding partner of the target protein. In some embodiments, the agent lacks antagonistic activity for the binding partner of the target protein. In some embodiments, the agent has antagonistic activity for the binding partner of the target protein. In some embodiments, the agent binds to the target binding site of the binding partner. In a specific embodiment, the agent further binds to at least one residue of a binding partner of the target protein, and the at least one residue is involved in the binding between the target protein and the binding partner. In a more specific embodiment, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein, and the one or more binding sites and / or domains are involved in the binding between the target protein and the binding partner.

[0058] In some embodiments, the agent modulates (eg, activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination of the foregoing.

[0059] In some embodiments, the agent enhances the signal involved in T cell activation and / or survival. In certain embodiments, the agent activates stimulatory checkpoint molecules. Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, inducible T cell co-stimulator (ICOS). In a specific embodiment, the agent is an agonist for CD28.

[0060] In some embodiments, the agent reduces signals involved in T cell anergy and / or exhaustion. In certain embodiments, the agent inhibits inhibitory checkpoint molecules. Non-limiting examples of inhibitory checkpoint molecules include PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTLA-4, A2AR, CD276, B7-H4, BTLA, IDO, KIR, NOX2, VISTA, SIGLEC 7, and SIGLEC9.

[0061] In certain embodiments, the agent reduces the function of (eg, a blocking antibody) directed against TNFα.

[0062] In certain embodiments, the agent regulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of a variant of a target protein disclosed herein. In some embodiments, the variant comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of the target protein disclosed herein. For example, the sequence identity to the variant may be at least about: 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the sequence identity is about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the sequence identity is about: 70%-99%, 75%-99%, 75%-95%, 80%-99%, 80%-98%, 80%-95%, 80%-90%, 85%-98%, 85%-97%, 85%-90%, 90%-97%, 90%-96%, 90%-85%, 90%-80%, or 95%-99%. In some embodiments, the variant comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to the amino acid sequence of the target protein disclosed herein.

[0063] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity (expressed as a percentage). For sequence alignment and comparison, a sequence is typically designated as a reference sequence to which a test sequence is compared. The sequence identity between a reference sequence and a test sequence is expressed as the percentage of the position over the entire length of the reference sequence, at which the reference sequence and the test sequence have the same nucleotide or amino acid when aligning the reference sequence and the test sequence to achieve the maximum level of identity. As an example, when the test sequence has the same nucleotide or amino acid residue at 70% of the same position over the entire length of the reference sequence when the alignment is to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0064] Sequence alignment for comparison to achieve maximum identity level can be easily performed by those of ordinary skill in the art using appropriate alignment methods or algorithms. In some cases, alignment can include the introduction of gaps to provide maximum identity level. Examples include the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).

[0065] When using a sequence comparison algorithm, a test sequence and a reference sequence are input into a computer, subsequent coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of one or more test sequences relative to a reference sequence based on the specified program parameters. A common tool for determining sequence identity percentage is the protein basic local alignment search tool (BLASTP), which can be obtained by the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health (National Library of Medicine) of the United States. (Altschul et al., 1990).

[0066] In some embodiments, the amino acid sequence of the variant of the target polypeptide disclosed herein comprises at least one amino acid substitution relative to the amino acid sequence of the target protein. In some embodiments, the number of amino acid substitutions in the variant is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 relative to the amino acid sequence of the target protein disclosed herein. In some embodiments, the number of amino acid substitutions is at least about: 5, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60. In some embodiments, the number of amino acid substitutions is at most about: 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 6, or 5. In some embodiments, the number of amino acid substitutions is about: 1-60, 1-55, 2-55, 2-50, 3-50, 3-45, 4-45, 4-40, 5-40, 5-35, 6-35, 6-30, 7-30, 7-25, 8-25, 8-20, 9-20, 9-15, 10-15, 5-60, 10-60, 10-55, 15-55, 15-50, 20-50, 20-45, 25-45, 25-40, or 30-40. In some embodiments, the number of amino acid substitutions is about: 10-35, 10-33, 11-33, 11-31, 12-31, 12-29, 13-29, 13-27, 14-27, or 14-25.

[0067] One or more amino acid substitutions in a variant can be substitutions with classical amino acids or non-classical amino acids. Non-classical amino acids include, but are not limited to, D amino acids, such as the D forms of classical L-amino acids.

[0068] In some embodiments, the amino acid substitution is a conservative substitution.The term "one or more conservative amino acid substitutions" or "one or more conservative substitutions" refers to an amino acid substitution that has a value of 0 or greater in BLOSUM62.

[0069] In some embodiments, the amino acid substitutions are highly conservative substitutions.The term "one or more highly conservative amino acid substitutions" or "one or more highly conservative substitutions" refers to an amino acid substitution that has a value of at least 1 (eg, at least 2) in BLOSUM62.

[0070] In some embodiments, relative to the amino acid sequence of the target protein disclosed herein, the variant of the target protein disclosed herein comprises about 5-60 amino acid substitutions. In some embodiments, the amino acid substitutions include at least one conservative substitution. In some embodiments, the amino acid substitutions include at least one highly conservative substitution. A. Peptide Agents

[0071] The term "polypeptide", "peptide" or "protein" refers to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Proteins, peptides or polypeptides can contain any suitable L- and / or D-amino acids, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine) and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl and / or other functional groups on the peptide can be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups and methods for adding or removing protecting groups are known in the art and are disclosed, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. Functional groups of proteins, peptides, or polypeptides may also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorophore or a hapten) using methods known in the art. If desired, the protein, peptide, or polypeptide may include one or more modifications (e.g., an amino acid linker, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization modification), N-methyl-α-amino group substitution). In addition, the protein, peptide, or polypeptide may be an analog of a known and / or naturally occurring peptide, such as a peptide analog having one or more conservative amino acid residue substitutions.

[0072] In some embodiments, the agent comprises a polypeptide. In some embodiments, the polypeptide is an isolated polypeptide (e.g., isolated or extracted from a biological sample or source). In some embodiments, the polypeptide is a recombinant polypeptide. In some embodiments, the polypeptide is an inhibitor of expression and / or activity of a target protein disclosed herein (e.g., a direct inhibitor or an indirect inhibitor). In some embodiments, the polypeptide is an activator of expression and / or activity of a target protein disclosed herein (e.g., a direct activator or an indirect activator). In some embodiments, the polypeptide reduces the expression or activity of a target protein disclosed herein. In other embodiments, the polypeptide increases the expression or activity of a target protein disclosed herein. In some embodiments, the polypeptide is a target protein disclosed herein or a portion thereof (e.g., a biologically active portion thereof, such as a biologically active fragment of a target protein).

[0073] In some embodiments, the polypeptide is an immunoglobulin molecule, such as an antibody (e.g., a whole antibody, an intact antibody) or an antigen-binding fragment of an antibody. In some embodiments, the antibody or its antigen-binding fragment binds to a target protein. In some embodiments, the antibody or its antigen-binding fragment binds to a protein that can modulate the expression or activity of a target protein.

[0074] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" refers to a full-length antibody or multimer thereof (e.g., IgM) comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (including domains CH1, hinge, CH2 and CH3). Each light chain contains a light chain variable region (V L ) and the light chain constant region (CL). H and V L The V region can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed within framework regions (FRs). H and V L Each comprises three CDRs and four FR segments arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The antibody may be of any species, such as a rodent (e.g., mouse, rat, guinea pig) antibody, a human antibody, or the antibody may be a humanized antibody or a chimeric antibody.

[0075] In some embodiments, the antibody comprises an IgA (e.g., IgA1 or IgA2) heavy chain constant region, an IgD heavy chain constant region, an IgE heavy chain constant region, an IgG (e.g., IgG1, IgG2 (e.g., IgG2a, IgG2b, or IgG2c), IgG3, or IgG4) heavy chain constant region, or an IgM heavy chain constant region. In some embodiments, the antibody comprises an IgG heavy chain constant region. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the antibody comprises a lambda light chain constant region.

[0076] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is human or chimeric. In some embodiments, the antibody is primatized (e.g., humanized). In some embodiments, the antibody is multispecific, such as bispecific, trispecific, or tetraspecific. In some embodiments, the antibody is a heterologous conjugate antibody.

[0077] In some embodiments, the polypeptide agent is an antigen-binding fragment of an immunoglobulin molecule (e.g., an antibody). The term "antigen-binding fragment" refers to a portion of an immunoglobulin molecule (e.g., an antibody) that retains the antigen-binding properties of the parent full-length antibody. Non-limiting examples of antigen-binding fragments include V H Area, V L region, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment and a V H domain or a V L Domain antibodies (dAbs) composed of domains, etc. H and V L The domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, where V H / V L The domains can be paired intramolecularly or intermolecularly, in the latter case, V H and V L The domains are expressed by separate chains to form monovalent antigen binding sites, such as single-chain Fv (scFv) or diabodies. In some embodiments, the polypeptides disclosed herein are antigen binding fragments selected from the following: Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv (sdFv, such as diabodies, triabodies or tetrabodies), scFv, SMIP or rIgG. In some embodiments, the polypeptide is a scFv. Antigen binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases, by chemical peptide synthesis procedures known in the art.

[0078] Polypeptide agents (e.g., monoclonal antibodies) can be monovalent, bivalent, or multivalent. Monoclonal antibodies can be monospecific or multispecific (e.g., bispecific). Monospecific antibodies bind to one antigenic epitope. Multispecific antibodies (such as bispecific antibodies or trispecific antibodies) are included in the term monoclonal antibody.

[0079] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen (e.g., three, four, or five different antigens or epitopes). "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen.

[0080] An "isolated antibody" refers to an antibody or antigen-binding fragment thereof that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated anti-target protein antibody is substantially free of antibodies that specifically bind to antigens other than the target protein). In the case of a bispecific antibody, the bispecific antibody specifically binds to two antigens of interest and is substantially free of antibodies that specifically bind to antigens other than the two antigens of interest. In some embodiments, a polypeptide agent (e.g., a monoclonal antibody) is at least 80% pure, such as about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0081] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity increases in a cancer state relative to a reference state). In some embodiments, the antibodies described herein are antagonist antibodies that bind to proteins that can regulate the expression or activity of a target protein. As used herein, the term "antagonist antibody" refers to an antibody that reduces (e.g., inhibits) the function of an antigen after binding to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein). In some embodiments, the antigen is a receptor, and the antagonist antibody binds to the ligand binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein, and the antagonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule, and the antagonist antibody reduces the activity of the enzyme or weakens the signal transduction pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody reduces antigen function by at least about 10%, such as by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99%.

[0082] In some embodiments, the polypeptide is an agonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is reduced in a cancer state relative to a reference state). In some embodiments, the antibody is an agonist antibody that binds to a protein that can regulate the expression or activity of a target protein. As used herein, the term "agonist antibody" refers to an antibody that increases the function of an antigen after binding to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of a target protein). In some embodiments, the antigen is a receptor, and the agonist antibody binds to the ligand binding domain of the receptor. In some embodiments, the antigen is a transmembrane protein, and the agonist antibody binds to the extracellular region of the transmembrane protein. In some embodiments, the antigen is an enzyme or a signaling molecule, and the agonist antibody increases the activity of the enzyme or activates a signal transduction pathway mediated by a signaling molecule. In some embodiments, the agonist antibody increases antigen function by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1,000%.

[0083] In some embodiments, the agonist antibody does not exert at least one of the following functional properties: reducing (e.g., inhibiting) the activity of an antigen; inducing antibody-dependent cellular killing of cells expressing the antigen (e.g., by natural killer (NK) cells, monocytes, macrophages, neutrophils, dendritic cells, or eosinophils); inducing phagocytosis of cells expressing the antigen (e.g., by macrophages); inducing opsonization of cells expressing the antigen; and inducing down-regulation of the antigen on the cell surface (e.g., by hyper-crosslinking or clustering the antigen to induce internalization and degradation).

[0084] Suitable techniques, assays, and reagents for preparing and using therapeutic antibodies directed against an antigen are known in the art. For methods of making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques, see, e.g., Therapeutic Monoclonal Antibodies: From Bench to Clinic (Edited by Zhiqiang An, 1st edition 2009); Antibodies: A Laboratory Manual (Edited by Edward A. Greenfield, 2d edition 2013); Ferrara et al., Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker, PLoS ONE 7(11):e49535 (2012).

[0085] In some embodiments, the polypeptide is an antibody mimetic that binds to a target protein disclosed herein. The term "antibody mimetic" refers to a polypeptide that can simulate the ability of an antibody to bind an antigen but is structurally different from a natural antibody structure. Non-limiting examples of antibody mimetic include Adnectin, Affibody, Affilin, Affimer, Affitin, Alphabody, Anticalin, Avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and Versabody.

[0086] In some embodiments (e.g., when the expression or activity of the target protein is reduced in a disease state relative to a reference state), the agent is a polypeptide (e.g., an isolated polypeptide) comprising an amino acid sequence that is at least 70% identical to at least a portion (e.g., a biologically active portion or fragment) of the target protein. For example, the percent identity to the full-length target protein or its biologically active portion or fragment may be at least about: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the polypeptide comprises the amino acid sequence of the full-length target protein. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a recombinant polypeptide. In some embodiments, the polypeptide comprising the amino acid sequence of the full-length target protein is a synthetic polypeptide.

[0087] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to the target protein. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a highly conservative substitution.

[0088] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence that is at least 70% identical to at least a portion of a protein that is capable of regulating the expression or activity of a target protein. For example, the identity percentage may be at least about: 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the polypeptide comprises an amino acid sequence of a protein that is capable of regulating the expression or activity of a target protein.

[0089] In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises an amino acid sequence having at least one amino acid substitution relative to a protein capable of regulating the expression or activity of a target protein. For example, the number of amino acid substitutions can be at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or about: 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11 or 9-11. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the amino acid substitution is a highly conservative substitution.

[0090] In some embodiments, polypeptide is a cell penetrating peptide. In certain embodiments, polypeptide is connected to cell penetrating peptide. Suitable cell penetrating peptide sequence can be protein derived, designed or chimeric (modified). See, for example, Regberg et al., Applications of cell-penetrating peptides for tumor targeting and future cancer therapies [cell penetrating peptides for tumor targeting and future cancer therapy applications], Pharmaceuticals [Pharmaceuticals] 5 (9): 991-1007 (2012). Non-limiting examples of cell penetrating peptides include TAT (48-60), transmembrane peptides, pVEC, MPG8, transporter (Transportan), transporter 10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearoyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine and PTD4.

[0091] In some embodiments, the polypeptide is a circulating factor (eg, a cytokine).

[0092] In some embodiments, the biological properties (e.g., biological activity or half-life) of the polypeptide (e.g., separated polypeptide) and the target protein are similar. Non-limiting examples of biological activity include enzyme activity or properties (e.g., selectivity, steady state or kinetics), binding activity (e.g., nucleic acid (DNA, RNA) binding protein binding) or properties (e.g., specificity, affinity or kinetics), cell signaling activity, immunological activity and structural activity (e.g., cell adhesion). Non-limiting examples of enzyme activity include transferase activity (e.g., functional groups are transferred from one molecule to another molecule), oxidoreductase activity (e.g., catalytic oxidation-reduction reaction), hydrolase activity (e.g., cutting chemical bonds via hydrolysis), lyase activity (e.g., producing double bonds), ligase activity (e.g., connecting two molecules via covalent bonds) and isomerase activity (e.g., catalyzing the structural change from one isomer to another isomer within a molecule).

[0093] In some embodiments, the polypeptide (e.g., an isolated polypeptide) is a recombinant protein. In other embodiments, the polypeptide (e.g., an isolated polypeptide) is a synthetic protein. Methods for producing therapeutic polypeptides are known in the art. See, for example, Therapeutic Proteins: Methods and Protocols [Therapeutic Proteins: Methods and Protocols] (Mark C. Smales and David C James, ed., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications [Drug Biotechnology: Basics and Applications] (Daan JACrommelin, Robert D. Sindelar and Bernd Meibohm, ed., 2013). Mammalian cells, insect cells, yeast or bacteria, etc. can be used to recombinantly express polypeptides under the control of appropriate promoters.

[0094] In some embodiments, the polypeptide comprises a post-translational modification or other chemical modification. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0095] In some embodiments, the polypeptides described herein (e.g., target proteins or portions thereof, polypeptide agents regulating target proteins) are modified, for example, by cleavage (e.g., protease cleavage) or post-translational modification. In certain embodiments, one or more modifications will affect the activity of the polypeptide, for example, by making an inactive polypeptide active or by changing (e.g., increasing, reducing) the activity level of the polypeptide. In a particular embodiment, the polypeptides described herein are provided as prodrugs, for example, they can be converted (e.g., by proteolytic cleavage, post-translational modification) into active polypeptides in vivo. In some embodiments, the polypeptides include post-translational modifications or other chemical modifications. Non-limiting examples of post-translational modifications include acetylation, amidation, formylation, glycosylation, hydroxylation, methylation, myristoylation, phosphorylation, deamidation, prenylation (e.g., farnesylation, geranylation, etc.), ubiquitination, ribosylation, and sulfation. Phosphorylation can occur on amino acids such as tyrosine, serine, threonine, or histidine.

[0096] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing, Table A, or variants thereof. As used herein, the term "neoantigen" refers to a tumor antigen produced by a target protein described herein. In some embodiments, the neoantigen is a cancer-specific neoantigen. There are a variety of ways to produce neoantigens. For example, the neoantigen can be produced in vitro as a polypeptide before being formulated into a tumor vaccine or an immunogenic pharmaceutical composition. In some embodiments, the immunogenic pharmaceutical composition comprises an effective amount of one or more neoantigens or one or more pharmaceutically acceptable salts thereof. In some embodiments, the immunogenic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, adjuvant or additive.

[0097] Alternatively, a new antigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding a new antigen into a cell or tissue (e.g., a subject in need thereof). In certain embodiments, the polypeptide comprises at least two new antigens. In some embodiments, the polypeptide comprises a T cell enhancer amino acid sequence. In some embodiments, the T cell enhancer is selected from the group consisting of: a constant chain, a leader sequence of a tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal. B. Polynucleotide Agents

[0098] In some embodiments, the medicament comprises a polynucleotide or its analog or derivative. In some embodiments, the polynucleotide or its analog or derivative is an inhibitor of the target protein. In some embodiments, the polynucleotide or its analog or derivative is an activator of the target protein. In some embodiments, the polynucleotide or its analog or derivative reduces (e.g., reduces or neutralizes) the expression or activity of the target protein. In other embodiments, the polynucleotide or its analog or derivative increases the expression or activity of the target protein.

[0099] The polynucleotide may have a sequence containing naturally occurring ribonucleotides or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or a combination thereof. Thus, the polynucleotide may include, for example, nucleotides containing naturally occurring bases (e.g., A, G, C, or T) and nucleotides containing modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides (such as 5-methyl dCTP and 5-hydroxymethylcytosine)). In some embodiments, the polynucleotide includes at least one modified nucleotide. Non-limiting examples of modified nucleotides include 2'-fluoro, 2'-o-methyl, 2'-deoxy, non-locked nucleic acids, 2'-hydroxyl, thiophosphate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification increases nuclease resistance, increases serum stability, reduces immunogenicity, or a combination of the foregoing.

[0100] In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is an RNA molecule. In some embodiments, the polynucleotide is a vector (e.g., an expression vector, a plasmid).

[0101] In some embodiments, polynucleotides include analogs or derivatives of polynucleotides. In some embodiments, analogs or derivatives are peptide nucleic acids (PNA). In some embodiments, analogs or derivatives are locked nucleic acids (LNA). In some embodiments, analogs or derivatives are morpholino oligonucleotides. In some embodiments, analogs or derivatives include one or more thiophosphates. In some embodiments, medicaments include deoxyribonucleic acid guanidine (DNG) nucleotides. In some embodiments, medicaments include ribonucleic acid guanidine (RNG) nucleotides.

[0102] In some embodiments, the polynucleotide modulates the expression and / or activity of a nucleic acid encoding a target protein disclosed herein (e.g., a target protein in the sequence listing), Table A, or a portion thereof (e.g., a biologically active portion or fragment thereof).

[0103] In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to at least a portion of a gene or gene transcript encoding a target protein disclosed herein (e.g., fully complementary or partially complementary), such that the polynucleotide sequence is capable of hybridizing or annealing to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the polynucleotide comprises a nucleotide sequence complementary to at least a portion of a gene or gene transcript encoding a protein capable of regulating the expression or activity of a target protein disclosed herein.

[0104] In some embodiments, the polynucleotide encodes a target protein disclosed herein, or a variant thereof (e.g., a biologically active variant thereof), or a portion thereof (e.g., a biologically active portion or fragment thereof).

[0105] In some embodiments, the nucleic acid encoding the target protein or its variant or part (e.g., fragment) is a gene sequence or part thereof. In some embodiments, the coding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or part thereof (e.g., 5'UTR, 3'UTR, intron). In some embodiments, the coding nucleic acid is an mRNA molecule or part thereof. In some embodiments, the coding nucleic acid is present in a non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA) or miRNA).

[0106] The encoding nucleic acid may comprise a classical open reading frame (ORF) or a non-classical ORF. In certain embodiments, the encoding nucleic acid comprises a non-classical ORF.

[0107] The polynucleotide may be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the double-stranded polynucleotide is about 15-50 base pairs in length, such as about: 15-45, 15-40, 15-35, 15-30, 15-25, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, or 40-50 base pairs in length. In some embodiments, the polynucleotide is about 19-23 base pairs in length. In some embodiments, the polynucleotide is about 21 base pairs in length.

[0108] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the single-stranded polynucleotide has a length of about 15-50 nucleotides, such as about: 15-45, 15-40, 15-35, 15-30, 15-25, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, or 40-50 nucleotides.

[0109] In some embodiments, the polynucleotide prevents maturation of newly generated nuclear RNA transcripts into mRNA for transcription.In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to a sequence at an intron and exon boundary.

[0110] In some embodiments, the polynucleotide (e.g., antisense oligonucleotide) can hybridize to an mRNA encoding a target protein (e.g., under physiological conditions). In some embodiments, the polynucleotide is at least about 10 nucleotides in length, such as at least about: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or about: 10-30, 15-30, 15-25, 20-25 nucleotides. In some embodiments, the polynucleotide is at least 75% identical to the antisense sequence of the target transcript, such as at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0111] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., an unpaired overhang nucleotide that is not directly involved in the formation of a double helix structure with the core sequence). In some embodiments, the polynucleotide comprises a 3' overhang, a 5' overhang, or both. In some embodiments, the overhang is about 1-5 nucleotides. In some embodiments, the overhang comprises a modified ribonucleotide or deoxynucleotide, such as a thiophosphate, a phosphorothioate, or a deoxynucleotide reverse (3' to 3' linked) nucleotide.

[0112] Non-limiting examples of polynucleotide agents suitable for use in the compositions, kits and methods described herein include small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholino nucleic acids (MNA), locked nucleic acids (LNA), peptide nucleic acids (PNA), aptamers and guide RNA (gRNA).

[0113] In some embodiments, the polynucleotide inhibits gene expression (e.g., a biological process via RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and generated by one of ordinary skill using techniques, assays, and reagents known in the art, including computational tools. See, e.g., Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004.

[0114] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the length of the miRNA is about 22 nucleotides. The miRNA binds to a target site on an mRNA molecule and silences the mRNA, such as by causing the cutting of the mRNA, the destabilization of the mRNA, or the inhibition of mRNA translation.

[0115] In some embodiments, the polynucleotide is siRNA. In some embodiments, the siRNA comprises a nucleotide sequence identical to about 15-25 consecutive mRNA sequences encoding a target protein. In some embodiments, the siRNA is a double-stranded RNA molecule having about 19-25 base pairs. In some embodiments, the siRNA begins with the dinucleotide AA. In some embodiments, the GC content of the siRNA is about 30%-70%, for example, about: 30%-65%, 30%-60%, 30%-55%, 30%-50%, 40%-70%, 40%-65%, 40%-60%, 40%-55%, 45%-70%, 45%-65%, 45%-60% or 45%-55%.

[0116] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule comprising a hairpin turn that reduces target gene expression via RNAi. shRNA can be delivered to cells in the form of a plasmid (e.g., a viral or bacterial vector), for example, by transfection, electroporation, or transduction.

[0117] siRNA and shRNA are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (see, e.g., Bartel, Cell 116:281-97 (2004)). In some embodiments, siRNA acts as miRNA; in other embodiments, miRNA acts as siRNA (see, e.g., Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., Genes Dev 17:438-42 (2003)). Like siRNA, microRNA uses RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cleave mRNA. Instead, miRNA reduces protein output by translational inhibition or poly A removal and mRNA degradation (see, e.g., Wu et al., Proc Natl Acad Sci USA 103:4034-39 (2006)). The known miRNA binding sites are within the mRNA 3'UTR; miRNAs appear to target sites that have almost complete complementarity with 2-8 nucleotides at the 5' end of the miRNA (see, e.g., Rajewsky, Nat Genet [Natural Genetics] 38 Suppl: S8-13 (2006) and Lim et al., Nature [Natural] 433: 769-73 (2005)). This region is referred to as the seed region. Because siRNA and miRNA are interchangeable, exogenous siRNAs downregulate mRNAs that have seed complementarity with the siRNA (see, e.g., Birmingham et al., Nat Methods [Natural Methods] 3: 199-204 (2006)). Multiple target sites within the 3'UTR produce stronger downregulation (see, e.g., Doench et al., Genes Dev [Genes and Development] 17: 438-42 (2003)).

[0118] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding a target protein disclosed herein or a variant thereof (e.g., at least about 70% identical (e.g., at least about: 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the wild-type protein). In some embodiments, the mRNA is codon-optimized (e.g., to improve the efficiency of protein synthesis and limit mRNA destabilization caused by rare codons (see, e.g., Presnyak et al., Cell. 160(6): 1111-24 (2015) and Thess et al., Mol Ther. 23(9): 1456-64 (2015)).

[0119] In some embodiments, polynucleotides including RNA are chemically synthesized. In some embodiments, polynucleotides including RNA are recombinantly expressed. In some embodiments, RNA is transcribed in vitro. The preparation and use of RNA therapeutics are known in the art. See, for example, RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud, ed., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60 (2017).

[0120] In some embodiments, mRNA is produced by in vitro transcription. In some embodiments, mRNA is modified to optimize its activity. In some embodiments, mRNA comprises modified bases, 5' caps, 5' cap analogs, anti-reverse cap analogs (ARCA) or a combination thereof.

[0121] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100-200 nucleotides. In some embodiments, the poly(A) tail improves the expression and / or stability of the mRNA (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)).

[0122] In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the mRNA comprises a 5' cap analog. In some embodiments, the 5' cap analog is a 1,2-dithiodiphosphate-modified cap (see, e.g., Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0123] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), a 5'UTR, or both. In some embodiments, the modified UTR comprises sequences responsible for recruiting RNA binding proteins (RBPs) and miRNAs (e.g., to enhance the level of protein products (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)). In some embodiments, the 3'UTR, the 5'UTR, or both are modified to encode regulatory elements. In some embodiments, the regulatory elements comprise a K-turn motif, a miRNA binding site, or a combination thereof to control RNA expression in a cell-specific manner (see, e.g., Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0124] In some embodiments, the mRNA comprises RNA base modifications. In some embodiments, the mRNA comprises pseudouridine. In some embodiments, the mRNA comprises N1-methyl-pseudouridine (e.g., to mask immunostimulatory activity and enhance translation initiation (see, e.g., Andries et al., J Control Release [Control Release Magazine] 217: 337-44 (2015) and Svitkin et al., Nucleic Acids Res. [Nucleic Acids Research] 45: 6023-36 (2017)).

[0125] In some embodiments, the RNA (e.g., mRNA) is a circular RNA.

[0126] Compositions and methods for producing mRNA are disclosed in, for example, WO 2016011306, WO 2016014846, WO 2016022914, WO 2016077123, WO 2016164762, WO 2016201377, WO 2017049275, US9937233, US 8710200, US10022425, US 9878056, US 9572897, WO 2010084371, US 9353153, WO 2015034925, and WO 2019236673. See also, e.g., Jemielity et al., RNA 9(9):1108-22 (2003); Mockey et al., Biochem Biophys Res Commun. 340:1062-88 (2006); Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016); Presnyak et al., Cell 160:1111-24 (2015) and Kaczmarek et al., Genome Medicine 9:60 (2017). In some embodiments, the mRNA is prepared in a lipid nanoparticle (LNP) formulation (e.g., for in vivo delivery, see, e.g., U.S. Pat. No. 9,764,036, U.S. Pat. No. 9,682,139, Kauffman et al., Nano Lett. 15:7300-6 (2015), and Fenton et al., Adv Mater. 28:2939-43 (2016)).

[0127] In some embodiments, the polynucleotide is an aptamer. In certain embodiments, the aptamer binds to a target protein disclosed herein. In specific embodiments, the aptamer binds to a binding partner of a target protein disclosed herein.

[0128] In some embodiments, the polynucleotide is connected to the delivery polymer (e.g., covalently). In some embodiments, the connection between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is connected to the delivery polymer via a physiologically unstable linker. In some embodiments, the physiologically unstable linker is a disulfide bond.

[0129] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of excess polymer. In some embodiments, excess polymer is removed prior to administration (eg, to a cell or subject).

[0130] One of ordinary skill in the art can readily prepare suitable polynucleotide agents for use in the compositions, kits and methods described herein using the locus information (such as chromosomal location, start and end nucleotide positions, and polymorphism identification) of the protein sequences contained in the Sequence Listing and Table A incorporated herein.

[0131] C. Agents containing gene editing systems

[0132] In some embodiments, the medicament comprises a gene editing system. In some embodiments, the gene editing system produces a nucleotide deletion, a nucleotide substitution, a nucleotide addition, or a combination thereof in a gene encoding a target protein.

[0133] In some embodiments, the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, or a transcription activator-like effector nuclease (TALEN) system. In some embodiments, the gene editing system is a CRISPR / Cas system. In some embodiments, the gene editing system is a Class II CRISPR / Cas system.

[0134] In some embodiments, the gene editing system (e.g., CRISPR / Cas system) reduces (e.g., reduces, inhibits) or eliminates (e.g., via gene knockout) the expression of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) reduces (e.g., reduces, inhibits) or eliminates (e.g., via gene knockout) the expression of a protein that can regulate the expression or activity of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of the target protein. In some embodiments, the gene editing system (e.g., CRISPR / Cas system) increases (e.g., via gene knock-in or gene replacement) the expression of a protein that can regulate the expression or activity of the target protein.

[0135] In certain embodiments, the CRISPR system specifically catalyzes the cutting in the gene encoding the target protein, thereby inactivating the gene. Repairing nucleic acid chain breakage by non-homologous end joining (NHEJ) often results in the change of DNA sequence at the cleavage site, resulting in a small insertion or deletion (insertion and deletion (Indel)). In certain embodiments, NHEJ is used to knock out the gene encoding the target protein. In certain embodiments, homology directed repair (HDR) is used to simultaneously inactivate the gene encoding the target protein and insert a heterologous sequence into the inactivated locus. The cell in which knockout and / or knock-in events have occurred can be identified and / or selected by methods well known in the art.

[0136] In some embodiments, the gene editing system comprises a single Cas endonuclease or a polynucleotide encoding a single Cas endonuclease. In some embodiments, the single Cas endonuclease is Cas9, Cpf1, C2C1 or C2C3. In some embodiments, the single Cas endonuclease is Cas9 (e.g., of Streptococcus Pyogenes). In some embodiments, the single Cas endonuclease is Cpf1. In some embodiments, Cpf1 is AsCpf1 (from Acidaminococcus sp.) or LbCpf1 (from Lachnospiraceae sp.). The selection of nuclease and one or more gRNAs will typically be determined based on whether a deletion, substitution or addition of one or more nucleotides of the target sequence is required.

[0137] In some embodiments, the type II Cas endonuclease is Cas9 (e.g., Streptococcus pyogenes). In some embodiments, the modified Cas9 is a nickase Cas9, an inactive Cas9 (dCas9) or an eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, dCas9 is D10A or H840A. In some embodiments, the gene editing system comprises a double nickase Cas9 (e.g., to achieve more accurate genome editing, see, e.g., Ran et al., Cell [Cell] 154: 1380-89 (2013)). Wild-type Cas9 produces double-strand breaks (DSBs) at specific DNA sequences targeted by gRNA. Nickase Cas9 only produces single-strand breaks. dCas9 is catalytically inactive. In some embodiments, dCas9 is fused to a nuclease (e.g., FokI to produce DSBs at target sequences homologous to two gRNAs). Various CRISPR / Cas9 plasmids are publicly available from the Addgene repository (Addgene, Cambridge, MA: addgene.org / crispr / ).

[0138] In some embodiments, the gene editing system comprises: a) a wild-type or modified type II Cas endonuclease or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; b) CRISPR RNA ("crRNA"); and c) trans-activating crRNA ("tracrRNA").

[0139] In some embodiments, the crRNA comprises at least one "guide RNA" (sgRNA), for example, at least: 2, 3 or 4 gRNAs. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of the target protein. In some embodiments, the gRNA comprises a sequence identical to a portion of the gene sequence of a protein capable of regulating the expression or activity of the target protein. In some embodiments, the gRNA is at least about 16 nucleotides, for example, at least about: 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; or about: 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides; or about: 16-24, 17-24, 17-23, 18-23, 18-22, 19-22 or 19-21 or 19, 20 or 21 nucleotides. In some embodiments, the sgRNA is chemically modified.

[0140] The design of gRNA sequences for gene editing is known in the art. See, for example, Cong et al., Science [Science], 339: 819-23 (2013) and Ran et al., Nature Protocols [Natural · Program] 8: 2281-308 (2013). Cas9 requires at least about 16 or 17 nucleotides of the gRNA sequence to cut DNA, and Cpf1 requires at least about 16 nucleotides of the gRNA sequence to cut DNA. In fact, the length of the gRNA sequence is about 17-24 nucleotides (for example, about: 19, 20 or 21 nucleotides), and is complementary to the target gene. Custom gRNA generators and algorithms are commercially available. Chemically modified sgRNA has also been shown to be effective in genome editing (see, for example, Hendel et al., Nature Biotechnol. [Natural · Biotechnology], 985-91 (2015)).

[0141] In some embodiments, crRNA further comprises a sequence capable of binding to tracrRNA.After binding, the partial double-stranded structure is cut by RNase III, and the resulting crRNA / tracrRNA hybrid guides the Cas9 endonuclease to recognize and cut the target DNA sequence.

[0142] In some embodiments, the target DNA sequence is close to a "protospacer adjacent motif" ("PAM") that is specific for the Cas endonuclease. PAM sequences appear to be found throughout a given genome. CRISPR endonucleases of various prokaryotic species have unique PAM sequence requirements. Non-limiting examples of PAM sequences include: 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases (e.g., Cas9 endonucleases) associate with G-rich PAM sites (e.g., 5'-NGG) and blunt-end cleave the target DNA at a position 3 nucleotides upstream (5') of the PAM site.

[0143] In some embodiments, the gene editing system comprises: a) a wild-type or modified type II Cas endonuclease or a polynucleotide encoding a wild-type or modified type II Cas endonuclease; and b)crRNA.

[0144] Cpf1 related CRISPR arrays are processed into mature crRNA without the need for tracrRNA. Cpf1 endonucleases associate with T-rich PAM sites (e.g., 5'-TTN). Cpf1 can also recognize 5'-CTA PAM motifs. Cpf1 cuts target DNA by introducing misplaced or staggered double-strand breaks with 5' overhangs of 4 or 5 nucleotides, such as cutting such target DNA, where the misplaced or staggered cutting of 5 nucleotides is located at 18 nucleotides downstream (3') of the PAM site on the coding strand and 23 nucleotides downstream of the PAM site on the complementary strand. The overhangs of 5 nucleotides produced by such misplaced cutting allow DNA insertion by homologous recombination to be more accurately edited than the insertion at the DNA of blunt end cutting. See, for example, Zetsche et al., Cell [cell] 163: 759-71 (2015).

[0145] In some embodiments, the gene editing system activates or inhibits transcription of the target gene. In some embodiments, the gene editing system comprises: a) a chimeric protein comprising dCas9 and one or more effector domains; and b) One or more sgRNAs.

[0146] In some embodiments, the chimeric protein inhibits the expression of the target protein (CRISPRi). In some embodiments, the chimeric protein activates the expression of the target protein (CRISPRa). In some embodiments, the chimeric protein methylates the DNA sequence recognized by the sgRNA. In some embodiments, the chimeric protein demethylates the DNA sequence recognized by the sgRNA.

[0147] The effector domain comprises a biologically active portion of an effector protein (e.g., a transcriptional activator or repressor). In some embodiments, the gene editing system comprises 1 effector domain. In some embodiments, the gene editing system comprises at least 2 effector domains, such as 2, 3, or 4 effector domains. In some embodiments, the effector domain comprises KRAB. In some embodiments, the effector domain comprises VP64. In some embodiments, the effector domain comprises VP64, p65, and Rta. In some embodiments, dCas9 is D10A. In some embodiments, dCas9 is H840A.

[0148] Because dCas9 is catalytically inactive, dCas9 does not cut the target DNA, but interferes with transcription through steric hindrance. The dCas9 chimeric protein (e.g., dCas9-VPR) (guided by one or more gRNAs to the upstream sequence of the transcription start site (TSS) of the target gene) regulates the transcription of the target gene. See, e.g., Gilbert et al., CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes, Cell, 154, 442-51 (2013); Cheng et al., Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system, Cell Res. 23: 1163-71 (2013); Gilbert et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation, Cell, 159: 647-61 (2014); Tanenbaum et al., A protein-tagging system for signal amplification in gene expression and fluorescenceimaging [Protein labeling system for signal amplification of gene expression and fluorescence imaging], Cell [Cell] 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex [Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex], Nature [Nature] 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming [Highly efficient Cas9-mediated transcriptional programming], Nat. Methods.[Nature Methods] 12:326-28 (2015); Zalatan et al., Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds, Cell 160:339-50 (2015); Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation, eLife. 5:e19760 (2016); Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7:563-67 (2016). .

[0149] CRISPR technology for editing genes in eukaryotic organisms is disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1 and U.S. Patent Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965 and 8,906,616. Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1. CRISPR technology for generating mtDNA dysfunction in the mitochondrial genome is disclosed in Jo et al., BioMed Res. Int. [International Biomedical Research] 2015:305716 (2015). Co-delivery of Cas9 and sgRNA using nanoparticles is disclosed in Mout et al., ACS Nano [American Chemical Society Nano] 11 (3): 2452-58 (2017).

[0150] In some embodiments, the agent comprises a transposon-based gene editing system. An example of a suitable transposon-based gene editing system for use in the present disclosure provided herein is a gene writer system described in International Publication No. WO 2020 / 047124, published on March 5, 2020, the contents of which are incorporated herein by reference in their entirety.

[0151] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. The TALEN-based system comprises a protein containing a TAL effector DNA binding domain and an enzyme domain. They are prepared by fusing the TAL effector DNA binding domain with a DNA cleavage domain (a nuclease that cuts the DNA chain). The above-mentioned FokI restriction enzyme is an exemplary enzyme domain suitable for use in a TALEN-based gene regulation system.

[0152] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when the bacteria infects plants. The DNA binding domain contains a repeated, highly conserved sequence of 33-34 amino acids, with the 12th and 13th amino acids being different. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly associated with specific nucleotide recognition. Therefore, TAL effector domains can be engineered to bind to specific target DNA sequences by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificity of the RVD combinations is as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (although, in some embodiments, NN can also bind to adenine with lower specificity).

[0153] In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, a target DNA sequence of a target protein. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, a target DNA sequence defined by a set of genomic coordinates.

[0154] In some embodiments, the gene regulation system comprises two or more TAL effector fusion proteins (each comprising a TAL effector domain), wherein at least one TAL effector domain binds to a target DNA sequence of a target protein. In some embodiments, at least one of the two or more TAL effector domains binds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a target DNA sequence defined by a set of genomic coordinates, or 100% identical thereto.

[0155] Methods and compositions for assembling TAL effector repeat sequences are known in the art. See, for example, Cermak et al., Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic Acids Res 39(12):e82 (2011). Plasmids for constructing TAL effector repeat sequences are commercially available from, for example, Aide Gene Corporation.

[0156] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. Commercially available plasmids (e.g., plasmid pair (CSTZFN-1KT) from Sigma Aldrich (St. Louis, Missouri) can be used. Custom zinc finger nuclease (ZFN) (R-3257609)) to generate ZFN domains. Plasmids can be prepared using a commercial system according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (Cat. No. T1010), New England Biolabs, Ipswich, MA).

[0157] In some embodiments, the medicament comprises a vector intended to deliver conventional gene therapy (e.g., gene knockout or knock-in via homologous recombination). Non-limiting examples of the vector include retroviruses (e.g., lentivirus 5), adenoviruses, adeno-associated viruses, herpes simplex viruses, nanoparticles, and DNA transposons. D. Small molecule drugs

[0158] In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule binds to a target protein. In some embodiments, the small molecule binds to a protein that can regulate the expression or activity of a target protein. In some embodiments, the small molecule is an inhibitor (e.g., a direct inhibitor, an indirect inhibitor) of a target protein. In some embodiments, the small molecule is an activator (e.g., a direct activator and an indirect activator) of a target protein.

[0159] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic compounds, organometallic compounds or inorganic compounds. The atoms in small molecules are typically linked together via covalent bonds and / or ionic bonds. In certain embodiments, small molecules are small organic molecules. The arrangement of atoms in small organic molecules can represent chains (e.g., carbon-carbon chains or carbon-heteroatom chains), or can represent rings containing carbon atoms (e.g., benzene or polycyclic systems) or rings of combinations of carbon and heteroatoms (i.e., heterocycles, such as pyrimidine or quinazoline). Although small molecules can have a wide range of molecular weights, they generally include molecules less than about 5,000 daltons. For example, such small molecules can be less than about 1000 daltons, and preferably less than about 750 daltons, or more preferably less than about 500 daltons. Small molecules can be found in nature (e.g., identification, separation, purification) and / or synthetically produced (e.g., by traditional organic synthesis, biologically mediated synthesis or a combination thereof). See, e.g., Ganesan, Drug Discov. Today 7(1):47-55 (January 2002); Lou, Drug Discov. Today 6(24):1288-1294 (December 2001). Examples of naturally occurring small molecules include, but are not limited to, hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids, and their derivatives.

[0160] In certain embodiments, the agent comprises a proteolysis targeting chimera (PROTAC).

[0161] Small molecules suitable for use in the compositions, kits, and methods of the present disclosure can be identified by one of ordinary skill in the art using any of the screening methods disclosed herein. E. Therapeutic cells and cell-based therapies

[0162] In some embodiments, the medicament comprises a therapeutic cell. In certain embodiments, the therapeutic cell expresses and / or is engineered to express a target protein as described herein (e.g., a target protein in a sequence table, Table A, or a variant thereof), a polypeptide (e.g., an antibody, an antigen binding fragment, or a polypeptide comprising an amino acid sequence at least 70% identical to at least a portion of a target protein), a polynucleotide (e.g., a recombinant DNA, an RNA (such as an mRNA or an siRNA)), and / or a gene editing system (e.g., a CRISPR / Cas system).

[0163] In some embodiments, the polypeptides disclosed herein (e.g., antibodies or antigen binding fragments) are incorporated into cell-based therapies. In some embodiments, the polypeptide is an engineered T cell receptor. In some embodiments, the polypeptide is a chimeric antigen receptor (CAR) (e.g., expressed on T (CAR-T) cells, natural killer (CAR-NK) cells, or macrophages (CAR-M)). In some embodiments, CAR comprises a transmembrane domain and an antigen recognition portion, wherein the antigen recognition portion binds to a target protein.

[0164] Therapeutic cells suitable for use in the compositions, kits and methods disclosed herein can be produced, identified and / or enriched using methods known to those of ordinary skill in the art. Non-limiting examples of the method include purifying, propagating and / or differentiating cells from subjects (e.g., humans) into specific cell products; engineering somatic cells for gene therapy; cell immortalization; ex vivo genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technology); in vivo genetic modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technology); genome editing; cell plasticity technology; genetic modification; and flow cytometry. In some embodiments, therapeutic cells are autologous or isogenic. In other embodiments, therapeutic cells are allogenic. Expression vectors and hosts

[0165] In another aspect, the present disclosure provides an expression vector comprising a polynucleotide described herein.

[0166] The term "expression vector" refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into an appropriate expression host cell.

[0167] In certain embodiments, the expression vector comprises an expression control polynucleotide sequence operably connected to the polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In certain embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In certain embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to the DNA region to which RNA polymerase binds and initiates gene transcription. The term "operably connected" means that nucleic acid is located in a recombinant polynucleotide (e.g., a vector) in such a way that nucleic acid can be expressed under the control of the element to which it is connected (e.g., a promoter). The term "selectable marker element" is an element that imparts a trait that is suitable for artificial selection. The selectable marker element can be a negative or positive selection marker. Non-limiting examples of expression vectors used for use with bacteria, fungi, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual] (Michael R. Green and Joseph Sambrook edited, 4th edition 2012).

[0168] In another aspect, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0169] The term "expression host cell" refers to a cell that can be used to receive, maintain, replicate and / or amplify a vector.

[0170] Non-limiting examples of expression host cells include mammalian cells, such as hybridoma cells, baby hamster kidney fibroblasts (BHK cells), Chinese hamster ovary (CHO) cells, COS cells, HeLa cells, and human embryonic kidney (HEK) cells; yeast cells, such as Pichia pastoris cells; or bacterial cells, such as DH5α, etc. For methods of host cell culture for producing protein therapeutics, see, e.g., Mammalian Cell Cultures for Biologics Manufacturing (Weichang Zhou and Anne Kantardjieff, eds., 2014); for purification of protein therapeutics, see, e.g., Protein Biotechnology: Isolation, Characterization, and Stabilization (Felix Franks, ed., 2013) and Protein Purification Protocols (Paul Cutler, ed., 2010); and for formulation of protein therapeutics, see, e.g., Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic (Brian K Meyer, ed., 2012).

[0171] Techniques known in the art (including transformation, electroporation and transduction) can be used to introduce polynucleotides or expression vectors described herein into suitable or desired host cells. The introduced nucleic acid can be extrachromosomal in the host cell, or can be integrated into the genome of the host cell. Pharmaceutical composition

[0172] In another aspect, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutical agent disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition or its final dosage form or formulation having pharmacological activity or other direct effect in alleviating, treating or preventing cancer.

[0173] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent or tonifier (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents or supplements include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose or dextrin); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); nonionic surfactants (e.g., Tween), PLURONICS TM and polyethylene glycol (PEG).

[0174] In some embodiments, the agent (e.g., polypeptide, polynucleotide, or small molecule) of the pharmaceutical composition is modified, for example, conjugated to a heterologous moiety. The term "conjugated" refers to attachment via covalent or non-covalent interactions. Conjugation can employ any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linkers.

[0175] In some embodiments, the heterologous moiety is a label (e.g., a fluorescent label or a radioactive label), a molecule that stabilizes the agent, a molecule that targets the agent (e.g., to specific cells or tissues to promote or prevent crossing the blood-brain barrier), or a combination thereof.

[0176] In some embodiments, the heterologous part is polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticle, polymerized domain and carrier peptide. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is a polymer nanoparticle. In some embodiments, the polymer is an amphipathic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. The non-limiting examples of polymers include poly (lactic acid)-poly (ethylene glycol), poly (lactic acid-co-glycolic acid)-poly (ethylene glycol), poly (lactic acid-co-glycolic acid) (PLGA), poly (lactic acid-co-glycolic acid)-d-α-tocopheryl polyethylene glycol succinate, poly (lactic acid-co-glycolic acid)-ethylene oxide fumarate, poly (glycolic acid)-poly (ethylene glycol), polycaprolactone-poly (ethylene glycol) or any salt thereof. In some embodiments, the polymer nanoparticle comprises poly (lactic acid-co-glycolic acid) (PLGA).

[0177] In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for suitable administration regimens and routes. Non-limiting examples of routes of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the composition (e.g., pharmaceutical composition) is stored in the form of an aqueous solution or a dry formulation (e.g., lyophilized). In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion).

[0178] In some embodiments, the composition is formulated to be administered as a combination therapy with one or more additional therapeutic agents (e.g., with a second therapeutic agent). As used herein, "combination therapy" or "combined administration" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a specific disease or condition. Non-limiting examples of additional agents or treatments include biological agents (e.g., antibodies, peptides), cell therapy, gene therapy, immunotherapy, and small molecules that affect a disease or condition described herein (e.g., cancer).

[0179] The treatment regimen defines the dosage and cycle of each medicament used so that the effect of a single medicament on a subject overlaps. In certain embodiments, two or more medicaments are used in a sequential manner as a part of a prescription regimen. In other embodiments, the delivery of two or more medicaments is simultaneous or parallel. In certain embodiments, two or more medicaments are co-prepared. In certain embodiments, two or more medicaments or treatments are used in combination so that the decline of symptoms or other parameters related to the disorder is greater than the situation observed when a single medicament or treatment is delivered or when there is no other. The effects of the two treatments can be partially accumulated, completely accumulated or greater than accumulated (for example, collaboratively). Each of the two or more therapeutic agents can be administered by any appropriate route including but not limited to oral route, intravenous route, intramuscular route and directly absorbed by mucosal tissue. Two or more therapeutic agents can be administered by the same route or by different routes.

[0180] In some embodiments, the pharmaceutical agents or compositions of the present disclosure are delivered via a viral vector (e.g., by contacting a cell with a viral vector), administered locally (e.g., injected) to a tumor, or administered systemically (e.g., intravenously or orally) to a subject (e.g., a human patient).

[0181] Viral genomes provide a rich source of vectors that can be used to effectively deliver foreign genes to mammalian cells. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the nuclear genome of mammalian cells by universal or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Non-limiting examples of viral vectors include retroviruses (e.g., Retroviridae virus vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus)), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus, replication-defective herpes virus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox virus)). Additional non-limiting examples include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Non-limiting examples of retroviruses include: avian leukosis-sarcoma virus, avian C virus, mammalian C virus, B virus, D virus, oncorretrovirus, HTLV-BLV group virus, lentivirus, alpharetrovirus, gammaretrovirus, foamy virus (see, e.g., Coffin JM. Retroviridae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM, et al., eds. Fundamental Virology. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996: 763-843). Additional non-limiting examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, monkey sarcoma virus, Rous sarcoma virus, and lentivirus. Additional non-limiting examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the teachings of which are incorporated herein by reference.

[0182] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered in vivo, in vitro, ex vivo or in situ by a membrane-based carrier. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammalian (such as a human) cell). In some embodiments, the membrane-based carrier is a vesicle-based carrier. In some embodiments, the membrane-based carrier comprises one or more vectors described herein (e.g., a plasmid, a virus, a virus-like particle, or a virosome).

[0183] In certain embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more liposomes. Liposomes are spherical vesicle structures consisting of a monolayer or multilayer lipid bilayer around an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from the degradation of plasma enzymes, and are loaded and transported across biomembranes and blood-brain barriers (BBB) ​​(see, e.g., Spuch and Navarro, J Drug Deliv. [Drug Delivery Magazine] 2011: 469679 (2011)).

[0184] Vesicles can be made of several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicle lipids). Although vesicle formation can be spontaneous when the lipid film is mixed with an aqueous solution, it can also be accelerated by applying force in the form of oscillation using a homogenizer, sonicator or extrusion device (see, e.g., Spuch and Navarro, J Drug Deliv. [Drug Delivery Magazine] 2011: 469679 (2011)). Extruded lipids can be prepared by extrusion through a filter with a gradually decreasing size, such as Templeton et al., Nature Biotech [Natural Biotechnology], 15: 647-52 (1997), which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

[0185] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by lipid nanoparticles (LNP). In one embodiment, the LNP preparation comprising the medicament or pharmaceutical composition of the present disclosure has one or more of the following features: (a) the LNP preparation comprises a cationic lipid, a neutral lipid, cholesterol and a PEG lipid, (b) the average particle size of the LNP preparation is between 80nm and 160nm.

[0186] Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of SLNs, improve drug stability and drug loading, and prevent drug leakage. Polymer nanoparticles (PNPs) are important components of drug delivery. These nanoparticles can effectively guide drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), i.e., novel carriers that combine liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Therefore, the two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials [Nanomaterials] 7 (6): 122 (2017).

[0187] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by a carbohydrate carrier (e.g., anhydride-modified phytoglycogen or glycogen-based material). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin.

[0188] In certain embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by a protein carrier (e.g., a protein covalently linked to a cyclic polyribonucleotide). Non-limiting examples of protein carriers include human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and globulin.

[0189] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by a cationic carrier (e.g., a cationic lipopolymer or a transfection reagent). Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-( The following are some examples of the compounds: 1,2-diisopropylaminoethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol hydrochloride), diheptadecylamido glycylidene spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).

[0190] In some embodiments, the pharmaceutical agents or compositions of the present disclosure are formulated for delivery via exosomes, adipocytes, and / or erythrocytes. See, e.g., Ha et al., Acta Pharm Sin B. [Pharmaceutical Journal B] 6(4): 287-96 (2016).

[0191] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more fusosomes. Fusosomes have been engineered to confer target cell specificity for fusion and payload delivery, allowing the generation of delivery vehicles with programmable cell specificity. See, for example, patent application WO 2020014209, which is incorporated herein by reference in its teachings on fusosome design, preparation and use.

[0192] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated for delivery via ex vivo differentiated erythrocytes. See, e.g., WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 111(28):10131-36 (2014); U.S. Patent 9,644,180; Huang et al., Nature Communications [Nature Communications] 8:423 (2017).

[0193] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more microsomes, virus-like particles (VLPs) or plant nanovesicles and plant messenger packages (PMPs). See, for example, WO 2011097480, WO2013070324, WO 2017004526 and WO 2020041784.

[0194] In some embodiments, the medicament or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more anellosomes. The preparation and use of anellosomes for delivering therapeutic products are described in U.S. Patent No. 11,166,996, which is incorporated herein by reference for its teachings on anellosome design, preparation and use. Methods for regulating target proteins

[0195] On the other hand, the present disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in the sequence listing, Table A, in a cell (target cell, cell of a target tissue), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent or a pharmaceutical composition comprising an agent, the agent comprising the target protein identified herein and / or regulating the expression or activity of the target protein.

[0196] The target cell can be any mammalian (eg, human) cell.

[0197] In some embodiments, the target cell is a cell involved in a disease or disorder described herein.

[0198] In some embodiments, the target cell is a cancer cell (e.g., a metastatic cancer cell), a cell in a tumor microenvironment (e.g., a stromal cell), or a combination thereof. In specific embodiments, the target cell is a metastatic cancer cell.

[0199] In some embodiments, the target cell is involved and / or participates in inflammation. In certain embodiments, the target cell is an epithelial cell, an endothelial cell, a stem cell, a non-immune cell, or a combination thereof.

[0200] In some embodiments, the target cell is involved and / or participates in fibrosis, aging and / or senescence. In certain embodiments, the target cell is an epithelial cell, an endothelial cell, a stem cell, a non-immune cell, or a combination thereof.

[0201] In certain embodiments, target cell is an immune cell. In certain embodiments, target cell is effector T cell, helper T cell, Th1 cell, Th2 cell, Th17 cell, B cell, natural killer (NK) cell, innate lymphocyte (for example, ILC1 cell, ILC2 cell, ILC3 cell), macrophage (for example, M1 macrophage, M2 macrophage), monocyte and / or antigen presenting cell (for example, dendritic cell) or aforesaid combination.

[0202] In some embodiments, the target protein of the present disclosure is used to mediate the depletion of a cell population (e.g., a cancer cell (such as a tumor cell) population; an immune cell population). In some embodiments, the target protein of the present disclosure facilitates cell targeting (e.g., delivering a therapeutic agent in a cell type-specific manner), for example as a conjugate of a surface marker.

[0203] The target tissue can be any tissue in the body.

[0204] In some embodiments, the target tissue is a tissue involved in a disease or condition described herein.

[0205] In certain embodiments, the target tissue comprises a tumor, a tumor microenvironment, a metastatic site, or a combination thereof.

[0206] In some embodiments, the target tissue is an immune tissue. In some embodiments, the target tissue is a non-immune tissue. In some embodiments, the target tissue comprises a lymph node, a spleen, a secondary lymphoid organ, a tertiary lymphoid organ, a barrier tissue, skin, an intestinal tract, an airway, a wound, an immune tissue, a non-immune tissue, or a combination thereof.

[0207] In certain embodiments, the effective amount is sufficient to reduce the expression of the target protein in the target cell and / or target tissue. In some embodiments, the reduction is at least about 10%, for example, at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction is about 10%-99%, for example, about 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75% or 70%-75%.

[0208] In certain embodiments, the effective amount is sufficient to increase the expression of the target protein in the target cell and / or target tissue. In some embodiments, the increase is at least about 10%, for example, at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the increase is about 10%-99%, for example, about 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75% or 70%-75%. In some embodiments, the increase is about 1-100 fold, such as about: 1-75, 1-50, 1-25, 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, or 1-2 fold.

[0209] In some embodiments, the effective amount is sufficient to modulate nuclear factor kappa B (NF-κB) signaling, growth factor signaling, cell death (eg, apoptosis), cell cycle (eg, mitosis), cell migration, inflammation, or a combination of the foregoing. Diagnosis and treatment methods

[0210] In another aspect, the present disclosure provides a method for detecting a disease or condition in a subject or predicting the likelihood (or risk level) of a subject developing a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood that the subject develops the disease or condition.

[0211] In certain embodiments, disease or illness is a disease targeted by a drug and / or diagnostic test approved by the U.S. Food and Drug Administration (FDA). In certain embodiments, disease or illness is a disease targeted by a drug and / or diagnostic test for clinical research. In certain embodiments, disease or illness is an immune-mediated disease, an idiopathic disease, a hereditary disease, a cancer (e.g., a tumor), a metabolic disease, or a combination thereof. In a specific embodiment, disease or illness is related to GWAS, TCGA, whole genome sequencing, PheWAS, eQTL research, or a combination thereof (e.g., diseases, illnesses, or pre-diseases listed in paragraphs

[0219] and

[0220] ).

[0212] In another aspect, the present disclosure provides a method for classifying a subject based on a predicted likelihood of developing a disease or condition, the method comprising quantifying the expression or activity of a target protein in a sample from the subject; predicting the likelihood of developing the disease or condition based on the expression or activity of the target protein in the sample; and classifying the patient based on the predicted likelihood.

[0213] In another aspect, the present disclosure provides a method for stratifying a group of subjects having a disease or condition, the method comprising: quantifying the expression and / or activity of a target protein in samples from individual subjects in the group; and stratifying the group of subjects for treatment according to the expression and / or activity level of the target protein in the samples from the individual subjects.

[0214] In some embodiments, a higher level of expression or activity of a target protein in a sample from a subject relative to an appropriate control (e.g., a reference standard) indicates a disease or disorder or the likelihood of developing a disease or disorder. In some embodiments, a lower level of expression or activity of a target protein in a sample from a subject relative to an appropriate control (e.g., a reference standard) indicates a disease or disorder or the likelihood of developing a disease or disorder.

[0215] In some embodiments, the method further comprises administering an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein to a subject determined to have or predicted to have a likelihood (or risk) of developing a disease or condition.

[0216] In some embodiments, the method further comprises administering an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein to a subject determined to have or predicted to have a likelihood of developing the disease or condition.

[0217] In another aspect, the present disclosure provides a method for preparing a sample that can be used to detect the likelihood of a subject suffering from a disease or condition, the method comprising: a) obtaining or having obtained a sample from a subject; b) adding a protease inhibitor, a control peptide, a standard peptide, or a combination thereof to the sample to prepare a sample that can be used to detect the possibility of having cancer; and c) quantifying the expression or activity of the target protein in the sample prepared in step b).

[0218] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein.

[0219] In another aspect, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent disclosed herein or a pharmaceutical composition disclosed herein, wherein the expression and / or activity level of a target protein disclosed herein in the subject is altered.

[0220] As used herein, "treatment" and "treating" refer to the medical management of a subject that is intended to improve, alleviate, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, or disorder. "Treatment" includes active treatment (treatment intended to improve a disease, pathological condition, or disorder), etiological treatment (treatment directed at the cause of the relevant disease, pathological condition, or disorder), palliative treatment (treatment intended to relieve symptoms), preventive treatment (treatment intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition, or disorder); and supportive treatment (treatment used to supplement another therapy). Treatment also includes reducing the extent of a disease or disorder; preventing the spread of a disease or disorder; delaying or slowing the progression of a disease or disorder; alleviating or relieving a disease or disorder; and relief (whether partial or complete), whether detectable or undetectable. "Relieve" or "mitigate" a disease or disorder means that the extent of the disease, disorder, or disorder and / or the reduction of undesirable clinical manifestations and / or the time course of progression are slowed or prolonged compared to the extent or time course in the absence of treatment. "Treatment" also includes prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0221] In some embodiments, the subject is an animal. In other embodiments, the subject is a bird, such as a hen, a rooster, a turkey, or a parrot. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. Non-limiting examples of non-human mammals include cattle (e.g., dairy cows or beef cattle), sheep, goats, pigs, horses, dogs, cats, mice, rats, etc. In some embodiments, the subject is a human. In some embodiments, the human is a neonate. In some embodiments, the human is a pediatric patient. In some embodiments, the human is a teenager. In some embodiments, the human is an adult. In some embodiments, the human is less than 18 years old. In some embodiments, the human is at least 18 years old. In some embodiments, the human is between 18 and 25 years old. In some embodiments, the human is at least 25 years old, for example, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old.

[0222] As used herein, the term "effective amount", "therapeutically effective amount" or "sufficient amount" refers to an amount sufficient to achieve treatment (e.g., produce a beneficial or desired result) (including effects at the cellular, tissue or clinical level, etc.) when administered to a subject (e.g., a mammal, such as a human cancer patient). Therefore, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of drug sufficient to achieve a response compared to a response obtained without the administration of the agent. The amount of a given composition described herein corresponding to such an amount will vary according to various factors such as the following: a given agent, a pharmaceutical formulation, an administration route, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or the host receiving treatment, etc., but can still be routinely determined by those skilled in the art. In some embodiments, the "therapeutically effective amount" of the composition disclosed herein is an amount that produces a beneficial or desired result (e.g., compared to a control) in a subject. The therapeutically effective amount of the composition disclosed herein can be easily determined by ordinary technicians by conventional methods known in the art. The dosage regimen can be adjusted to provide the best therapeutic response.

[0223] The therapeutic agents described herein can be administered via a variety of routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the compound and the specific disease or condition to be treated. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the specific compound selected.

[0224] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more additional therapeutic agents (eg, a second therapeutic agent).

[0225] Administration of two or more therapeutic agents encompasses co-administration of therapeutic agents in a substantially simultaneous manner (such as in a drug combination). Alternatively, such administration encompasses co-administration of each therapeutic agent in multiple containers or separate containers (e.g., capsules, powders, and liquids). Such administration also encompasses the use of therapeutic agents in a sequential manner at approximately the same time or at different times. When two or more therapeutic agents are administered, the therapeutic agents may be administered via the same route of administration or via different routes of administration.

[0226] In another aspect, the disclosure provides a method for regulating the expression or activity of a target protein or a variant thereof identified in the sequence listing, Table A, in a cell, the method comprising contacting the cell with an agent disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is in a subject.

[0227] In another aspect, the present disclosure provides a method for identifying an agent that modulates the expression and / or activity of a target protein (e.g., a target protein in the sequence listing), the method comprising: a) contacting a sample (e.g., a biological sample, such as a cell or tissue) comprising a target protein with an agent (e.g., a candidate agent to be tested for its ability to modulate expression and / or activity of the target); and b) determining whether the agent modulates the expression or activity of a target protein, A difference in the expression or activity of a target protein that has been contacted with the agent compared to a reference indicates that the agent modulates the expression or activity of the target protein.

[0228] In some embodiments, a difference of at least about 10% in the expression or activity of a protein that has been contacted with an agent compared to a reference indicates that the agent modulates the expression or activity of the protein. In some embodiments, the difference is at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more.

[0229] In some embodiments, a decrease in the expression or activity of a target protein that has been contacted with an agent compared to a reference indicates that the agent inhibits the expression or activity of the target protein. In some embodiments, an increase in the expression or activity of a protein compared to a reference indicates that the agent activates the expression or activity of the protein. Non-limiting examples of indications GWAS-related diseases or conditions

[0230] In some embodiments, the GWAS-associated disease or condition is: abdominal aortic aneurysm, chromosomal segregation abnormalities, achalasia, acne, acute lymphoblastic leukemia, acute myeloid leukemia, Addison's disease, juvenile idiopathic scoliosis, adult-onset asthma, age-related hearing impairment, age-related macular degeneration, age-related nuclear cataract, AIDS, alcohol abuse, alcohol dependence, alcoholic cirrhosis, alcoholic pancreatitis, allergic rhinitis, allergy, hair loss, alopecia areata, Alzheimer's disease, amblyopia, amyotrophic lateral sclerosis, androgenic alopecia, anemia, true heart pain, ankle injury, ankylosing spondylitis, anorectal malformation, anorexia nervosa, anterior uveitis, anti-neutrophil antibody-associated vasculitis, anti-topoisomerase -I antibody positive systemic sclerosis, anxiety, asparaginase-induced acute pancreatitis, Asperger's syndrome, aspirin-induced asthma, asthma, atopic asthma, atopic eczema, atrial fibrillation, atrophic gastritis, atrophic macular degeneration, attention deficit hyperactivity disorder, atypical femoral fracture, autism spectrum disorder, autoimmune disease, autoimmune hepatitis, autoimmune thyroid disease, azoospermia, back pain, bacterial meningitis, baldness, basal cell carcinoma, B-cell acute lymphoblastic leukemia, Behcet's syndrome, Bell's palsy, benign prostatic hyperplasia, biliary cirrhosis, bipolar disorder, bladder cancer, fracture, cerebral aneurysm, cerebral infarction, breast cancer, bulimia nervosa, bullous pemphigoid, cancer, arrhythmia, cardiac hypertrophy , cardiotoxicity, cardiovascular disease, cardiovascular disease myocardial infarction, carpal tunnel syndrome, cataracts, celiac disease, central nervous system cancer, central nervous system non-Hodgkin lymphoma, cerebral amyloid angiopathy, cervical cancer, grade 2 / 3 cervical intraepithelial neoplasia, Chagas cardiomyopathy, childhood onset asthma, cholecystitis, cholelithiasis, choroidal melanoma, chronic central serous retinopathy, chronic hepatitis B virus infection, chronic hepatitis C virus infection, chronic kidney disease, chronic lymphocytic leukemia, chronic obstructive pulmonary disease, chronic sinusitis, smoking addiction, cirrhosis, cleft lip, cleft palate, clonal hematopoiesis, Clostridium difficile infection, cocaine dependence, colon polyps, colorectal adenoma, colorectal cancer, conduct disorder, congenital left heart disease, conus arteriosus Visceral malformation, coronary artery aneurysm, coronary artery disease, corticobasal degeneration, new coronavirus pneumonia, Creutzfeldt-Jakob disease, Crohn's disease, skin mastocytosis, skin melanoma, skin squamous cell carcinoma, dementia, dengue hemorrhagic fever, dental caries, depression, dermatomyositis, developmental hip dysplasia, diabetes, diabetic nephropathy, diabetic retinopathy, diffuse large B-cell lymphoma, digestive system infectious diseases, dilated cardiomyopathy, diverticulosis, diverticulitis, drug-induced agranulocytosis, drug-induced liver injury, duodenal ulcer, Dupuytren's contracture, eating disorders, eczema, endometrial cancer, endometrioma, endometriosis, eosinophilic esophagitis, epithelioid cell uveal melanoma,Epstein-Barr virus infection, esophageal adenocarcinoma, esophageal varices, essential tremor, estrogen receptor-negative breast cancer, Ewing's sarcoma, exfoliation syndrome, exploratory eye movement measurement, extrahepatic bile duct cancer, facial wrinkles, familial long QT syndrome, familial sick sinus syndrome, febrile seizures (in the age range of 3 months to 6 years), female reproductive system diseases, focal segmental glomerulosclerosis, frontal fibrosis and alopecia, frozen shoulder, gallstones, gastric adenocarcinoma, gastric cancer, gastric ulcer, gastroesophageal reflux disease, glaucoma, glioma, glucosuria, Graves' disease, Hashimoto's thyroiditis, head and neck malignancies, head and neck squamous cell carcinoma, hearing loss, heart failure, hematuria, hemorrhoids, Henlein-Schonlein purpura, hepatitis B virus infection, hepatitis C virus infection, heroin dependence, herpes zoster, hippocampal atrophy, Hirschsprung disease, HIV-1 infection, Hodgkin lymphoma, human papillomavirus infection, Huntington's disease, hypermetropia, hyperopia, hypertension, hyperthyroidism, hypertriglyceridemia, hypertrophic cardiomyopathy, hyperuricemia, hypoalbuminemia, hypospadias, hypothyroidism, idiopathic pulmonary fibrosis, IgA glomerulonephritis, immune system disease, infantile hypertrophic pyloric stenosis, inflammatory bowel disease, influenza A (H1N1) infection, inguinal hernia, insomnia, interstitial lung disease, cerebral hemorrhage, irritable bowel syndrome, ischemic stroke, juvenile idiopathic arthritis, keratinocyte carcinoma, keratoconus, knee pain, speech Speech injury, large artery stroke, late-onset autoimmune diabetes in adults, late-onset Alzheimer's disease, late-onset myasthenia gravis, leprosy, leukopenia, dementia with Lewy bodies, localized scleroderma, liver disease, liver tumors, lumbar disc degeneration, lung adenocarcinoma, lung cancer, lupus nephritis, macrovascular complications of diabetes, macular telangiectasia type 2, major depressive disorder, malaria, male infertility, marginal zone B-cell lymphoma, mastocytosis, melanoma, membranous glomerulonephritis, menorrhagia, mental decline, metabolic rate measurement, metabolic syndrome, metastasis measurement, metastatic colorectal cancer, migraine disorder, moderate albuminuria, molar-incisor hypomineralization, monoclonal gammopathy, mood disorders, moyamoya disease, mucocutaneous lymphadenopathy Syndrome, multiple myeloma, multiple sclerosis, multiple system atrophy, myeloproliferative disorder, myocardial infarction, myopia, myositis, narcolepsy with cataplexy, nasal polyps, nasopharyngeal tumors, neonatal systemic lupus erythematosus, nephrotic syndrome, neuroblastoma, neurotic disorders, nicotine dependence, tuberous sclerosis Hodgkin lymphoma, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, non-lobar hemorrhage, non-melanoma skin cancer, non-obstructive coronary artery disease, non-small cell lung cancer, obesity, obesity esophageal cancer, obsessive-compulsive disorder, ocular sarcoidosis, oligoarticular juvenile idiopathic arthritis, open-angle glaucoma, opioid use disorder, oral cancer, oral ulcers, oropharyngeal cancer, deforming osteitis, osteoarthritis, hip osteoarthritis, knee osteoarthritis,Osteonecrosis, osteoporosis, osteosarcoma, ovarian cancer, ovarian endometrioid cancer, ovarian serous cancer, pain, pancreatic cancer, panic disorder, Parkinson's disease, pathological myopia, pemphigus vulgaris, peptic ulcer disease, periodontitis, peripheral arterial disease, peripheral neuropathy, periprosthetic osteolysis, pneumonia, polymyositis, gallbladder polyps, postoperative sensory impairment, preeclampsia, primary angle-closure glaucoma, primary biliary cirrhosis, prion disease, progressive supranuclear palsy, proliferative diabetic retinopathy, prostate cancer, proteinuria, pseudotumor cerebri, psoriasis, psoriasis vulgaris, nontuberculous mycobacterial infection of the lungs, refractory Atopic dermatitis, renal cell carcinoma, respiratory diseases, retinal detachment with a hole, rheumatic heart disease, rheumatoid arthritis, rotator cuff tear, SAPHO syndrome (synovitis, acne, impetigo, hyperostosis, osteitis syndrome), sarcoidosis, scarlet fever, schizophrenia, sclerosing cholangitis, seasonal allergic rhinitis, selective IgA deficiency, sepsis, shoulder impingement syndrome, Sjögren's syndrome, skin cancer, skin tumors, skin sensitivity to sunlight, small cell lung cancer, small vessel stroke, spontaneous premature birth, sporadic amyotrophic lateral sclerosis, sporadic Creutzfeldt-Jakob disease Creutzfeld Jacob disease, sporadic Creutzfeldt Jacob disease, squamous cell carcinoma, squamous cell lung cancer, stenosing tenosynovitis, Stevens-Johnson syndrome, gastric polyps, stress-related disorders, stroke, substance abuse, cardiac arrest, sunburn, systemic juvenile idiopathic arthritis, systemic lupus erythematosus, systemic mastocytosis, systemic sclerosis, Takayasu's arteritis, testicular cancer, testicular germ cell tumor, thyroid cancer, thyrotoxic periodic palsy, tinnitus, tooth loss, tooth decay, Tourette syndrome, triple-negative breast cancer, tropical spastic paraparesis, tuberculosis, type 1 diabetes mellitus, type 2 diabetes mellitus, ulcerative colitis, unipolar depression, upper aerodigestive tract cancer, urolithiasis, uterine fibroids, uveal melanoma, varicose veins, vascular dementia, venous thromboembolism, vitamin D deficiency, vitiligo, or wet macular degeneration, or a combination thereof. TCGA-related diseases or conditions

[0231] In some embodiments, the TCGA-associated disease or disorder is: biliary tract cancer, bladder urothelial carcinoma, bone cancer, glioblastoma multiforme, brain low-grade glioma, breast cancer, cervical squamous cell carcinoma, chronic lymphocytic leukemia, chronic myeloid disorder, colon adenocarcinoma, early-onset prostate cancer, esophageal adenocarcinoma, gastric adenocarcinoma, gastric cancer, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, lymphocytic neoplasms diffuse large B-cell lymphoma, malignant lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma or uterine corpus endometrial carcinoma or a combination thereof.

[0232] In some embodiments, treatment slows (eg, inhibits) the progression of (eg, inhibits) a disease or condition described herein (eg, inhibits cancer cell growth, proliferation, metastasis, invasion, and / or migration).

[0233] In some embodiments, an effective amount is sufficient to slow (eg, inhibit) the progression of a disease or disorder described herein.

[0234] In some embodiments, the effective amount is sufficient to slow the progression of the disease or condition by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the effective amount is sufficient to slow the progression of the disease or condition by about 10%-99%, for example, about 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75%, or 70%-75%.

[0235] In some embodiments, treatment reduces symptoms of a disease or disorder described herein.

[0236] In some embodiments, the effective amount is sufficient to reduce the symptoms of a disease or condition described herein. In some embodiments, the reduction is at least about 10%, such as at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the reduction is about 10%-99%, for example, about 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75% or 70%-75%.

[0237] In some embodiments, the effective amount is sufficient to prevent the death of the subject, thereby reducing the mortality rate. In certain embodiments, the reduction in mortality rate is at least about 10%, such as at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In certain embodiments, the reduction in mortality is about 10%-99%, for example, about 10%-98%, 15%-98%, 15%-97%, 20%-97%, 20%-96%, 25%-96%, 25%-95%, 30%-95%, 30%-94%, 35%-94%, 35%-93%, 40%-93%, 40%-92%, 45%-92%, 45%-91%, 50%-91%, 50%-90%, 55%-90%, 55%-85%, 60%-85%, 60%-80%, 65%-80%, 65%-75% or 70%-75%.

[0238] In some embodiments, the effective amount is sufficient to modulate (eg, increase or decrease) expression of the target protein in a cell involved in a disease or disorder described herein.

[0239] In some embodiments, an effective amount is sufficient to modulate the body's response to a disease or condition described herein.

[0240] In some embodiments, the present disclosure provides treatments utilizing the immune system (e.g., immuno-oncology). In still other embodiments, the immune system is an innate immune system. In some embodiments, the immune system is an adaptive immune system. In still other embodiments, treatment involves humoral immunity or antibody-mediated immunity. In some embodiments, treatment involves cell-mediated immunity, such as cancer. In some embodiments, the method involves treatment at or before early disease progression, while in other embodiments, the method involves treatment at or before advanced disease progression or prevention of advanced disease progression.

[0241] In some embodiments (e.g., immuno-oncology-specific treatments), the effective amount is sufficient to modulate (e.g., increase or decrease) immune cell-related readouts, migration of immune cells (e.g., antigen presenting cells (such as dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen presenting cells (such as dendritic cells and / or macrophages), monocytes, T cells and / or B cells), lymph node innervation, lymph node homing of immune cells (e.g., dendritic cells and / or T cells), lymph node egress of immune cells (e.g., dendritic cells and / or T cells), differentiation of immune cells, activation of immune cells, polarization of immune cells, cytokine production (e.g., increasing pro-inflammatory cytokine production, decreasing pro-inflammatory cytokine production, increasing anti-inflammatory cytokine production, decreasing anti-inflammatory cytokine production), degranulation of immune cells, maturation of immune cells, ADCC of immune cells, ADCP of immune cells, antigen presentation, target protein expression, or a combination of the foregoing.

[0242] Unless otherwise defined, all art terms, symbols and other scientific terms or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this disclosure belongs. In some cases, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not necessarily be interpreted as representing substantial differences from what is commonly understood in the art. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having the meanings in the context of the relevant art and / or as otherwise defined herein.

[0243] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0244] As used herein, the indefinite articles “a,” “an,” and “the” should be understood to include plural referents unless the context clearly indicates otherwise.

[0245] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of, for example, one stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced by the terms "contains" or "includes".

[0246] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel features of the claim. Whenever used herein in the context of a certain aspect or embodiment of the present disclosure, any of the terms "comprising," "containing," "including," and "having" may be replaced in some embodiments by the terms "consisting of" or "consisting essentially of" to change the scope of the present disclosure.

[0247] As used herein, the connection term "and / or" between multiple listed elements is understood to cover both single and combined options. For example, where two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any of these options are understood to fall within the meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option is also understood to fall within the meaning and therefore meet the requirements of the term "and / or".

[0248] When presenting a list, it is understood that each individual element of the list and each combination of the list is a separate embodiment unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C." Examples Example 1: Validation of target protein as genetic driver of disease

[0249] This example demonstrates validation of a target protein of the present disclosure that contains one or more mutations associated with a disease with an as-yet unknown mechanism of action (determined by a genome-wide association study (GWAS)). Specifically, a GWAS associated with Th17 autoimmune diseases was mapped to an intergenic locus without annotated proteins but with a target protein identified by the methods of the present disclosure.

[0250] Transient knockout and knockin cell lines were generated by CRISPR-Cas9 in primary PHA blast T cells, in which the target protein was knocked out or the alternative SNP allele of the target protein replaced the wild type. STAT3, STAT1, and IL-6 activation levels were assessed before and after transient stimulation with IL-23, IL-12, and IL-6. IL-23 and IL-17 are Th17 pathway inducing cytokines that stimulate STAT3 activation (doi:10.1038 / cmi.2017.128). Hypothesis: The SNP allele of the target protein induces STAT3 (but not STAT1 or NF-kb) at levels significantly higher than the wild-type allele. Knockout of the target protein reduces STAT3 activity.

[0251] Materials and Methods

[0252] PHA blast generation: Fresh or frozen primary human PBMCs were placed in PBMax Karyotyping Medium supplemented with 10 ng / ml rhIL-2 for 3-4 days.

[0253] Knockout and knockin of target proteins were accomplished by CRISPR-Cas9Alt-R knockout (Idtdna.com) via RNP particles transfected via electroporation with guides specific for the target protein.

[0254] STAT3, STAT1 and NF-kB activation assays: PHA blasts were plated at approximately 10 million cells / ml in 96-well plates (80 μl). Cells were serum starved for 3-4 hours and then IL-23, IL-12 or IL-6 were added for 30 minutes. Cells were harvested and cell extracts were assayed by phospho-STAT3, STAT1 or NF-kB ELISA.

[0255] Together, these experiments demonstrate that target proteins containing GWAS alleles associated with autoimmune diseases have a more pronounced activation effect on STAT3 rather than STAT1 or NF-kB, strongly suggesting that the target proteins are potential targets for ameliorating autoimmune diseases. Example 2: Validation of a target protein as a genetic driver of a disease. Overexpression of a predicted TM protein leads to increased proliferation of tumor cells. A known TCGA mutant of this protein has a more potent effect.

[0256] This example shows the validation of the target protein (SEQ ID NO:5391) as a genetic driver of the disease. Specifically, SEQ ID NO:5391 is a target protein containing known TCGA mutants, which appear in a large proportion of patients with colon adenocarcinoma. We demonstrate that SEQ ID NO:5391 overexpressed in colon cancer cell line HCT116 cells significantly increases the proliferation of these cells. Importantly, the mutant form of SEQ ID NO:5391 containing the TCGA P31L mutation has a significantly stronger effect than the wild-type SEQ ID NO:5391, which is consistent with the TCGA mutant being a genetic driver of the cancerous phenotype.

[0257] Experimental design:

[0258] Protein overexpression plasmids encoding target proteins or controls were transiently transfected into multiple cell lines. After 24 hours, proliferation was measured using the WST-1 assay.

[0259] Materials and Methods

[0260] The target cells are inoculated into 100 μL cell culture medium in a 96-well plate at a density of 4,000 cells / well. The cells are incubated in a 37°C humidified incubator with 5% CO2 for 24 hours. Lipofectamine3000 (Thermo Fisher) is diluted to a final concentration of 30 μL / mL in Opti-MEM culture medium (Thermo Fisher). DNA (pcDNA3.1 overexpression plasmid (Thermo Fisher) encoding related controls or target proteins) and P3000 are diluted to a final concentration of 10 μg / mL (DNA) and 20 μL / mL (P3000) in Opti-MEM culture medium. The diluted DNA is mixed with the diluted Lipofectamine 3000 reagent, and the mixture is incubated at room temperature for 10 minutes. The mixture is added dropwise to the cells, and the plate is gently vortexed to evenly distribute the mixture. The cells are incubated in a CO2 incubator at 37°C for 48 hours. 10 μL of WST-1 reagent (Abcam) was added directly to the wells, and the plates were incubated in a humidified incubator with 5% CO2 at 37°C in the dark for 45 minutes. The absorbance at 450 nm was measured using a microplate reader. A reference wavelength of 650 nm can be used to correct for background absorbance. The percentage of live cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the results as a percentage of the control group.

[0261] in conclusion:

[0262] SEQ ID NO: 5391 is a novel unannotated protein that increases the proliferation of colon cancer cells (HCT116 cell line) under high serum conditions. Interestingly, SEQ ID NO: 5391 contains a TCGA mutation (P31L), which is known to occur in many cancer patients, but its significance to carcinogenic processes and mechanisms was previously unknown. We show that when the P31L mutation is included in the expressed target protein, its effect on proliferation is significantly more significant. Therefore, SEQ ID NO: 5391 and SEQ ID NO: 5391_P31L are both genetic drivers of cancer and target proteins for anticancer therapy ( Figure 1 ).

[0263] Example 3: Validation of target protein as genetic driver of disease Overexpression of predicted TM protein leads to increased proliferation of tumor cells.

[0264] This example demonstrates the validation of multiple target proteins (SEQ ID NO: 4538, SEQ ID NO: 5392, SEQ ID NO: 4335) as genetic drivers of disease. We demonstrate that genetic overexpression of these transmembrane proteins in multiple cell lines significantly increases the proliferation of these cells. Therefore, these extracellular transmembrane proteins become attractive targets for anti-proliferative cancer drugs.

[0265] Experimental Design: Protein overexpression plasmids encoding target proteins or controls were transiently transfected into multiple cell lines. After 24 hours, proliferation was measured using the WST-1 assay.

[0266] Materials and methods: The target cells were seeded at a density of 4,000 cells / well in 100 μL of cell culture medium in a 96-well plate. The cells were incubated in a humidified incubator at 37°C with 5% CO2 for 24 hours. Lipofectamine 3000 (Thermo Fisher Scientific) was diluted to a final concentration of 30 μL / mL in Opti-MEM medium (Thermo Fisher Scientific). DNA (pcDNA3.1 overexpression plasmid encoding the relevant control or target protein (Thermo Fisher Scientific)) and P3000 were diluted to a final concentration of 10 μg / mL (DNA) and 20 μL / mL (P3000) in Opti-MEM medium. The diluted DNA was mixed with the diluted Lipofectamine 3000 reagent, and the mixture was incubated at room temperature for 10 minutes. The mixture was added dropwise to the cells, and the plate was gently vortexed to evenly distribute the mixture. The cells were incubated at 37°C in a CO2 incubator for 48 hours. 10 μL of WST-1 reagent (Abcam) was added directly to the wells, and the plate was incubated in a humidified incubator with 5% CO2 at 37°C in the dark for 45 minutes. The absorbance at 450 nm was measured using a microplate reader. A reference wavelength of 650 nm can be used to correct the background absorbance. The percentage of live cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the results as a percentage of the control group.

[0267] SEQ ID NO:5391 is a novel unannotated protein that increases the proliferation of colon cancer cells (HCT116 cell line) under high serum conditions ( FIG. 2 , see also Table C). Table C Example 4. Validation of target proteins as genetic drivers of disease.

[0268] Purpose: The examples listed verify the regulation of circulating factors achieved by genetic variants that change the amino acid sequence of novel ORFs. SEQ ID NO:4371 contains a human genetic variant (rs221797) associated with metabolic diseases (i.e., type 2 diabetes). SEQ ID NO:4371's 4 genetic forms (V, A, G, D) are novel peptides that show variant-dependent differences in secretion using HiBiT assays. This demonstrates how disease-related forms of novel peptides regulate secretion levels, which has potential therapeutic relevance in identifying secretory forms of genetic disease modulators.

[0269] Experimental design: Promega The HiBiT cleavage detection system (Cat. No. N3030) assesses the impact of GWAS variants on modulating secretion. HiBiT expression vectors containing different versions of the sequence were transfected into HEK293T cells, and secretion was determined by measuring the luminescence of the HiBiT-tagged protein in the cell lysate.

[0270] Methods and Materials: Each genetic form of SEQ ID NO: 4371 was cloned into the HiBiT expression vector. HEK293T cells were plated at 50k / well in 96-well plates and transfected with Lipofectamin 3000 and DNA (HiBiT expression vector containing the sequence). Secretion was measured using the HiBiT Lysis Detection System (Cat. No. N3030). The HiBiT cleavage detection system uses a simple add-mix-read assay protocol to sensitively quantify HiBiT-labeled proteins in cell lysates. HiBiT is an 11-amino acid peptide tag that is fused to the N-terminus or C-terminus of the target protein or inserted into an accessible position within the protein structure. By adding a substrate containing furimazine and large BiT (LgBiT) ( The large subunit used in the binary technique ( 1)) cleavage detection reagent to determine the amount of HiBiT-tagged protein expressed in cells. HiBiT binds tightly to LgBiT (KD = 0.7nM), promoting complex formation in cell lysates to produce brightly luminescent enzymes. The amount of luminescence is proportional to the amount of HiBiT-tagged protein in the cell lysate. The target protein can be tagged with HiBiT at the N-terminus or C-terminus using a HiBiT expression vector.

[0271] Conclusion: If Figure 3 Shown, the human genetic variant in SEQ ID NO:4371 changes the secretion level of these peptides.rs221797V to A has no effect on secretion (a hydrophobic amino acid is replaced by another hydrophobic amino acid), while rs221797V to G and V to D significantly reduce secretion, and the replacement of charged amino acid (D) has the greatest effect.This proves how disease-related genetic variation can regulate the relative level of these peptides in circulation. Example 5. Validation of the target protein as a genetic driver of the disease. Stable overexpression of the novel ORF leads to increased proliferation of tumor cells.

[0272] Purpose: This example demonstrates the validation of a target protein (SEQ ID NO: 5404) as a genetic driver of a disease. Specifically, SEQ ID NO: 5404 is a target protein that significantly increases the proliferation of breast cancer cell line MCF-7 when overexpressed in these cells.

[0273] Experimental Design: Protein overexpression plasmids encoding target proteins or controls were transfected into multiple cell lines. After selection of stable expressing cells, proliferation was measured using the WST-1 assay.

[0274] Materials and methods: Stable cell lines: MCF-7 cell lines were inoculated into 2 mL of cell culture medium without antibiotics in 6-well plates at a density of 500,000 cells / well. Cells were incubated in a 37°C humidified incubator with 5% CO2. Lipofectamine 3000 (Thermo Fisher Scientific) was diluted to a final concentration of 30 μL / mL in Opti-MEM medium (Thermo Fisher Scientific). DNA (pcDNA3.1 overexpression plasmid encoding SEQ ID NO:5404) and P3000 (Thermo Fisher Scientific) were diluted to a final concentration of 10 μg / mL (DNA) and 20 μL / mL (P3000) in Opti-MEM medium. The diluted DNA was mixed with the diluted Lipofectamine 3000 reagent, and the mixture was incubated at room temperature for 10 minutes. The mixture was added dropwise to the cells, and the plate was gently vortexed to evenly distribute the mixture. The cells were incubated at 37° C. in a CO 2 incubator for 48 hours. The cells were selected with puromycin (2 μg / mL) for 10 days.

[0275] WST-1 assay: The target cells were seeded at a density of 4,000 cells / well in 100 μL cell culture medium in a 96-well plate. The cells were incubated in a 37°C humidified incubator with 5% CO2 for 24 hours. 10 μL WST-1 reagent (Abcam) was added directly to the wells, and the plates were incubated in a humidified incubator with 5% CO2 at 37°C in the dark for 45 minutes. The absorbance at 450 nm was measured using a microplate reader. A reference wavelength of 650 nm can be used to correct for background absorbance. The percentage of live cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the results as a percentage of the control group.

[0276] Conclusion: If Figure 4 As shown, SEQ ID NO: 5404 is a novel unannotated protein that increases the proliferation of breast cancer cells (MCF-7 cell line) under low and high serum conditions. Therefore, SEQ ID NO: 5404 is a genetic driver of cancer and a target protein for anticancer therapy.

[0277] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0278] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as encompassed by the appended claims.

Claims

1. A pharmaceutical agent comprising a target protein or a variant thereof identified in the sequence listing or in Table A and / or regulating the expression or activity of the target protein or a variant thereof.

2. The agent of claim 1, wherein the agent comprises the target protein.

3. The agent of claim 1, wherein the agent modulates the expression of the target protein.

4. The agent of claim 2 or 3, wherein the agent modulates the expression of a gene or gene transcript encoding the target protein. The agent of claim 1 , wherein the agent modulates the activity of the target protein.

6. The agent of any one of claims 2-5, comprising a polypeptide, a polynucleotide or a small molecule.

7. The agent of any one of claims 2-6, wherein the agent reduces the expression or activity of the target protein.

8. The agent of claim 7, wherein the agent comprises an inhibitor of the target protein.

9. The agent of claim 8, wherein the inhibitor is a polypeptide.

10. The agent of claim 9, wherein the polypeptide is an antagonist antibody or an antigen-binding fragment thereof that binds to the target protein.

11. The agent of claim 8, wherein the inhibitor is a polynucleotide. 12 . The agent of claim 11 , wherein the polynucleotide comprises a nucleotide sequence complementary to at least a portion of a gene or gene transcript encoding the target protein.

13. The agent of claim 11 or 12, wherein the polynucleotide comprises DNA.

14. The agent of claim 11 or 12, wherein the polynucleotide comprises RNA.

15. The agent of claim 11 or 12, wherein the polynucleotide is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense DNA, an antisense RNA, a micro RNA (miRNA), an antagonist, a guide RNA (gRNA), a locked nucleic acid (LNA), or a morpholino nucleic acid (MNA).

16. The agent of claim 8, wherein the inhibitor is a small molecule. The agent of claim 16 , wherein the small molecule binds to the target protein, thereby reducing the activity of the target protein.

18. The agent of any one of claims 1-6, wherein the agent increases the expression or activity of the target protein.

19. The agent of claim 18, wherein the agent is an isolated polypeptide comprising the amino acid sequence of the target protein, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of the target protein.

20. The agent of claim 19, wherein the isolated polypeptide or variant thereof is a recombinant protein or a synthetic protein.

21. The agent of claim 18, wherein the agent is a polynucleotide encoding the target protein or a variant thereof having at least 80% sequence identity with the amino acid sequence of the target protein.

22. The agent of claim 21, wherein the polynucleotide comprises DNA.

23. The agent of claim 21 or 22, wherein the polynucleotide comprises a vector.

24. The agent of claim 21, wherein the polynucleotide comprises RNA.

25. The agent of claim 24, wherein the RNA is messenger RNA (mRNA) and / or circular RNA (circRNA).

26. The agent of claim 18, wherein the agent comprises an activator of the target protein.

27. The agent of claim 26, wherein the activator is a polypeptide.

28. The agent of claim 27, wherein the polypeptide is an agonist antibody or an antigen-binding fragment thereof that binds to the target protein.

29. The agent of claim 26, wherein the activator is a small molecule.

30. The agent of claim 29, wherein the small molecule binds to the target protein, thereby increasing the activity of the target protein.

31. The agent of any one of claims 1-8, 18 and 26, comprising a gene editing system.

32. The agent of claim 31, wherein the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, and a transcription activator-like effector nuclease (TALEN) system.

33. The agent of any one of claims 1-32, wherein the target protein is translated from a non-coding RNA.

34. The agent of claim 33, wherein the non-coding RNA is a long intergenic non-coding RNA (lincRNA).

35. The agent of any one of claims 1-32, wherein the target protein is translated from a non-exonic element in an unprocessed precursor mRNA (pre-mRNA).

36. The agent of claim 35, wherein the non-exonic element is an intron in the pre-mRNA.

37. The agent of claim 35, wherein the non-exonic element is a 5' untranslated region (5'UTR) in pre-mRNA.

38. The agent of claim 35, wherein the non-exonic element is a 3' untranslated region (3'UTR) in pre-mRNA.

39. A pharmaceutical composition comprising the agent of any one of claims 1-38 and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable carrier is a carrier.

41. A method for regulating the expression or activity of a target protein or a variant thereof identified in the sequence listing, Table A, in a cell, the method comprising contacting the cell with an agent as described in any one of claims 1 to 38 or a pharmaceutical composition as described in claim 39 or 40.

42. The method of claim 41, wherein the agent reduces expression or activity of the target protein in the cell.

43. The method of claim 41, wherein the agent increases the expression or activity of the target protein in the cell.

44. The method of any one of claims 41-43, wherein the cell is in a subject.

45. The method of claim 44, wherein the subject suffers from a disease or condition associated with a genome-wide association study (GWAS), The Cancer Genome Atlas (TCGA), whole genome sequencing, a phenome-wide association study (PheWAS), an expression quantitative trait locus (eQTL) study, or a combination thereof.

46. ​​The method of any one of claims 41-45, wherein the agent modulates the expression or activity of the target protein by at least 10%.

47. The method of any one of claims 41-46, wherein the agent modulates the expression of the gene encoding the target protein by at least 10%, thereby modulating the expression or activity of the target protein.

48. A method for predicting the likelihood of a subject developing a disease or condition, the method comprising quantifying the expression or activity of a target protein identified in the sequence list, Table A, or a variant thereof in a sample from the subject, wherein the expression or activity level of the target protein in the sample indicates the likelihood of the subject developing cancer, wherein the disease or condition is associated with a genome-wide association study (GWAS), The Cancer Genome Atlas (TCGA), whole genome sequencing, a phenome-wide association study (PheWAS), an expression quantitative trait locus (eQTL) study, or a combination thereof.

49. A method of preparing a sample that can be used to detect the likelihood of a subject suffering from a disease or condition, the method comprising: a) obtaining or having obtained a sample from the subject; b) adding a protease inhibitor, a control peptide, a standard peptide or a combination thereof to the sample to prepare a sample that can be used to detect the possibility of suffering from cancer; and c) quantifying the expression or activity of the target protein identified in the sequence listing, Table A or the aforementioned variants in the sample prepared in step b), wherein the disease or condition is associated with a genome-wide association study (GWAS), The Cancer Genome Atlas (TCGA), whole genome sequencing, a phenome-wide association study (PheWAS), an expression quantitative trait locus (eQTL) study, or a combination thereof.

50. The method of claim 48 or 49, further comprising administering to the subject an effective amount of the agent of any one of claims 1-38 or the pharmaceutical composition of claim 39 or 40 if the subject is predicted to have a likelihood of developing cancer.

51. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent as described in any one of claims 1-38 or a pharmaceutical composition as described in claim 39 or 40, wherein the disease or condition is associated with a genome-wide association study (GWAS), The Cancer Genome Atlas (TCGA), whole genome sequencing, a phenome-wide association study (PheWAS), an expression quantitative trait locus (eQTL) study, or a combination thereof.

52. A method for identifying an agent that modulates the expression or activity of a target protein or variant thereof identified in Table A in the sequence listing, the method comprising: a) contacting a protein identified in the sequence listing or Table A or a variant thereof with a pharmaceutical agent; as well as b) determining whether the agent modulates the expression or activity of the target protein, wherein a difference in the expression or activity of the target protein that has been contacted with the agent compared to a reference for the expression or activity of the target protein indicates that the agent modulates the expression or activity of the target protein.

53. The method of claim 52, wherein a difference of at least 10% in the expression or activity of the target protein that has been contacted with the agent compared to the reference indicates that the agent modulates the expression or activity of the target protein.

54. The method of claim 52 or 53, wherein a decrease in the expression or activity of the target protein that has been contacted with the agent compared to the reference indicates that the agent inhibits the expression or activity of the target protein.

55. The method of claim 52 or 53, wherein an increase in the expression or activity of the target protein compared to the reference indicates that the agent activates the expression or activity of the target protein.

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