Compositions and methods for modulating genetic drivers

Abstract

The present disclosure provides compositions such as polypeptides, polynucleotides, gene editing systems, small molecules, vectors or host cells that include and / or modulate the expression or activity of immune regulatory related proteins such as cytokines in various embodiments. The present disclosure also provides methods for treating a disease or condition (e.g., a disease or condition related to genome-wide association studies (GWAS), The Cancer Genome Atlas (TCGA), whole genome sequencing, phenome-wide association studies (PheWAS), expression quantitative trait locus (eQTL) studies, or a combination thereof) using an agent that includes and / or modulates the expression or activity of an immune regulatory related protein, as well as methods for identifying said agent.

Description

Technical Field

[0001] Incorporation by Reference of Materials 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.

[0002] Incorporation of XML by Reference of Materials This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, which is filed concurrently 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 underlies unmet medical needs across many diseases and conditions. Many diseases and conditions have genetic components that can affect or directly cause dysfunction. The genetic predisposition to develop a disease or condition can result from changes at a single locus or as a result of changes at many loci. Identifying novel genetic drivers of diseases and conditions provides opportunities to develop and / or improve diagnostic, preventive, and / or treatment options for various diseases and conditions.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The disclosure provided herein is based in part on the identification of non-standard protein targets (e.g., proteins encoded by non-standard open reading frames (ORFs)) that are altered (e.g., mutated) in various diseases, conditions, or pre-diseases.

Means for Solving the Problems

[0005] In one aspect, the present disclosure relates to an agent that includes and / or modulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., the target proteins of the Sequence Listing, Table A, or the aforementioned variants). In some embodiments, the agent includes a target protein identified herein (e.g., the target proteins of the Sequence Listing, Table A, or the aforementioned variants). In certain embodiments, the agent modulates (e.g., increases or decreases) the expression and / or activity of a target protein identified herein (e.g., the target proteins of the Sequence Listing, Table A, or the aforementioned variants). In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy). The agent can be an inhibitor or activator of a target protein identified herein. In some embodiments, the agent modulates the expression of a target protein identified herein. In some embodiments, the agent modulates 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 present 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 of developing a disease or condition in a subject, the method comprising quantifying the expression or activity of a target protein in a sample from the subject, wherein the level of expression or activity of the target protein in the sample indicates the likelihood of developing a disease or condition in the subject. In certain embodiments, the disease or condition is related to 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 locus (eQTL) studies (e.g., Nica & Dermitzakis, Expression quantitative trait loci: present and future, Philos Trans R Soc Lond B Biol Sci. 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 for preparing a sample useful for determining the likelihood of developing a disease or condition in a subject, 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 useful for detecting the likelihood of developing cancer; and c) quantifying the expression or activity of a target protein in the sample prepared in step b). The present disclosure provides a method comprising the above steps.

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

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

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

[0014] In another aspect, the disclosure provides a method of modulating the expression or activity of a target protein identified in the Sequence Listing, or a variant as described above, in a cell (e.g., a cancer cell such as a cancer cell in a subject), the method comprising contacting the cell (e.g., in vitro, ex vivo, or in vivo) with an agent that comprises and / or modulates the expression or activity of a target protein identified herein, or a pharmaceutical composition comprising the agent.

[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, a) contacting the target protein with the agent; and b) determining whether the agent modulates the expression or activity of the target protein comprising Provided herein is a method indicating that a difference in the expression or activity of a target protein contacted with an agent, as compared to a reference with respect to the expression or activity of the target protein, indicates that the agent regulates the expression or activity of the target protein.

[0016] The foregoing will be apparent from the following more particular description of the exemplary 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 embodiments.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2-1

Figure 2-2

Figure 2-3

Figure 3

Figure 4

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The description of the exemplary embodiments continues below.

[0019] Target Protein In one aspect, the present disclosure provides the target proteins identified herein. As used herein, either of the expressions "the target proteins identified herein" and "the target proteins of the present disclosure" refers to the polypeptides disclosed in the sequence listing (e.g., target proteins comprising amino acid sequences 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 in Table C herein), variants thereof (e.g., target proteins comprising amino acid sequences selected from SEQ ID NO: 4335_64NS, SEQ ID NO: 4335_P43A, SEQ ID NO: 5391_P31L, SEQ ID NO: 4371 rs221797 V-to-A, V-to-G, or V-to-D), and the peptides disclosed in Table A herein. The target proteins can be produced recombinantly (e.g., via DNA or mRNA) or synthetically.

[0020] 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 certain embodiments, the target protein is membrane-bound and extracellular but not transmembrane. In more specific embodiments, the target protein is embedded in the membrane but not transmembrane.

[0021] In various embodiments, the target protein is a protein comprising the amino acid sequence set forth in the Sequence Listing or Table A. In some embodiments, the target protein consists of the amino acid sequence set forth in the Sequence Listing or Table A. In some embodiments, the target protein comprises an amino acid sequence having one amino acid substitution relative to the amino acid sequence set forth in the Sequence Listing or Table A, where the substitution is by a methionine (Met) residue at the N-terminal residue in the amino acid sequence of the Sequence Listing or Table A. In some embodiments, the target protein consists of an amino acid sequence having one amino acid substitution relative to the amino acid sequence set forth in the Sequence Listing or Table A, where the substitution is by a methionine (Met) residue at the N-terminal residue in the amino acid sequence of the Sequence Listing or Table A. In some embodiments, the target protein comprises the amino acid sequence set forth 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 the amino acid sequence set forth in the Sequence Listing or Table A and the methionine (Met) residue at its N-terminus.

[0022]

Table A

[0023] While not wishing to be bound by theory, the mutations of the specific target proteins disclosed herein are thought to be related to, contribute to, or result in a disease, condition, and / or pre-disease state, where the mutation results in a loss or gain of a biological function that contributes to or alleviates the disease, condition, and / or pre-disease state. In some embodiments, the mutation and / or target protein is used as a biomarker or surrogate for a disease, condition, and / or pre-disease state.

[0024] Some of the target proteins in the sequence listing and Table A are differentially expressed (e.g., upregulated or downregulated) in a disease and / or condition selected from a disease, condition, and / or pre-disease state (e.g., the diseases listed in paragraphs

[0219] and

[0220] ) related to GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or combinations thereof, as compared to a reference state (e.g., a normal state), such that regulation of the level and / or activity of the target protein acts to treat, improve, and / or prevent the onset of the disease or condition.

[0025] As used herein, the term "differentially expressed" refers to at least one recognizable difference in protein expression. It can be a quantitatively measurable, semi-quantitatively estimable, or qualitatively detectable difference in protein expression. Thus, a protein that is differentially expressed, i.e., a "DEP", can have a higher expression level in a reference state (e.g., a normal state) than in a disease state in which 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) in which the DEP has a lower expression level or is not expressed at all. Further, expression can be considered differential if the DEP changes recognizably (e.g., mutates) between the two states being compared. Recognizable changes can include amino acid substitutions, insertions, and / or deletions, including N-terminal and C-terminal cleavage, as well as modifications (e.g., post-translational modifications).

[0026] As used herein, the term "reference" refers to a standard used for comparison purposes. One of ordinary skill in the art can select an appropriate reference for a particular comparison purpose. Thus, for example, a reference for a disease state may be a normal healthy state; a reference for a mutant protein may be a non-mutant protein; a reference for disease treatment may be no treatment or a standard treatment. In some embodiments, particularly those including methods for identifying an agent that modulates 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 the agent. In some embodiments, the reference is based on a predetermined level, such as functional expression or an empirical assay. In some embodiments, the reference is obtained from one cell, sample, or subject (e.g., a cell or sample from a healthy subject, a subject without a particular disease; a healthy subject, a subject without a particular disease). In some embodiments, the reference is obtained from two or more cells, samples, or subjects (e.g., a population thereof) such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more (e.g., a cell or sample from a healthy subject, a subject without a particular disease; a healthy subject, a subject without a particular disease), or a statistically significant number of cells, samples, or healthy subjects. A reference obtained from two or more cells, samples, or subjects can be expressed as a statistic (e.g., an average or median).

[0027] In some embodiments, the target protein has an expression level that is at least about 0.5-fold higher, such as at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) than the target protein expression level in a reference (e.g., a sample from a cell or tissue of a subject without a disease or condition) in a disease or condition (e.g., a disease or condition associated with a GWAS, TCGA, whole genome sequencing, PheWAS, eQTL study, or a combination thereof) (e.g., as determined from a sample from a cell or tissue of a subject having the disease or condition).

[0028] In some embodiments, the target protein has an expression level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) than the target protein expression level in a reference (e.g., a sample derived from cells or tissue of a subject without a disease or condition), in a disease or condition (e.g., a disease such as those listed in paragraphs

[0219] and

[0220] , a disease or condition associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof) (e.g., determined from a sample comprising or obtained from cells or tissue of a subject having the disease or condition). In some embodiments, the target protein is not expressed or is expressed at a non-detectable level in a disease or condition (e.g., a disease such as those listed in paragraphs

[0219] and

[0220] , a disease or condition associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof) (e.g., when determined from a sample comprising or obtained from cells or tissue of a subject having the disease or condition).

[0029] In some embodiments, the target protein has a transcript level that is at least about 0.5-fold higher, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher (e.g., 50-fold higher, 100-fold higher) than the target protein transcript level in a reference (e.g., a sample from a cell or tissue of a subject without a disease or condition), in a disease or condition (e.g., a disease such as those listed in paragraphs

[0219] and

[0220] , a disease or condition associated with GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof), as determined from a sample from a cell or tissue of a subject having the disease or condition. In certain embodiments, the increase in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0030] In some embodiments, the target protein has a transcript level that is at least about 0.5-fold lower, e.g., at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold lower (e.g., 50-fold lower, 100-fold lower) than the target protein transcript level in a reference (e.g., a sample from a cell or tissue of a subject without a disease or condition), in a disease or condition (e.g., a disease or condition related to GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ) (e.g., determined from a sample comprising or obtained from a cell or tissue of a subject having the disease or condition). In some embodiments, the transcription of the target protein is not expressed or is expressed at undetectable levels in a disease or condition (e.g., when determined from a sample comprising or obtained from a cell or tissue of a subject having the disease or condition). In certain embodiments, the decrease in the transcription level of the target protein contributes to (e.g., results in) the disease or condition described herein.

[0031] In certain embodiments, the gene encoding the target protein comprises at least one mutation (e.g., a fusion of amino acid repeats, a deletion, an insertion, a point mutation, and / or an expansion) in the disease described herein.

[0032] In some embodiments, the mutation and / or the target protein is used as a marker for a disease or condition (e.g., a disease or condition related to GWAS, TCGA, whole genome sequencing, PheWAS, eQTL studies, or a combination thereof, such as the diseases listed in paragraphs

[0219] and

[0220] ).

[0033] In certain embodiments, the target protein has a higher expression level in cells and / or tissues associated with the diseases or conditions described herein. In some embodiments, the expression level of the target protein in cells and / or tissues is at least about 0.5-fold, for example, at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher (e.g., 50-fold higher, 100-fold higher) than the target protein expression level in a reference (e.g., a different cell / tissue type).

[0034] In certain embodiments, the target protein has a lower expression level in cells and / or tissues associated with the diseases or conditions described herein. In some embodiments, the expression level of the target protein in cells and / or tissues is at least about 0.5-fold, for example, at least about 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1.0-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold lower (e.g., 50-fold lower, 100-fold lower) than the target protein expression level in a reference (e.g., a different cell / tissue type).

[0035] 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 micro-biopsy), pancreatic juice, chorionic villus samples, and cells isolated from a subject.

[0036] 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).

[0037] 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 pre-mRNA. In some embodiments, the non-exon element is the 5' untranslated region (5'-UTR) in pre-mRNA. In some embodiments, the non-exon element is the 3' untranslated region (3'-UTR) in pre-mRNA.

[0038] In some embodiments, the target protein has a length of 2,000 amino acids or less, such as 1000 amino acids or less, 750 amino acids or less, 500 amino acids or less, 250 amino acids or less, 150 amino acids or less, or 100 amino acids or less. In some embodiments, the target protein has a length of 7 amino acids or more, such as 8, 9, 10, 15, 18, 25, 50, 75, or 100 amino acids or more. In certain embodiments, the target protein has a length of about 50 to about 200 amino acids, such as about 100 to about 150 amino acids. In certain embodiments, the target protein has a length of 7 amino acids or more. In more particular embodiments, the target protein has a length of about 18 amino acids.

[0039] 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.

[0040] The expression and / or activity of various GPCRs are associated with various diseases / disorders, conditions, and indications, including those shown in Table B (e.g., see Kenakin, T., Biased Receptor Signaling in Drug Discovery, Pharmacol Rev 71:267-315, April 2019; Harmar, A.J., 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, de Graaf C, Brown AJH, and Maguire JJ. Advances in therapeutic peptides targeting G protein-coupled receptors. Nat Rev Drug Discov. 2020 Jun. 19(6):389-413; the entire contents of each are hereby incorporated by reference in their entirety). Thus, in some embodiments, the target proteins disclosed herein that are modulators of GPCRs are useful for treating and / or diagnosing one or more diseases / disorders, conditions, and / or indications known to be associated with GPCR expression and / or activity, such as cancer or pre-cancerous conditions, or any of the diseases / disorders, conditions, and indications listed in Table B.

[0041]

Table B-1

[0042]

Table B-2

[0043]

Table B-3

[0044]

Table B-4

[0045]

Table B-5

[0046] Agents that regulate the target protein As used herein, provided are agents that regulate the expression of a target protein disclosed herein, such as a target protein in the Sequence Listing or Table A, or a variant or fragment thereof (e.g., a biologically active fragment of the target protein). The expression of the target protein or its variant or fragment can be regulated by a wide range of processes that directly or indirectly result in an increase or decrease in the level of the target protein. Non-limiting examples include changes in the copy number of the gene encoding the target protein, transcription initiation, elongation or termination, RNA processing, RNA stability (e.g., mRNA stability), RNA degradation, translation initiation, post-translational modification of the protein, protein stability, proteolysis (e.g., cleavage such as protease cleavage), or combinations thereof.

[0047] In some embodiments, the agent regulates (e.g., increases or decreases) the expression of the gene or gene transcript encoding the target protein. In some embodiments, the agent regulates the expression or activity of the target protein. In some embodiments, the agent decreases (e.g., inhibits, reduces or neutralizes) the activity of the target protein. In some embodiments, the agent increases (e.g., activates) the activity of the target protein. In some aspects, the agent decreases (e.g., inhibits or down-regulates) the expression of the target protein. In other embodiments, the agent increases (e.g., activates or up-regulates) the expression of the target protein.

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

[0049] As used herein, the term "lowering" or "lower" refers to an adjustment that results in a lower level of expression, activity, function, or a combination or metric thereof (e.g., cancer cell death or DNA methylation of a target site) of a target protein as compared to a reference (e.g., a level before or in the absence of modulation by an agent). In some embodiments, the agent lowers the expression or activity or metric of the target protein by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% as compared to the reference.

[0050] Non-limiting examples of metrics include energy production or energy conversion in the liver (e.g., regulation of ATP synthesis, β-oxidation, oxidation of metabolites derived from glycolysis, oxidation of metabolites derived from amino acids), mitochondrial transcription, mitochondrial ribosome assembly, mitochondrial translation, mitochondrial heat production, 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, endoplasmic reticulum stress response pathway, signaling processes (e.g., calcium signaling).

[0051] In some embodiments, the level of expression, activity, function, or combination thereof of the target protein, or the metric, is measured, for example, after initiating a treatment regimen, after contacting the agent (e.g., with cells) or administering the agent (e.g., to a subject) for at least about 1 day, such as 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.

[0052] In some embodiments, the agent comprises, consists essentially of, or consists of a polypeptide, polynucleotide, gene editing system, small molecule, or cell (e.g., cell therapy).

[0053] In some embodiments, the target protein activates immune cells, and the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or combinations 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 combinations thereof), and the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or combinations thereof of the target protein.

[0054] In certain embodiments, the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or combinations thereof of the target protein in cells and / or tissues associated with the disease or condition described herein, or performs the aforementioned combinations.

[0055] In some embodiments, the agent modulates (e.g., increases or decreases) the protein function and / or signaling pathway associated with the disease or condition described herein, or the aforementioned combinations.

[0056] In some embodiments, the agent induces downregulation of the target protein (e.g., increases target protein degradation); prevents multimerization (e.g., dimerization) of the target protein; sequesters the target protein (e.g., secreted target protein); modulates (e.g., agonizes, antagonizes, or disrupts) a known function of the target protein; decreases the binding between the target protein and a binding partner (e.g., via steric hindrance); induces antibody-dependent cell killing, phagocytosis, and / or opsonization of cells expressing the target protein; or combinations thereof. In certain embodiments, the agent lacks agonist activity against the target protein. In certain embodiments, the agent has agonist activity against the target protein. In some embodiments, the agent lacks antagonist activity against the target protein. In some embodiments, the agent has antagonist activity against the target protein. In certain embodiments, the agent binds to at least one residue of the target protein 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 involved in binding of the target protein to the binding partner.

[0057] The binding partner of the target protein of the present disclosure can be any known protein in the mammalian (e.g., human) genome.

[0058] In some embodiments, the agent induces downregulation of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); prevents multimerization (e.g., dimerization) of a binding partner of the target protein; sequesters a binding partner of the target protein (e.g., a secreted binding partner); induces antibody-dependent cell killing, phagocytosis and / or opsonization of cells expressing a binding partner of the target protein; modulates (e.g., agonizes, antagonizes, or disrupts) a known function of a binding partner of the target protein; reduces the binding between the target protein and the binding partner (e.g., via steric hindrance); or performs a combination of the above. In certain embodiments, the agent lacks agonist activity against a binding partner of the target protein. In certain embodiments, the agent has agonist activity against a binding partner of the target protein. In some embodiments, the agent lacks antagonist activity against a binding partner of the target protein. In some embodiments, the agent has antagonist activity against a binding partner of the target protein. In some embodiments, the agent binds to the target binding site of the binding partner. In certain embodiments, the agent further binds to at least one residue of a binding partner of the target protein involved in the binding between the target protein and the binding partner. In more specific embodiments, the agent further binds to one or more binding sites and / or domains of a binding partner of the target protein involved in the binding between the target protein and the binding partner.

[0059] In some embodiments, the agent modulates (e.g., activates or inhibits) immune signaling, cytokine signaling, inflammatory signaling, or a combination of the foregoing.

[0060] In some embodiments, the agent enhances signals 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 certain embodiments, the agent is an agonist for CD28.

[0061] 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 SIGLEC 9.

[0062] In certain embodiments, the agent reduces the function of (e.g., blocking antibody) against TNFα.

[0063] In certain embodiments, the agent modulates (e.g., increases or decreases) the level of expression, activity, function, or a combination thereof of a variant of the 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 can 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.

[0064] 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, typically one sequence is designated as the reference sequence and is compared to the test sequence. The sequence identity between the reference sequence and the test sequence is expressed as the percentage of positions over the full length of the reference sequence at which the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the maximum level of identity. As an example, when aligned to achieve the maximum level of identity, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0065] Alignment of the comparison arrays to achieve maximum level of identity can be readily implemented by one of ordinary skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment may include gaps introduced to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (generally, see Ausubel et al., Current Protocols in Molecular Biology).

[0066] When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, coordinates are then specified as necessary, and the sequence algorithm program parameters are specified. Next, the sequence comparison algorithm calculates the percent sequence identity of the test sequence to the reference sequence based on the specified program parameters. A commonly used tool for determining percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP) available from the National Center for Biotechnology Information of the National Library of Medicine of the National Institutes of Health. (Altschul et al., 1990).

[0067] In some embodiments, the amino acid sequence of a 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 relative to the amino acid sequence of the target protein disclosed herein 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. 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.

[0068] The amino acid substitution in the variant can be a substitution with a standard amino acid or a non - standard amino acid. Non - standard amino acids include, but are not limited to, D - amino acids such as the D - version of standard L - amino acids.

[0069] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution having a value of 0 or more in BLOSUM62.

[0070] In some embodiments, the amino acid substitutions are highly conservative substitutions. The term "highly conservative amino acid substitution" or "highly conservative substitution" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0071] In some embodiments, variants of the target proteins of the present disclosure contain about 5 to 60 amino acid substitutions relative to the amino acid sequences of the target proteins disclosed herein. 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.

[0072] A. Polypeptide agent The terms “polypeptide,” “peptide,” or “protein” mean a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Proteins, peptides, or polypeptides can include 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). Amino, carboxyl, and / or other functional groups on the peptide may be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, as well as 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 can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorophore or hapten) using methods known in the art. Proteins, peptides, or polypeptides can optionally include one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modification factors (e.g., cyclization modification), N-methyl-α-amino group substitution). Further, proteins, peptides, or polypeptides can be analogs of known and / or native peptides, such as peptide analogs having conservative amino acid residue substitutions.

[0073] 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 (e.g., a direct inhibitor or an indirect inhibitor) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide is an activator (e.g., a direct activator or an indirect activator) of the expression and / or activity of a target protein disclosed herein. In some embodiments, the polypeptide decreases 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., its biologically active portion, e.g., a biologically active fragment of the target protein).

[0074] 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 regulate the expression or activity of a target protein.

[0075] In some embodiments, the polypeptide is an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via 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 comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or multimers thereof (e.g., IgM). Each heavy chain has a heavy-chain variable region (V Hand a heavy chain constant region (including domains CH1, hinge, CH2 and CH3). Each light chain comprises a light chain variable region (V L ) and a light chain constant region (CL). V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed within framework regions (FRs). V H and V L each contain three CDRs and four FR segments and are arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibody can be of any species, e.g., a rodent (e.g., mouse, rat, guinea pig) antibody, a human antibody, or the antibody can be a humanized or chimeric antibody.

[0076] 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 κ light chain constant region. In some embodiments, the antibody comprises a λ light chain constant region.

[0077] 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, e.g., bispecific, trispecific or tetravalent. In some embodiments, the antibody is a heteroconjugate antibody.

[0078] 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 the V H region, the V L region, Fab fragment, F(ab’)2 fragment, Fd fragment, Fv fragment, and domain antibodies (dAbs) consisting of one V H domain or one V L domain. The VH and VL domains can be linked to each other via a synthetic linker to form various types of single-chain antibody designs where the V H / V L domains pair within the molecule or, when the V H and V L domains are expressed by separate chains, pair intermolecularly, forming a monovalent antigen-binding site such as a single-chain Fv (scFv) or a diabody. In some embodiments, the polypeptides disclosed herein are antigen-binding fragments selected from Fab, Fab’, F(ab’)2, Fd, Fv, disulfide-bonded Fv (sdFv, e.g., diabody, triabody or tetrabody), scFv, SMIP or rlgG. In some embodiments, the polypeptide is an scFv. Antigen-binding fragments can be generated by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.

[0079] The polypeptide agent (e.g., a monoclonal antibody) can be monovalent, bivalent or multivalent. A monoclonal antibody can be monospecific or multispecific (e.g., bispecific). A monospecific antibody binds to one antigen epitope. Multispecific antibodies such as bispecific or trispecific antibodies are included within the term monoclonal antibody.

[0080] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen, such as 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.

[0081] "Isolated antibody" refers to an antibody or an antigen-binding fragment thereof that is substantially free of other antibodies having different antigen 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 the 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, the polypeptide agent (e.g., a monoclonal antibody) is at least 80% pure, e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0082] In some embodiments, the polypeptide is an antagonist antibody that binds to a target protein (e.g., a target protein whose expression or activity is elevated in a cancer state compared to a reference state). In some embodiments, the antibodies described herein are antagonist antibodies that bind to a protein that can modulate the expression or activity of a target protein. As used herein, the term "antagonist antibody" refers to an antibody that, when bound to an antigen (e.g., a target protein or a protein that can modulate the expression or activity of a target protein), reduces (e.g., inhibits) the function of the antigen. 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 attenuates the signaling pathway mediated by the signaling molecule. In some embodiments, the antagonist antibody reduces the antigen function by at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 99%.

[0083] 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 cancerous state compared 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 the target protein. As used herein, the term "agonist antibody" refers to an antibody that, when bound to an antigen (e.g., a target protein or a protein that can regulate the expression or activity of the target protein), increases the function of the antigen. 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 signaling pathway mediated by the signaling molecule. In some embodiments, the agonist antibody increases the antigen function by at least about 10%, such as 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%.

[0084] In some embodiments, the agonist antibody does not exhibit at least one of the following functional properties: reducing (e.g., inhibiting) the activity of the antigen; inducing antibody-dependent cell death 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 downregulation of the antigen on the cell surface (e.g., by cross-linking or clustering the antigen to induce internalization and degradation).

[0085] Appropriate techniques, assays, and reagents for making and using therapeutic antibodies against antigens are known in the art. See, for example, methods for 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 in Therapeutic Monoclonal Antibodies: From Bench to Clinic (Zhiqiang An eds., 1st ed. 2009); Antibodies: A Laboratory Manual (Edward A. Greenfield eds., 2d ed. 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).

[0086] 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 mimic the ability of an antibody to bind an antigen but has a structure that is structurally different from the native antibody structure. Non-limiting examples of antibody mimetics include adnectin, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, nanobody, nanoCLAMP, and Versabody.

[0087] In some embodiments (e.g., when the expression or activity of the target protein is reduced in the disease state as compared to the 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 of the target protein (e.g., a biologically active portion or fragment). For example, the percent identity can 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% relative to the full-length target protein or a biologically active portion or fragment thereof. 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.

[0088] 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.

[0089] 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 can regulate the expression or activity of a target protein. For example, the percent identity can 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 the amino acid sequence of a protein that can regulate the expression or activity of a target protein.

[0090] 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 that can regulate 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 substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are highly conservative substitutions.

[0091] In some embodiments, the polypeptide is a cell-penetrating peptide. In certain embodiments, the polypeptide is linked to a cell-penetrating peptide. Suitable cell-penetrating peptide sequences 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, Pharmaceuticals 5(9):991-1007(2012). Non-limiting examples of cell-penetrating peptides include TAT(48-60), Penetratin, pVEC, MPG8, Transportan, Transportan10, PepFect3, PepFect 6, PepFect 14, polyarginine, stearyl-polyarginine, Pep-1, Pep-3, CADY, YTA2, YTA4, SynB1, SynB3, Maurocalcine, and PTD4.

[0092] In some embodiments, the polypeptide is a circulating factor (e.g., a cytokine).

[0093] In some embodiments, the polypeptide (e.g., an isolated polypeptide) and the biological properties (e.g., biological activity or half-life) of the target protein are similar. Non-limiting examples of biological activity include, inter alia, 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., transferring a functional group from one molecule to another), oxidoreductase activity (e.g., catalyzing an oxidation-reduction reaction), hydrolase activity (e.g., cleaving a chemical bond by hydrolysis), lyase activity (e.g., generating a double bond), ligase activity (e.g., joining two molecules via a covalent bond) and isomerase activity (e.g., catalyzing an intramolecular structural change from one isomer to another).

[0094] 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 (Mark C. Smales & David C James eds., 2005); Pharmaceutical Biotechnology: Fundamentals and Applications (Daan J.A. Crommelin, Robert D. Sindelar & Bernd Meibohm eds., 2013). The polypeptide can be recombinantly expressed using, for example, mammalian cells, insect cells, yeast or bacteria under the control of an appropriate promoter.

[0095] In some embodiments, the polypeptide comprises 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 polypeptides described herein (e.g., a target protein or a portion thereof, a polypeptide agent that modulates a target protein) are modified, for example, by cleavage (e.g., protease cleavage) or post-translational modification. In certain embodiments, the modification affects the activity of the polypeptide, for example, by activating an inactive polypeptide or by changing (e.g., increasing, decreasing) the activity level of the polypeptide. In certain embodiments, the polypeptides described herein are provided as prodrugs that can be converted, for example, in vivo (e.g., by proteolytic cleavage, post-translational modification) into an active polypeptide. In some embodiments, the polypeptide comprises 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.

[0097] In some embodiments, the polypeptide comprises one or more neoantigens selected from the sequence listing, Table A, or a variant as described above. As used herein, the term "neoantigen" refers to a tumor antigen arising from a target protein described herein. In some aspects, the neoantigen is a cancer-specific neoantigen. There are various methods of producing neoantigens. For example, a neoantigen can be produced in vitro as a polypeptide before being formulated into a neoadjuvant vaccine or an immunogenic pharmaceutical composition. In some embodiments, the immunogenic pharmaceutical composition comprises an effective amount of one or more neoantigens or a pharmaceutically acceptable salt thereof. In some embodiments, the immunogenic pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, adjuvant, or additive.

[0098] Alternatively, a neoantigen can be produced in vivo by introducing a polynucleotide or expression vector (e.g., a viral expression vector) encoding the neoantigen into a cell or tissue (e.g., of a subject in need thereof). In certain embodiments, the polypeptide comprises at least two neoantigens. 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 an invariant chain, a leader sequence of tissue-type plasminogen activator, a PEST sequence, a cyclin destruction box, a ubiquitination signal, and a SUMOylation signal.

[0099] B. Polynucleotide Agents In some embodiments, the agent comprises a polynucleotide or an analog or derivative thereof. In some embodiments, the polynucleotide or an analog or derivative thereof is an inhibitor of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof is an activator of a target protein. In some embodiments, the polynucleotide or an analog or derivative thereof reduces (e.g., reduces or neutralizes) the expression or activity of a target protein. In other embodiments, the polynucleotide or an analog or derivative thereof increases the expression or activity of a target protein.

[0100] A polynucleotide can have a sequence containing naturally occurring ribonucleotide or deoxyribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Thus, a polynucleotide can 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, locked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, and 2'-deoxyuridine. In some embodiments, the modification is to increase nuclease resistance, increase serum stability, decrease immunogenicity, or a combination thereof.

[0101] 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, plasmid).

[0102] In some embodiments, the polynucleotide includes a polynucleotide analog or derivative. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative includes one or more phosphorothioate linkages. In some embodiments, the agent includes deoxyribonucleoside guanidine (DNG) nucleotides. In some embodiments, the agent includes ribonucleoside guanidine (RNG) nucleotides.

[0103] In some embodiments, the polynucleotide regulates the expression and / or activity of a nucleic acid encoding a target protein (e.g., the target protein of the Sequence Listing) or a portion thereof (e.g., a biologically active portion or a fragment thereof) disclosed herein in Table A.

[0104] In some embodiments, the polynucleotide comprises a nucleotide sequence that is complementary (e.g., fully or partially complementary) to at least a portion of a gene or gene transcript encoding a target protein disclosed herein, such that the polynucleotide sequence can hybridize or anneal to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the polynucleotide comprises a nucleotide sequence that is complementary to at least a portion of a gene or gene transcript encoding a protein that can regulate the expression or activity of a target protein disclosed herein.

[0105] 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 a fragment thereof).

[0106] In some embodiments, the nucleic acid encoding the target protein, or a variant thereof, or a portion thereof (e.g., a fragment) is a gene sequence or a portion thereof. In some embodiments, the encoding nucleic acid is an unprocessed RNA transcript (e.g., pre-mRNA) or a portion thereof (e.g., 5'-UTR, 3'-UTR, intron). In some embodiments, the encoding nucleic acid is an mRNA molecule or a portion thereof. In some embodiments, the encoding nucleic acid is present in a non-coding RNA (e.g., long intergenic non-coding RNA (lincRNA), long non-coding RNA (lncRNA), or miRNA).

[0107] The encoding nucleic acid can include a standard open reading frame (ORF) or a non-standard ORF. In certain embodiments, the encoding nucleic acid includes a non-standard ORF.

[0108] The polynucleotide can be single-stranded (ss) or double-stranded (ds). In some embodiments, the polynucleotide is double-stranded (ds). In some embodiments, the length of the ds polynucleotide is about 15 to 50 base pairs, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 base pairs. In some embodiments, the length of the polynucleotide is about 19 to 23 base pairs. In some embodiments, the length of the polynucleotide is about 21 base pairs.

[0109] In some embodiments, the polynucleotide is single-stranded (ss). In some embodiments, the length of the ss polynucleotide is about 15 to 50 nucleotides, such as about 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40 or 40 to 50 nucleotides.

[0110] In some embodiments, the polynucleotide inhibits the maturation of a newly generated nuclear RNA transcript into mRNA for transcription. In some embodiments, the polynucleotide comprises a nucleotide sequence complementary to a sequence at the intron-exon boundary.

[0111] In some embodiments, a polynucleotide (e.g., an antisense oligonucleotide) can hybridize to an mRNA encoding a target protein (e.g., under physiological conditions). In some embodiments, the length of the polynucleotide is at least about 10 nucleotides, 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 same target transcript, such as at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0112] In some embodiments, the polynucleotide further comprises an overhang sequence (e.g., unpaired overhang nucleotides that are not directly involved in the formation of the duplex structure by 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 modified ribonucleotides or deoxynucleotides, such as phosphorothioates, phosphorothioates, or deoxynucleotide inversion (linked 3' to 3') nucleotides.

[0113] 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), microRNA (miRNA), antagomir, antisense DNA, antisense RNA, morpholino nucleic acid (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), aptamer, and guide RNA (gRNA).

[0114] In some embodiments, the polynucleotide inhibits gene expression (e.g., via the biological process of RNA interference (RNAi)). Polynucleotides suitable for RNA interference can be readily designed and made by those skilled in the art using techniques, assays, and reagents known in the art, including computational tools. See, for example, 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.

[0115] In some embodiments, the polynucleotide is a miRNA. In some embodiments, the miRNA is about 22 nucleotides in length. The miRNA binds to a target site on the mRNA molecule and silences the mRNA, for example, by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA.

[0116] In some embodiments, the polynucleotide is a siRNA. In some embodiments, the siRNA comprises a nucleotide sequence identical to about 15-25 contiguous mRNA sequences encoding the 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 siRNA has a GC content of about 30-70%, such as 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%.

[0117] In some embodiments, the polynucleotide is shRNA. shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene via RNAi. shRNA can be delivered to cells in the form of a plasmid, such as a viral vector or a bacterial vector, by, for example, transfection, electroporation or transduction.

[0118] siRNAs and shRNAs 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, siRNAs function as miRNAs; in other embodiments, miRNAs function as siRNAs (see, e.g., Zeng et al., Mol Cell 9:1327-33 (2002); Doench et al., and Genes Dev 17:438-42 (2003)). MicroRNAs such as siRNAs use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNA. Instead, miRNAs reduce protein output through translational repression or polyA removal and mRNA degradation (see, e.g., Wu et al., Proc Natl Acad Sci USA 103:4034-39 (2006)). Known miRNA binding sites are within the mRNA 3’UTR; miRNAs are thought to target sites with near-perfect complementarity to nucleotides 2-8 from the 5’ end of the miRNA (see, e.g., Rajewsky, Nat Genet 38 Suppl:S8-13 (2006) and Lim et al., Nature 433:769-73 (2005)). This region is known as the seed region. Since siRNAs and miRNAs are interchangeable, exogenous siRNAs downregulate mRNAs that have seed complementarity to the siRNA (see, e.g., Birmingham et al., Nat Methods 3:199-204 (2006)). Multiple target sites within the 3’UTR result in stronger downregulation (see, e.g., Doench et al., Genes Dev 17:438-42 (2003)).

[0119] In some embodiments, the polynucleotide is a messenger RNA (mRNA) or circular RNA (circRNA) encoding a target protein or a variant thereof (e.g., a variant that is at least about 70% identical to the wild-type protein, e.g., at least about 75%, 80%, 85%, 90%, 95%, 98% or 99% identical) disclosed herein. In some embodiments, the mRNA is codon-optimized (e.g., to improve the efficiency of protein synthesis and limit mRNA destabilization 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)).

[0120] In some embodiments, the polynucleotide comprising RNA is chemically synthesized. In some embodiments, the polynucleotide comprising RNA is recombinantly expressed. In some embodiments, the RNA is transcribed in vitro. The production and use of RNA therapeutics are known in the art. See, e.g., RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud eds., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60(2017).

[0121] In some embodiments, the mRNA is produced by in vitro transcription. In some embodiments, the mRNA is modified to optimize its activity. In some embodiments, the mRNA comprises modified bases, a 5' cap, a 5' cap analog, an anti-reverse cap analog (ARCA), or a combination thereof.

[0122] In some embodiments, the mRNA comprises a poly(A) tail. In some embodiments, the poly(A) tail is about 100 to 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)).

[0123] 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-dithiophosphate-modified cap (see, e.g., Strenkowska et al., Nucleic Acids Res. 44:9578-90 (2016)).

[0124] In some embodiments, the mRNA comprises a modified 3' untranslated region (UTR), 5' UTR, or both. In some embodiments, the modified UTR comprises sequences involved in the recruitment of RNA-binding proteins (RBPs) and miRNAs to enhance the level of protein production (see, e.g., Kaczmarek et al., Genome Medicine 9:60 (2017)). In some embodiments, the 3' UTR, 5' UTR, or both are modified to encode regulatory elements. In some embodiments, the regulatory elements comprise a K-turn motif, miRNA binding site, or a combination thereof for controlling RNA expression in a cell-specific manner (see, e.g., Wroblewska et al., Nat Biotechnol. 33:839-41 (2015)).

[0125] In some embodiments, the mRNA comprises an RNA base modification. 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 217:337-44 (2015) and Svitkin et al., Nucleic Acids Res. 45:6023-36 (2017)).

[0126] In some embodiments, the RNA (e.g., mRNA) is circular RNA.

[0127] Compositions and methods for producing mRNA are described, for example, in WO 2016 / 011306, WO 2016 / 014846, WO 2016 / 022914, WO 2016 / 077123, WO 2016 / 164762, WO 2016 / 201377, WO 2017 / 049275, US Patent No. 9,937,233, US Patent No. 8,710,200, US Patent No. 10,022,425, US Patent No. 9,878,056, US Patent No. 9,572,897, WO 2010 / 084371, US Patent No. 9,353,153, WO 2015 / 034925, and WO 2019 / 236673. See, for example, 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 (see, for example, for in vivo delivery, US Patent No. 9,764,036, US Patent No. 9,682,139, Kauffman et al., Nano Lett. 15:7300-6 (2015) and Fenton et al., Adv Mater. 28:2939-43 (2016)).

[0128] In some embodiments, the polynucleotide is an aptamer. In certain embodiments, the aptamer binds to a target protein disclosed herein. In certain embodiments, the aptamer binds to a binding partner of a target protein disclosed herein.

[0129] In some embodiments, the polynucleotide is linked (e.g., by a covalent bond) to a delivery polymer. In some embodiments, the linkage between the polynucleotide and the delivery polymer is reversible. In some embodiments, the polynucleotide is linked to the delivery polymer via a physiologically labile linker. In some embodiments, the physiologically labile linker is a disulfide bond.

[0130] In some embodiments, the polynucleotide is conjugated to the polymer in the presence of an excess of the polymer. In some embodiments, the excess polymer is removed prior to administration (e.g., to a cell or subject).

[0131] One of ordinary skill in the art can readily make appropriate polynucleotide agents for use in the compositions, kits, and methods described herein using the locus information of the protein sequences, e.g., chromosomal location, start nucleotide position, and end nucleotide position, and polymorphism identification, contained in the sequence listing and Table A incorporated herein.

[0132] C. Agent Comprising a Gene Editing System In some embodiments, the agent comprises a gene editing system. In some embodiments, the gene editing system results in a nucleotide deletion, nucleotide substitution, nucleotide addition, or a combination thereof in the gene encoding the 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, a gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of a target protein (e.g., via gene knockout). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) reduces (e.g., decreases, inhibits) or eliminates the expression of a protein capable of regulating the expression or activity of a target protein (e.g., via gene knockout). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) increases the expression of a target protein (e.g., via gene knock-in or gene replacement). In some embodiments, a gene editing system (e.g., a CRISPR / Cas system) increases the expression of a protein capable of regulating the expression or activity of a target protein (e.g., via gene knock-in or gene replacement).

[0135] In some embodiments, the CRISPR system specifically catalyzes the cleavage of the gene encoding the target protein, thereby inactivating the gene. Repair of nucleic acid strand breaks via non-homologous end joining (NHEJ) often results in changes in the DNA sequence at the cleavage site, resulting in small insertions or deletions (Indels). In some embodiments, NHEJ is used to knockout the gene encoding the target protein. In some embodiments, homologous recombination repair (HDR) is used to simultaneously inactivate the gene encoding the target protein and insert a heterologous sequence at the inactivated locus. Cells 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 (derived from Acidaminococcus species) or LbCpf1 (derived from Lachnospiraceae species). The selection of nuclease and gRNA is typically determined according to whether nucleotide deletions, substitutions or additions to the target sequence are desired.

[0137] In some embodiments, the type II Cas endonuclease is Cas9 from (e.g., Streptococcus pyogenes). In some embodiments, the modified Cas9 is nickase Cas9, dead Cas9 (dCas9), or eSpCas9. In some embodiments, the nickase Cas9 is Cas9 D10A. In some embodiments, the dCas9 is D10A or H840A. In some embodiments, the gene editing system comprises dual nickase Cas9 (see, e.g., Ran et al., Cell 154:1380-89 (2013) for achieving more accurate genome editing). Wild-type Cas9 generates a double-strand break (DSB) at a specific DNA sequence targeted by the gRNA. Nickase Cas9 generates only single-strand breaks. dCas9 is catalytically inactive. In some embodiments, the dCas9 is fused to a nuclease (e.g., FokI for generating a DSB at a target sequence 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) a CRISPR RNA ("crRNA"); and c) a trans-activating crRNA ("tracrRNA") and.

[0139] In some embodiments, the crRNA comprises at least one "guide RNA" (sgRNA), e.g., 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 that can regulate the expression or activity of the target protein. In some embodiments, the gRNA is at least about 16 nucleotides, e.g., 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, 339:819 - 23 (2013) and Ran et al., Nature Protocols 8:2281 - 308 (2013). Cas9 requires a gRNA sequence of at least about 16 or 17 nucleotides to cleave DNA, and Cpf1 requires a gRNA sequence of at least about 16 nucleotides to cleave DNA. In practice, the gRNA sequence has a length of about 17 - 24 nucleotides (e.g., about 19, 20, or 21 nucleotides) and is complementary to the target gene. Custom gRNA generators and algorithms are commercially available. Chemically modified sgRNAs have also been demonstrated to be effective for genome editing (see, for example, Hendel et al., Nature Biotechnol., 985 - 91 (2015)).

[0141] In some embodiments, the crRNA further comprises a sequence capable of binding to the tracrRNA. When bound, the partially double-stranded structure is cleaved by RNase III, and the resulting crRNA / tracrRNA hybrid instructs the Cas9 endonuclease to recognize and cleave the target DNA sequence.

[0142] In some embodiments, the target DNA sequence is proximal to a "protospacer adjacent motif" ("PAM") specific to the Cas endonuclease. The PAM sequence appears 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, such as Cas9 endonuclease, associate with a G-rich PAM site, such as 5'-NGG, and perform blunt-end cleavage of 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) a crRNA and comprises.

[0144] Cpf1-related CRISPR arrays are processed into mature crRNAs without the need for a tracrRNA. The Cpf1 endonuclease associates with a T-rich PAM site, such as 5'-TTN. Cpf1 can also recognize a 5'-CTA PAM motif. Cpf1 introduces offset or staggered double-strand breaks with 4- or 5-nucleotide 5' overhangs, for example, by cleaving target DNA having an offset or staggered cut of 5 nucleotides located 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 5-nucleotide overhangs resulting from such offset cuts allow for more precise genome editing by DNA insertion via homologous recombination rather than by insertion of blunt-ended cut DNA. See, e.g., Zetsche et al., Cell 163:759-71 (2015).

[0145] In some embodiments, the gene editing system activates or suppresses the transcription of a 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 comprising.

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

[0147] The effector domain comprises the biologically active portion of an effector protein (e.g., a transcriptional activator or a transcriptional repressor). In some embodiments, the gene editing system comprises one effector domain. In some embodiments, the gene editing system comprises at least two 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 aspects, dCas9 is H840A.

[0148] Since dCas9 is catalytically inactive, it does not cleave target DNA and instead interferes with transcription through steric hindrance. dCas9 chimeric proteins (e.g., dCas9-VPR) are guided by one or more gRNAs to sequences upstream of the transcription start site (TSS) of a target gene and regulate the transcription of the target gene. For example, 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 fluorescence imaging, Cell 159:635-46 (2014); Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Nature 517:583-88 (2015); Chavez et al., Highly efficient Cas9-mediated transcriptional programming, Nat.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.See Chavez et al., Comparison of Cas9 activators in multiple species, Nat Methods. 7:563-67 (2016); eLife. 5:e19760 (2016); Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.

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

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

[0151] In some embodiments, the agent comprises a transcription activator-like effector nuclease (TALEN) system. TALEN-based systems include proteins that contain a TAL effector DNA binding domain and an enzyme domain. They are created by fusing the TAL effector DNA binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). The above-mentioned FokI restriction enzyme is an exemplary enzyme domain suitable for use in TALEN-based gene regulatory systems.

[0152] TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when infecting plants. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence with different 12th and 13th amino acids. These two positions, called repeat variable diresidues (RVDs), are highly variable and strongly correlate with specific nucleotide recognition. Thus, the TAL effector domain can be engineered to bind to a specific target DNA sequence by selecting a combination of repeat segments containing appropriate RVDs. The nucleic acid specificity for combinations of RVDs is as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (however, 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, or 100% identical, to the target DNA sequence of the 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, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0154] In some embodiments, the gene regulatory system comprises two or more TAL effector fusion proteins, each comprising a TAL effector domain, and at least one of the TAL effector domains binds to a target DNA sequence of the 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, or 100% identical, to the target DNA sequence defined by a set of genomic coordinates.

[0155] Methods and compositions for assembling TAL-effector repeats 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 the construction of TAL-effector repeats are commercially available, for example, from Addgene.

[0156] In some embodiments, the agent comprises a zinc finger nuclease (ZFN) system. The ZFN domain can be generated using commercially available plasmids, for example, plasmid pairs from Sigma Aldrich (St. Louis, MO) (CSTZFN-1KT COMPOZR® Custom Zinc Finger Nuclease (ZFN) R-3257609). The plasmids can be prepared using commercially available systems according to the manufacturer's protocol (e.g., NEB Monarch Miniprep (Catalog No. T1010), New England Biolabs, Ipswich, MA).

[0157] In some embodiments, the agent comprises a vector designed to deliver conventional gene therapy (e.g., gene knockout or knock-in by homologous recombination). Non-limiting examples of such vectors include retroviruses (e.g., lentivirus 5), adenoviruses, adeno-associated viruses, herpes simplex viruses, nanoparticles, and DNA transposons.

[0158] D. Small molecule agents 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 the target protein. In some embodiments, the small molecule is an inhibitor of the target protein (e.g., a direct inhibitor, an indirect inhibitor). In some embodiments, the small molecule is an activator of the target protein (e.g., a direct activator, and an indirect activator).

[0159] Examples of small molecules include organic compounds, organometallic compounds, inorganic compounds, and salts of organic, organometallic or inorganic compounds. Atoms in a small molecule are typically linked to each other via covalent and / or ionic bonds. In certain embodiments, the small molecule is an organic small molecule. The arrangement of atoms in an organic small molecule may represent a chain (e.g., a carbon-carbon chain or a carbon-heteroatom chain), or a ring containing carbon atoms, such as benzene or a polycyclic system, or a combination of carbon and heteroatoms, i.e., a heterocycle such as pyrimidine or quinazoline. Small molecules can have a wide range of molecular weights, but generally include molecules with a molecular weight of less than about 5,000 Daltons. For example, such small molecules can be less than about 1000 Daltons, preferably less than about 750 Daltons, or more preferably less than about 500 Daltons. Small molecules can be found naturally (e.g., identified, isolated, purified) and / or produced synthetically (e.g., by traditional organic synthesis, biocatalyzed synthesis, or a combination thereof). See, for example, 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 derivatives thereof.

[0160] In certain embodiments, the agent includes 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.

[0162] E. Therapeutic Cells and Cell-Based Therapies In some embodiments, the agent comprises therapeutic cells. In certain embodiments, the therapeutic cells express and / or are engineered to express a target protein (e.g., a target protein described herein, such as a target protein of the Sequence Listing, Table A, or a variant described above), a polypeptide (e.g., an antibody, antigen-binding fragment, or polypeptide comprising an amino acid sequence that is at least 70% identical to at least a portion of the target protein), a polynucleotide (e.g., recombinant DNA, RNA, such as mRNA or siRNA), and / or a gene editing system (e.g., the CRISPR / Cas system).

[0163] In some embodiments, the polypeptides (e.g., antibodies or antigen-binding fragments) disclosed herein 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 macrophage (CAR-M) cells). In some embodiments, the CAR comprises a transmembrane domain and an antigen recognition portion that binds to a target protein.

[0164] Therapeutic cells suitable for use in the compositions, kits, and methods of the present disclosure can be generated, identified, and / or enriched by methods known to those of skill in the art. Non-limiting examples of such methods include purifying, expanding, and / or differentiating cells from a subject (e.g., a human) into specific cell products; engineering somatic cells for gene therapy; immortalizing cells; ex vivo gene modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); in vivo gene modification of cells (e.g., using viral vectors and / or lipid nanoparticle delivery technologies); genome editing; cell plasticity technologies; genetic modification; and flow cytometry. In some embodiments, the therapeutic cells are autologous or syngeneic. In other embodiments, the therapeutic cells are allogeneic.

[0165] Expression Vectors and Hosts In another aspect, the present disclosure provides an expression vector comprising the polynucleotide described herein.

[0166] The term "expression vector" refers to a replicable nucleic acid that can express one or more proteins when the expression vector is transformed into a suitable expression host cell.

[0167] In some embodiments, the expression vector comprises an expression control polynucleotide sequence operably linked to the polynucleotide, a polynucleotide sequence encoding a selectable marker, or both. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to a region of DNA to which RNA polymerase binds to initiate transcription of a gene. The term "operably linked" means that a nucleic acid is arranged in a recombinant polynucleotide, such as a vector, in such a way that it enables expression of the nucleic acid under the control of an element (e.g., a promoter) to which it is linked. The term "selectable marker element" is an element that confers a trait suitable for artificial selection. A selectable marker element can be a negative or positive selectable marker. Non-limiting examples of expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Molecular Cloning: A Laboratory Manual (Michael R. Green & Joseph Sambrook eds., 4th ed. 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 useful for receiving, maintaining, replicating, and / or amplifying a vector.

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

[0171] The polynucleotides or expression vectors described herein can be introduced into appropriate or desired host cells using techniques known in the art, including transformation, electroporation, and transduction. The introduced nucleic acid can be extrachromosomal within the host cell or integrated into the genome of the host cell.

[0172] Pharmaceutical composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an agent disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a composition having a pharmacological activity or other direct effect in the alleviation, treatment, or prevention of cancer, or a finished dosage form or formulation thereof.

[0173] In some embodiments, the composition (e.g., pharmaceutical composition) includes a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or tonic (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or tonics 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® and polyethylene glycol (PEG).

[0174] In some embodiments, the agent of the pharmaceutical composition (e.g., polypeptide, polynucleotide, or small molecule) is modified, e.g., conjugated to a heterologous moiety. The term "conjugated" refers to being joined via a covalent or non-covalent interaction. Conjugation can be carried out using any suitable linker; non-limiting examples include peptide linkers, compound linkers, and chemical cross-linking agents.

[0175] In some embodiments, the heterologous moiety is a marker (e.g., a fluorescent marker or a radioactive marker), a molecule that stabilizes an agent, a molecule that targets an agent (e.g., to a specific cell or tissue, such as to facilitate or prevent passage through the blood-brain barrier), or a combination thereof.

[0176] In some embodiments, the heterologous moiety is polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticles, multimerization domains, and carrier peptides. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymer nanoparticles. In some embodiments, the polymer is an amphiphilic polymer. In other embodiments, the polymer is a hydrophobic or hydrophilic polymer. 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 nanoparticles comprise poly(lactic acid-co-glycolic acid) (PLGA).

[0177] In some embodiments, the composition (e.g., a pharmaceutical composition) is formulated for an appropriate dosing schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the composition (e.g., a 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 "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. Non-limiting examples of additional agents or treatments include biologics (e.g., antibodies, peptides), cell therapies, gene therapies, immunotherapies, and small molecules that affect the diseases or conditions described herein (e.g., cancer).

[0179] The treatment regimen defines the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, two or more agents are administered sequentially as part of a prescription regimen. In other embodiments, the delivery of two or more agents is simultaneous or co-temporaneous. In some embodiments, two or more agents are co-formulated. In some embodiments, by administering two or more agents or treatments in combination, a reduction in other parameters associated with the symptom or disorder is greater than that observed when one agent or treatment is delivered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Each of the two or more therapeutic agents can be administered by any suitable route, including but not limited to oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The two or more therapeutic agents can be administered by the same route or different routes.

[0180] In some embodiments, the agent or pharmaceutical composition of the present disclosure is delivered by a viral vector, e.g., by contacting cells with the viral vector, administered locally to a tumor (e.g., by injection), or systemically to a subject (e.g., a human patient) (e.g., by intravenous or oral administration).

[0181] The viral genome provides a rich source of vectors that can be used for the efficient delivery of exogenous genes into mammalian cells. The viral genome is a particularly useful vector for gene delivery because the polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Non-limiting examples of viral vectors include retroviruses (e.g., retroviridae viral 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), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia virus Ankara (MVA), fowlpox, and canarypox). Further 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, avian C-type virus, mammalian C-type, B-type virus, D-type virus, oncovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (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). Further non-limiting examples include murine leukemia virus, murine sarcoma virus, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, chimpanzee endogenous virus, gibbon ape leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Further non-limiting examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the teachings of which are incorporated herein by reference.

[0182] In some embodiments, the agents or pharmaceutical compositions of the present disclosure are formulated to be delivered by a membrane-based carrier in vivo, in vitro, ex vivo, or in situ. In some embodiments, the membrane-based carrier is a cell-based carrier (e.g., a mammal 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 (e.g., plasmids, viruses, virus-like particles, or virions) described herein.

[0183] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more liposomes. Liposomes are spherical vesicular structures composed of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible and non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)).

[0184] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate 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, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). Vesicle formation can be spontaneous when lipid membranes are mixed with an aqueous solution, but can also be facilitated by applying force in the form of agitation using a homogenizer, sonicator, or extrusion device (see, e.g., Spuch and Navarro, J Drug Deliv. 2011:469679 (2011)). Extruded lipids can be prepared by extrusion through a small-sized filter as described in Templeton et al., Nature Biotech, 15:647-52 (1997), the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference).

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

[0186] Nanostructured lipid carriers (NLCs) are SLNs that retain the characteristics of modified solid lipid nanoparticles (SLNs), improve drug stability and loading capacity, and prevent drug leakage. Polymeric nanoparticles (PNPs) are important components of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier combining 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. Thus, the two components enhance drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs. See, for example, Li et al., Nanomaterials 7(6):122 (2017).

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

[0188] In some embodiments, the agent 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 polynucleotide). 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 agent or pharmaceutical composition of the present disclosure is formulated to be delivered by a cationic carrier (e.g., a cationic lipopolymer or transfection reagent). Non-limiting examples of cationic carriers include Lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimer, chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC).

[0190] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by exosomes, adipocytes, and / or red blood cells. See, for example, Ha et al., Acta Pharm Sin B. 6(4):287-96(2016).

[0191] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more fusosomes. The fusosomes are engineered to confer target cell specificity for fusion and payload delivery, thereby enabling the generation of a delivery vehicle with programmable cell specificity. See, for example, WO 2020014209 pamphlet, the teachings of which regarding the design, preparation, and use of fusosomes are incorporated herein by reference.

[0192] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by ex vivo differentiated red blood cells. See, for example, WO 2015073587 pamphlet; WO 2017123646 pamphlet; WO 2017123644 pamphlet; WO 2018102740 pamphlet; WO 2016183482 pamphlet; WO 2015153102 pamphlet; WO 2018151829 pamphlet; WO 2018009838 pamphlet; Shi et al., PNAS, 111(28):10131-36(2014); US Patent No. 9,644,180; Huang et al., Nature Communications 8:423(2017).

[0193] In some embodiments, the agent 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 packs (PMPs). See, for example, WO 2011 / 097480, WO 2013 / 070324, WO 2017 / 004526, and WO 2020 / 041784.

[0194] In some embodiments, the agent or pharmaceutical composition of the present disclosure is formulated to be delivered by one or more anellosomes. The preparation and use of anellosomes for the delivery of therapeutic products are described in U.S. Patent No. 11,166,996, and its teachings regarding the design, preparation, and use of anellosomes are incorporated herein by reference.

[0195] Method for regulating a target protein In another aspect, the present disclosure provides a method for regulating the expression or activity of a target protein identified in the Sequence Listing, Table A, or a variant thereof 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 that includes and / or regulates the expression or activity of the target protein identified herein, or a pharmaceutical composition comprising the agent.

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

[0197] In some embodiments, the target cell is a cell associated with the disease or condition described herein.

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

[0199] In some embodiments, the target cells are related to and / or involved in inflammation. In certain embodiments, the target cells are epithelial cells, endothelial cells, stem cells, non-immune cells, or combinations thereof.

[0200] In some embodiments, the target cells are related to and / or involved in fibrosis, aging, and / or senescence. In certain embodiments, the target cells are epithelial cells, endothelial cells, stem cells, non-immune cells, or combinations thereof.

[0201] In some embodiments, the target cells are immune cells. In certain embodiments, the target cells are effector T cells, helper T cells, Th1 cells, Th2 cells, Th17 cells, B cells, natural killer (NK) cells, innate lymphoid cells (e.g., ILC1 cells, ILC2 cells, ILC3 cells), macrophages (e.g., M1 macrophages, M2 macrophages), monocytes, and / or antigen-presenting cells (e.g., dendritic cells), or combinations thereof.

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

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

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

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

[0206] In some embodiments, the target tissue is immune tissue. In some embodiments, the target cells are non-immune tissue. In some embodiments, the target tissue includes lymph nodes, spleen, secondary lymphoid organs, tertiary lymphoid organs, barrier tissues, skin, intestine, airway, wound, immune tissue, non-immune tissue, or combinations thereof.

[0207] In certain embodiments, the effective amount is sufficient to reduce the expression of the target protein in the target cells 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 cells 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, for example, 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 κB (NF-κB) signaling, growth factor signaling, cell death (e.g., apoptosis), cell cycle (e.g., mitosis), cell migration, inflammation, or combinations thereof.

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

[0211] In some embodiments, the disease or condition is a disease targeted by a drug and / or diagnostic test approved by the U.S. Food and Drug Administration (FDA). In certain embodiments, the disease or condition is a disease targeted by a drug and / or diagnostic test in clinical trials. In some embodiments, the disease is an immune-mediated disease, a sporadic disease, a genetic disease, cancer (e.g., a tumor), a metabolic disease, or a combination thereof. In certain embodiments, the disease or condition is associated with a GWAS, TCGA, whole-genome sequencing, PheWAS, eQTL study, or a combination thereof (e.g., the diseases, conditions or pre-diseases listed in paragraphs

[0219] and

[0220] ).

[0212] In another aspect, the present disclosure provides a method of classifying a subject based on a predicted likelihood of developing a disease or condition, comprising quantifying the expression or activity of a target protein in a sample derived from the subject; predicting a 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 of stratifying a set of subjects having a disease or condition, comprising quantifying the expression and / or activity of a target protein in a sample derived from an individual subject within the set; and stratifying the set of subjects with respect to treatment according to the level of expression and / or activity of the target protein in the sample for each individual subject.

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

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

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

[0217] In another aspect, the disclosure provides a method of preparing a sample useful for detecting the likelihood of developing a disease or condition in a subject, 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 useful for detecting the likelihood of developing cancer; and c) quantifying the expression or activity of a target protein in the sample prepared in step b). The method is provided.

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

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

[0220] As used herein, the terms "treatment" or "treating" refer to the medical management of a subject with the purpose of improving, alleviating, stabilizing (i.e., not worsening), preventing or curing a disease, medical condition or disorder. "Treatment" includes active treatment (treatment directed at improving a disease, medical condition or disorder), causal treatment (treatment directed at the cause of the associated disease, medical condition or disorder), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment directed at minimizing or partially or completely inhibiting the onset of the associated disease, medical condition or disorder); and adjuvant treatment (treatment utilized to supplement another therapy). Treatment also includes a detectable or undetectable decrease in the degree of a disease or condition; prevention of the spread of a disease or condition; delay or deceleration of the progression of a disease or condition; improvement or alleviation of a disease or condition; and remission (partial or complete). "Improving" or "alleviating" a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disorder or condition are reduced, and / or the time course of progression is decelerated or lengthened as compared to the degree or time course in the absence of treatment. "Treatment" also includes prolonging survival as compared to survival expected in the absence of treatment. Persons in need of treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, or those in whom the condition or disorder should be prevented.

[0221] In some embodiments, the subject is an animal. In other embodiments, the subject is a bird, such as a hen, rooster, turkey or 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 cows (e.g., dairy cows or beef cattle), sheep, goats, pigs, horses, dogs, cats, mice, rats, and the like. 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 young person. In some embodiments, the human is an adult. In some embodiments, the human is under 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, e.g., at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 years old.

[0222] As used herein, the terms “effective amount,” “therapeutically effective amount,” or “sufficient amount” refer to an amount sufficient to effect a treatment (e.g., to produce a beneficial or desired result), including effects at the cellular, tissue, or clinical level, when administered to a subject (e.g., a mammal such as a human cancer patient). Thus, the term depends on the context in which it is applied. For example, in the context of treating cancer, it is an amount of an agent sufficient to achieve a response as compared to the response obtained without administration of the agent. The amount of a given composition described herein corresponding to such an amount will vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject (e.g., age, sex, weight, etc.), or identity of the host being treated, but can nonetheless be routinely determined by one of ordinary skill in the art. In some embodiments, a “therapeutically effective amount” of a composition of the present disclosure is an amount that produces a beneficial or desired result in a subject (e.g., as compared to a control). A therapeutically effective amount of a composition of the present disclosure can be readily determined by one of ordinary skill in the art by routine methods known in the art. The dosing regimen can be adjusted to provide an optimal therapeutic response.

[0223] The therapeutic agents described herein can be administered via various 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., intratracheal, intranasal, or oral inhalation, intranasal instillation), depending on the compound and the particular disease or condition being treated. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the particular 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 (e.g., a second therapeutic agent).

[0225] Administration of two or more therapeutic agents includes co-administering the therapeutic agents substantially simultaneously, such as in a combination pharmaceutical. Alternatively, such administration includes co-administering each therapeutic agent in multiple or separate containers (e.g., capsules, powders, and liquids). Such administration also includes using the therapeutic agents in a sequential manner, either substantially simultaneously or at different times. When two or more therapeutic agents are administered, the therapeutic agents can be administered via the same route of administration or via different routes of administration.

[0226] In another aspect, the present disclosure provides a method of modulating 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 within a subject.

[0227] In another aspect, the present disclosure provides a method of identifying an agent that modulates the expression and / or activity of a target protein (e.g., the target protein of the Sequence Listing), the method comprising: a) contacting a sample containing the target protein (e.g., a biological sample such as a cell or tissue) with an agent (e.g., a candidate agent being tested for its ability to modulate the expression and / or activity of the target); and b) determining whether the agent modulates the expression or activity of the target protein comprising There is provided a method showing that a difference in the expression or activity of a target protein contacted with an agent, as 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 contacted with an agent, as 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 contacted with an agent, as 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, as compared to a reference, indicates that the agent activates the expression or activity of the protein.

[0230] Non-limiting examples of indications GWAS-related diseases or conditions In some embodiments, the GWAS-related disease or condition is: abdominal aortic aneurysm, abnormal chromosome segregation, achalasia, acne, acute lymphoblastic leukemia, acute myeloid leukemia, Addison's disease, adolescent idiopathic scoliosis, adult-onset asthma, age-related hearing loss, age-related macular degeneration, age-related nuclear cataract, AIDS, alcohol abuse, alcohol dependence, alcoholic cirrhosis, alcoholic pancreatitis, allergic rhinitis, allergy, alopecia, alopecia areata, Alzheimer's disease, amblyopia, amyotrophic lateral sclerosis, androgenetic alopecia, anemia, angina, ankle injury, ankylosing spondylitis, anorectal malformation, anorexia nervosa, anterior uveitis, anti-neutrophil antibody-related vasculitis, anti-topoisomerase I antibody-positive systemic sclerosis, anxiety disorder, asparaginase-induced acute pancreatitis, Asperger 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, Behçet's syndrome, Bell's palsy, benign prostatic hyperplasia, biliary cirrhosis, bipolar disorder, bladder cancer, fracture, cerebral aneurysm, cerebral infarction, breast cancer, bullous pemphigoid, bullous pemphigoid, cancer, cardiac arrhythmia, cardiac hypertrophy, cardiotoxicity, cardiovascular disease, cardiovascular disease, myocardial ischemia infarction, carpal tunnel syndrome, cataract, celiac disease, central nervous system cancer, central nervous system non-Hodgkin lymphoma, cerebrovascular disorder, cervical cancer, cervical intraepithelial neoplasia grade 2 / 3 neoplastic malignancy, Chagas cardiomyopathy, childhood-onset asthma, cholecystitis, cholestasis, 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 rhinosinusitis, tobacco addiction, cirrhosis, cleft lip, cleft palate, clonal hematopoiesis, Clostridium difficile infection, cocaine dependence, colon polyp, colorectal adenoma, colorectal cancer, conduction disorder, congenital left heart lesion, congenital left heart malformation, coronary aneurysm, coronary artery disease, corticobasal degeneration, COVID-19, Creutzfeldt-Jakob disease, Crohn's disease, cutaneous mastocytosis, cutaneous melanoma, cutaneous squamous cell carcinoma, dementia, dengue hemorrhagic fever, dental caries, depression, dermatomyositis, femoral joint dysplasia, diabetes, diabetic nephropathy, diabetic retinopathy,Diffuse large B-cell lymphoma, digestive system infectious diseases, dilated cardiomyopathy, diverticular disease, diverticulitis, drug-induced agranulocytosis, drug-induced liver injury, duodenal ulcer, Dupuytren's contracture, eating disorders, eczema, endometrial carcinoma, endometrial neoplasm, endometriosis, eosinophilic esophagitis, epitheloid cell choroidal melanoma, Epstein-Barr virus infection, esophageal adenocarcinoma, esophageal varices, essential tremor, estrogen receptor-negative breast cancer, Ewing sarcoma, detachment syndrome, saccadic eye movement measurement, extrahepatic bile duct cancer, facial wrinkles, familial QT prolongation syndrome, familial atrial incomplete syndrome, febrile seizures (within the age range of 3 months to 6 years), female genital system diseases, focal segmental glomerulosclerosis, frontal fibrosing alopecia, frozen shoulder, gallstones, gastric adenocarcinoma, gastric carcinoma, gastric ulcer, gastroesophageal reflux disease, glaucoma, glioma, hyperglycemia, Graves' disease, Hashimoto's thyroiditis, head and neck malignant neoplasms, head and neck squamous cell carcinoma, hearing loss, heart failure, hematuria, hemorrhoids, Henoch-Schönlein purpura, hepatitis B virus infection, hepatitis C virus infection, heroin dependence, herpes zoster, hippocampal atrophy, Hirschsprung's disease, HIV-1 infection, Hodgkin lymphoma, human papillomavirus infection, Huntington's disease, color vision abnormality, myopia, hypertension, hyperthyroidism, hypertriglyceridemia, hypertrophic cardiomyopathy, hyperuricemia, hypoalbuminemia, hypotension, hypothyroidism, idiopathic pulmonary fibrosis, IgA glomerulonephritis, immune system diseases, infantile hypertrophic pyloric stenosis, inflammatory bowel disease, influenza A (H1N1) infection, inguinal hernia, insomnia, interstitial lung disease, intracerebral hemorrhage, irritable bowel syndrome, ischemic stroke, juvenile idiopathic arthritis, keratinocyte carcinoma, keratoconus, knee pain, language disorders, large artery stroke, adult latent autoimmune diabetes, late-onset Alzheimer's disease, late-onset myasthenia gravis, Hansen's disease, leukopenia, Lewy body disease, localized scleroderma, liver diseases, liver neoplasms, lumbar intervertebral disc degeneration, lung adenocarcinoma, lung carcinoma, lupus nephritis, macrovascular complications of diabetes, type 2 macular telangiectasia, major depressive disorder, malaria, male infertility, marginal zone B-cell lymphoma, obesity, melanoma, membranous glomerulonephritis, menorrhagia, mental retardation, metabolic rate measurement, metabolic syndrome, metastasis measurement, metastatic colorectal cancer, migraine disorder, moderate albuminuria, MIH (Molar Incisor Hypomineralization), monoclonal gammopathy of undetermined significance, mood disorders, moyamoya disease, mucocutaneous lymph node syndrome, multiple myeloma, multiple sclerosisMultiple system atrophy, myeloproliferative disorders, myocardial infarction, myopia, myositis, narcolepsy with cataplexy, nasal polyps, nasopharyngeal neoplasms, neonatal generalized erythematosus, nephrotic syndrome, neuroblastoma, neuropathy, nicotine dependence, tuberous sclerosis, Hodgkin lymphoma, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-intralobular hemorrhage, non-melanoma skin cancer, non-occlusive coronary artery disease, non-small cell lung cancer, obesity, obese esophageal cancer, obsessive-compulsive disorder, ocular sarcoidosis, oligoarticular juvenile idiopathic arthritis, open-angle glaucoma, opioid use disorder, oral cancer, oral ulcers, oropharyngeal cancer, Paget's disease of bone, osteoarthritis, hip osteoarthritis, knee osteoarthritis, osteonecrosis, osteosarcoma, ovarian carcinoma, endometrial carcinoma, ovarian serous carcinoma, pain, pancreatic carcinoma, 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 disturbances, preeclampsia, primary angle-closure glaucoma, primary biliary cirrhosis, prion disease, progressive supranuclear palsy, proliferative diabetic retinopathy, prostate carcinoma, proteinuria, pseudotumor cerebri, psoriasis, psoriasis vulgaris, pulmonary nontuberculous mycobacterial infection, recalcitrant atopic dermatitis, renal cell carcinoma, respiratory diseases, rhegmatogenous retinal detachment, rheumatic heart disease, rheumatoid arthritis, tendon plate rupture, SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis syndrome), sarcoidosis, scarlet fever, schizophrenia, sclerosing cholangitis, seasonal allergic rhinitis, selective IgA deficiency, sepsis, scapulohumeral syndrome, Sjögren syndrome, skin carcinoma, skin neoplasms, skin sensitivity to sunlight, small cell lung cancer, small vessel stroke, spontaneous preterm birth, sporadic amyotrophic lateral sclerosis, sporadic Creutzfeldt-Jakob disease, sporadic Creutzfeldt-Jakob disease, squamous cell carcinoma, squamous cell lung carcinoma, stenosing tenosynovitis, Stevens-Johnson syndrome, gastric polyps, stress-related disorders, stroke, substance abuse, sudden cardiac arrest, sunburn, systemic juvenile idiopathic arthritis, systemic lupus erythematosus, systemic mastocytosis, systemic sclerosis, Takayasu arteritis, testicular carcinoma, testicular germ cell tumor, thyroid carcinoma, thyrotoxic periodic paralysis, tinnitus, tooth agenesis, dental caries, Tourette syndrome, triple-negative breast cancer, tropical spastic paraparesis, tuberculosis, type 1 diabetes, type 2 diabetes, ulcerative colitis, unipolar depression, upper aerodigestive neoplasms, urolithiasis, uterine leiomyoma, uveal melanoma, venous aneurysm, vascular dementiaIt is venous thromboembolism, vitamin D deficiency, vitiligo, or wet age-related macular degeneration, or a combination thereof.

[0231] TCGA-related diseases or conditions In some embodiments, the TCGA-related diseases or conditions are cholangiocarcinoma, bladder urothelial carcinoma, bone cancer, glioblastoma multiforme, low-grade glioma, breast cancer, cervical squamous cell carcinoma, chronic lymphocytic leukemia, chronic myeloid disorder, colorectal adenocarcinoma, early-onset prostate cancer, esophageal adenocarcinoma, gastric adenocarcinoma, gastric cancer, head and neck squamous cell carcinoma, renal chromophobe cell, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, diffuse large B-cell lymphoma, malignant lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, cutaneous melanoma, or uterine corpus endometrial carcinoma, or a combination thereof.

[0232] In some embodiments, the treatment delays (e.g., inhibits) the onset of the diseases or conditions described herein (e.g., inhibits the growth, proliferation, metastasis, invasion, and / or migration of cancer cells).

[0233] In some embodiments, the effective amount is sufficient to delay (e.g., inhibit) the onset of the diseases or conditions described herein.

[0234] In some embodiments, the effective amount is sufficient to delay the onset of the disease or condition by at least about 10%, e.g., 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 about 10 - 99%, e.g., 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%, sufficient to delay the onset of the disease or condition.

[0235] In some embodiments, the treatment alleviates the symptoms of the disease or condition described herein.

[0236] In some embodiments, the effective amount is sufficient to reduce the symptoms of the 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%, such as 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 and thereby reduce the mortality rate. In certain embodiments, the reduction in the 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 the mortality rate is about 10 - 99%, such as 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 (e.g., increase or decrease) the expression of the target protein in cells related to the disease or condition described herein.

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

[0240] In some embodiments, the present disclosure provides treatments that harness the immune system (e.g., immuno - oncology). In still further embodiments, the immune system is the innate immune system. In some embodiments, the immune system is the adaptive immune system. In still further embodiments, the treatment relates to humoral immunity or antibody - mediated immunity. In some embodiments, the treatment relates to cell - mediated immunity such as cancer. In some embodiments, the method relates to treatment at the time of initial disease progression or prior to initial disease progression, while in other embodiments, the method relates to treatment or prevention of late - stage disease progression at the time of late - stage 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 (e.g., dendritic cells and / or macrophages) and / or T cells), proliferation of immune cells, recruitment of immune cells (e.g., antigen - presenting cells (e.g., 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 combinations of the foregoing.

[0242] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those skilled in the art related to this disclosure. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. It should be further understood that terms defined in commonly used dictionaries, etc., should be construed to have meanings consistent with their meanings in the context of the relevant art and / or as otherwise defined in this specification.

[0243] The terms used in this specification are for the sole purpose of describing particular embodiments and are not intended to be limiting.

[0244] As used in this specification, the indefinite articles "a", "an", and "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0245] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", mean, for example, including the recited integer or step or group of integers or steps, but not excluding other integers or steps or group of integers or steps. As used in this specification, the term "comprising" can be replaced by the term "containing" or "including".

[0246] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims. Whenever used in the context of an aspect or embodiment of the present disclosure, the terms "comprising," "containing," "including," and "having" can all, in some embodiments, be replaced with "consisting of" or "consisting essentially of" so as to change the scope of the disclosure.

[0247] As used herein, the connective term "and / or" between a plurality of recited elements is understood to encompass both individual choices and combinations of choices. For example, when two elements are connected by "and / or," the first choice refers to the applicability of the first element without the second element. The second choice refers to the applicability of the second element without the first element. The third choice refers to the applicability of the first element and the second element together. Any one of these choices falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or" as used herein. The ability to apply multiple choices simultaneously also falls within the scope of meaning and is thus understood to meet the requirements of the term "and / or."

[0248] When a list is presented, it should be understood that each individual element of the list, and all combinations of the list, are separate embodiments, unless otherwise stated. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

Examples

[0249] Example 1: Verification of a target protein as a genetic driver of a disease This example demonstrates the validation of a target protein of the present disclosure that has a mechanism of action not previously known and includes mutations associated with diseases (determined by Genome Wide Association Studies (GWAS)). Specifically, GWAS associated with Th17 autoimmune diseases map to intergenic loci that do not have annotated proteins but have the target proteins identified by the methods of the present disclosure.

[0250] Transient knockout and knock-in cell lines are generated by CRISPR-Cas9 in primary PHA blasts T cells where the target protein is knocked out or an alternative SNP allele target protein replaces the wild type. STAT3, STAT1, and IL-6 activation levels are evaluated before and after short-term 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 at a significantly higher level than the wild type allele and does not induce STAT1 or NF-kb. Knockout of the target protein reduces STAT3 activity.

[0251] Materials and methods: Generation of PHA blasts: Fresh or frozen primary human PBMCs are placed in PBMax Karyotyping medium and 10 ng / mL of rhIL-2 is added for 3 - 4 days.

[0252] Knockout and knock-in of the target protein are achieved by CRISPR-Cas9 Alt-R knockout via RNP particles transfected by electroporation (Idtdna.com) using a guide specific to the target protein.

[0253] STAT3, STAT1, and NF-κB Activation Assay: Put about 10 million cells / mL of PHA blast cells into 80 μL in a 96-well plate. After starving the cells in a serum-free state for 3 - 4 hours, add IL-23, IL-12, or IL-6 for 30 minutes. Collect the cells and assay the cell extracts by phosphorylated-STAT3, STAT1, or NF-κB ELISA.

[0254] In summary, these experiments demonstrate that target proteins containing GWAS alleles related to autoimmune diseases have a more significant activation effect on STAT3 but not on STAT1 or NF-κB, which strongly suggests that the target proteins are potential targets for the improvement of autoimmune diseases.

[0255] Example 2: Verification of Target Protein as a Genetic Driver of Disease. Overexpression of the predicted TM protein leads to an increase in the proliferation of tumor cells. The known TCGA variants of that protein have a stronger effect. This example demonstrates the verification of target protein, SEQ ID NO: 5391, as a genetic driver of disease. Specifically, SEQ ID NO: 5391 is a target protein containing known TCGA variants that occur in a significant portion of patients suffering from colon adenocarcinoma. The inventors demonstrate that SEQ ID NO: 5391 overexpressed in the colon cancer cell line HCT116 cells significantly increases the proliferation of those cells. Importantly, the variant 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 mutation being a genetic driver of the cancerous phenotype.

[0256] Experimental Design: Transiently transfected a protein overexpression plasmid encoding the target protein or a control into multiple cell lines. Measured the proliferation after 24 hours using the WST-1 assay.

[0257] Materials and Methods: In 100 μL of cell culture medium, the target cells were seeded in a 96-well plate at a density of 4,000 cells / well. The cells were incubated in a humidified incubator at 37 °C and 5% CO2 for 24 hours. Lipofectamine 3000 (Thermo Fisher) was diluted in Opti-MEM medium (Thermo Fisher) to a final concentration of 30 μL / mL. The DNA (pcDNA3.1 overexpression plasmid) encoding the relevant control or target protein (Thermo Fisher) and P3000 were diluted in Opti-MEM medium to final concentrations of 10 μg / mL (DNA) and 20 μL / mL (P3000). 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 swirled to distribute the mixture evenly. The cells were 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 plate was incubated at 37 °C for 45 minutes in a 5% CO2 humidified incubator protected from light. The absorbance at 450 nm was measured using a microplate reader. The background absorbance can be corrected using a reference wavelength of 650 nm. The percentage of viable cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the result as a percentage of the control group.

[0258] Conclusion: 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 the TCGA mutation (P31L) that is known to occur in a large number of cancer patients, but whose importance for the carcinogenesis process and mechanism was previously unknown. The inventors show that when the overexpressed target protein contains the P31L mutation, its effect on proliferation is significantly more prominent. Therefore, both SEQ ID NO: 5391 and SEQ ID NO: 5391_P31L are genetic drivers of target proteins for cancer and anti-cancer therapy (Figure 1).

[0259] Example 3: Verification of target proteins as genetic drivers of diseases. Overexpression of the predicted TM proteins results in increased proliferation of tumor cells. This example demonstrates the verification of multiple target proteins, SEQ ID NO: 4538, SEQ ID NO: 5392, SEQ ID NO: 4335 as genetic drivers of diseases. The inventors demonstrate that gene overexpression of these transmembrane proteins in multiple cell lines significantly increases the proliferation of these cells. Therefore, these extracellular transmembrane proteins are attractive targets for anti-proliferative cancer drugs.

[0260] Experimental design: Protein overexpression plasmids encoding the target protein or a control were transiently transfected into multiple cell lines. Proliferation after 24 hours was measured using the WST-1 assay.

[0261] Materials and methods: The target cells were seeded in a 96-well plate at a density of 4,000 cells / well in 100 μL of cell culture medium. The cells were incubated in a humidified incubator at 37 °C and 5% CO2 for 24 hours. Lipofectamine 3000 (Thermo Fisher) was diluted in Opti-MEM medium (Thermo Fisher) to a final concentration of 30 μL / mL. The DNA (pcDNA3.1 overexpression plasmid) encoding the relevant control or target protein (Thermo Fisher) and P3000 were diluted in Opti-MEM medium to final concentrations of 10 μg / mL (DNA) and 20 μL / mL (P3000). 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 swirled to distribute the mixture evenly. The cells were 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 plate was incubated at 37 °C for 45 minutes in a 5% CO2 humidified incubator protected from light. The absorbance at 450 nm was measured using a microplate reader. The background absorbance can be corrected using a reference wavelength of 650 nm. The percentage of viable cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the result as a percentage of the control group.

[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 (see also Figure 2, Table C).

[0263]

Table C

[0264] Example 4. Verification of target protein as a genetic driver of disease. Objective: The examples listed verify the regulation of circulating factors by genetic variants that alter the amino acid sequence of a novel ORF. SEQ ID NO: 4371 contains a human genetic variant (rs221797) associated with a metabolic disease, namely type 2 diabetes. Four gene versions (V, A, G, D) of SEQ ID NO: 4371 are novel peptides that show variant-dependent differences in secretion using the HiBiT assay. This demonstrated how the disease-related versions of the novel peptides regulate the secretion levels that have potential therapeutic relevance in the identification of secreted versions of hereditary disease regulators.

[0265] Experimental design: The effect of GWAS variants on secretion regulation was evaluated using Promega's Nano-Glo® HiBiT Lytic Detection System (Catalog No. N3030). HEK293T cells were transfected with HiBiT expression vectors containing different versions of the sequence, and secretion was determined by measuring the luminescence of the HiBiT-tagged protein in cell lysates.

[0266] Methods and Materials: Each of the gene versions of SEQ ID NO: 4371 was cloned into a HiBiT expression vector. HEK293T cells were seeded in 96-well plates at 50k / well and transfected with Lipofectamin 3000 and DNA (HiBiT expression vector containing the sequence). Secretion was measured using the Nano-Glo® HiBiT Lytic Detection System from Promega (Catalog No. N3030). The Nano-Glo® HiBiT Lytic Detection System quantifies HiBiT-tagged proteins in cell lysates with high sensitivity using a simple add-mix-read assay protocol. HiBiT is an 11-amino acid peptide tag that is fused to the N-terminus or C-terminus of the protein of interest or inserted at an accessible position within the protein structure. The amount of HiBiT-tagged protein expressed intracellularly is determined by adding a lysis detection reagent containing the substrate furimazine and the large subunit, Large BiT (LgBiT), used in NanoLuc® Binary Technology (NanoBIT®; 1). HiBiT binds strongly to LgBiT (KD = 0.7 nM), promoting complex formation in cell lysates to produce a bright luminescent enzyme. The amount of luminescence is proportional to the amount of HiBiT-tagged protein in the cell lysate. The protein of interest can be tagged with HiBiT at the N-terminus or C-terminus using a HiBiT expression vector.

[0267] Conclusions: As seen in Figure 3, the human genetic variants in SEQ ID NO: 4371 alter the secretion levels of these peptides. rs221797 V-to-A has no effect on secretion (one hydrophobic amino acid is replaced by another hydrophobic one), while rs221797 V-to-G and V-to-D significantly reduce secretion, with the substitution of a charged amino acid (D) having the greatest effect. This demonstrated how disease-related genetic mutations can regulate the relative levels of these peptides in circulation.

[0268] Example 5. Verification of a target protein as a genetic driver of a disease. Stable overexpression of the novel ORF results in increased proliferation of tumor cells. Objective: This example demonstrates the verification 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, when overexpressed in the breast cancer cell line MCF-7, significantly increases the proliferation of those cells.

[0269] Experimental design: Protein overexpression plasmids encoding the target protein or a control were transfected into multiple cell lines. After selecting the stably expressing cells, proliferation was measured using the WST-1 assay.

[0270] Materials and methods: Stable cell line: The MCF-7 cell line was seeded in a 6-well plate at a density of 500,000 cells / well in 2 mL of cell culture medium without antibiotics. The cells were incubated in a humidified incubator at 37 °C and 5% CO2. Lipofectamine 3000 (Thermo Fisher) was diluted in Opti-MEM medium (Thermo Fisher) to a final concentration of 30 μL / mL. DNA (pcDNA3.1 overexpression plasmid encoding SEQ ID NO: 5404) and P3000 (Thermo Fisher) were diluted in Opti-MEM medium to final concentrations of 10 μg / mL (DNA) and 20 μL / mL (P3000). 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 swirled to distribute the mixture evenly. The cells were incubated in a CO2 incubator at 37 °C for 48 hours. The cells were selected with puromycin (2 μg / mL) for 10 days.

[0271] WST-1 assay: The target cells were seeded in a 96-well plate at a density of 4,000 cells / well in 100 μL of cell culture medium. The cells were incubated in a humidified incubator at 37 °C and 5% CO2 for 24 hours. 10 μL of WST-1 reagent (Abcam) was added directly to the wells, and the plate was incubated at 37 °C for 45 minutes in a 5% CO2 humidified incubator protected from light. The absorbance at 450 nm was measured using a microplate reader. The background absorbance can be corrected using a reference wavelength of 650 nm. The percentage of viable cells in each well was calculated by subtracting the absorbance of the blank (medium only) from the absorbance of each well and expressing the result as a percentage of the control group.

[0272] Conclusion: As shown in Figure 4, SEQ ID NO: 5404 is a novel unannotated protein that increases the proliferation of breast cancer cells (MCF-7 cell line) under low serum and high serum conditions. Therefore, SEQ ID NO: 5404 is a genetic driver of target proteins for cancer and anti-cancer therapy.

[0273] The teachings of all patents, published applications, and references cited herein are hereby incorporated by reference in their entirety.

[0274] Although exemplary embodiments have been specifically shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

[Claim 1] The invention described in the specification.

Images