Polypeptides, constructs, libraries and methods for identifying modulators of aberrant protein condensates

Abstract

Described herein are polypeptide useful for identifying compound for modulating aberrant protein condensates. The polypeptide comprise a condensate-modulating protein, a first biomarker polypeptide, a barcoding biomarker polypeptide. When inside a cell, the non-natural polypeptide exist as multiple fragments, including a first fragment comprising the condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide. The barcoding biomarker polypeptide is uniquely associated with and identifies the cell expressing associated condensate-modulating protein. Also described is a polynucleotide encoding the non-natural polypeptide, a library of the polynucleotide, and a method of identifying compound for modulating aberrant protein condensates using the non-natural polypeptide, the construct, and / or the library.

Description

[0001] Attorney Docket No. 047162-7546WOl(02844)

[0002] POLYPEPTIDES, CONSTRUCTS, LIBRARIES AND METHODS FOR IDENTIFYING MODULATORS OF ABERRANT PROTEIN CONDENSATES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 761,506, filed February 21, 2025, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under W81XWH2110717 and HT9425-25-1-0888 awarded by the U.S. Department of Defense. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Studies have indicated up to one third of human diseases may be related to the formation of aberrant aggregates by condensate-forming proteins (Vendruscolo et al., Nature Communications, 13, 5550 (2022)).

[0009] For example, Alzheimer’s disease involves condensates formed by P-amyloid and Tau, Parkinson’s disease involves condensates formed by a -synuclein, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) involve condensates formed by FUS, TDP-43, HNRNPA1, TIA1, VCP, and G3BP1, and Huntington disease involves condensates formed by HTT.

[0010] Reversing the aberrant condensates is a promising approach to tackling these diseases. However, the tools and methods for identifying compounds that can modulate the aberrant condensates remain limited.

[0011] There is a need for tools and methods for screening compounds that can modulate these aberrant condensates. The present invention addresses this need.

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[0014] SUMMARY

[0015] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0016] Polypeptides

[0017] In some aspects, the present invention is directed to a polypeptide.

[0018] In some embodiments, the polypeptide comprises: a condensate-modulating protein; a first biomarker polypeptide; a barcoding biomarker polypeptide; and a first cleavage site separating the barcoding biomarker polypeptide from the condensate-modulating protein and the first biomarker polypeptide.

[0019] In some embodiments, when inside a cell, the polypeptide is cleaved by an endogenous enzyme, thereby producing a first fragment comprising the condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide.

[0020] In some embodiments, the condensate-modulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

[0021] In some embodiments, the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a viral infection, and the viral infection is selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.

[0022] In some embodiments, the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a cancer, and the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.

[0023] In some embodiments, the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1,

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[0026] MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoV NP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

[0027] In some embodiments, the first biomarker polypeptide comprises a first fluorescent protein.

[0028] In some embodiments, the barcoding biomarker polypeptide comprises one or more fluorescent protein emitting a different signal from the first biomarker polypeptide.

[0029] In some embodiments, the barcoding biomarker polypeptide comprises two or more second biomarkers.

[0030] In some embodiments, the two or more second biomarkers are not separated by a cleavage site.

[0031] In some embodiments, the two or more second biomarkers are separated by one or more second cleavage sites.

[0032] In some embodiments, the two or more second biomarkers are separated by one or more second cleavage sites, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

[0033] In some embodiments, the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.

[0034] In some embodiments, the polypeptide comprises a plurality of condensate-modulating proteins and a plurality of first biomarker polypeptides. In some embodiments, when in a cell, the polypeptide produces a plurality of first fragments, each comprising a unique condensatemodulating protein and a unique first biomarker polypeptide.

[0035] Polynucleotide

[0036] In some aspects, the present invention is directed to a polynucleotide.

[0037] In some embodiments, the polynucleotide encodes: a first fragment and a second fragment.

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[0040] In some embodiments, the first fragment comprises a condensate-modulating protein; and a first biomarker polypeptide.

[0041] In some embodiments, the second fragment comprises a barcoding biomarker polypeptide.

[0042] In some embodiments, the polynucleotide further encodes a first cleavage site separating the first fragment and the second fragment.

[0043] In some embodiments, when inside a cell, the polypeptide is cleaved by an endogenous enzyme, thereby producing the first fragment and the second fragment.

[0044] In some embodiments, the polynucleotide comprises a ribosomal skipping sequence separating the sequence encoding the first fragment from the sequence encoding the second fragment, such that the polynucleotide is translated to the first fragment and the second fragment, such as directly during the translation process.

[0045] In some embodiments, the condensate-modulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

[0046] In some embodiments, the viral infection selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.

[0047] In some embodiments, the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.

[0048] In some embodiments, the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1,

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[0051] PRKAR1 A, MLL4, HSPA1 A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoV NP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

[0052] In some embodiments, the first biomarker polypeptide comprises a first fluorescence protein.

[0053] In some embodiments, the barcoding biomarker polypeptide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide.

[0054] In some embodiments, the barcoding biomarker polypeptide comprises two or more second biomarkers, and the two or more second biomarkers are neither separated by a cleavage site nor belong to two or more different fragments.

[0055] In some embodiments, the barcoding biomarker polypeptide comprises two or more second biomarkers, and the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different sequences separated by a ribosomal skipping sequence.

[0056] In some embodiments, the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different sequences separated by a ribosomal skipping sequence, and one or more of the two or more second biomarkers comprise a localization signal peptide.

[0057] In some embodiments, the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.

[0058] In some embodiments, the polynucleotide a viral construct.

[0059] In some embodiments, the polynucleotide a lentiviral construct.

[0060] In some embodiments, the transcription of one or more polypeptides encoded by the polynucleotide is controlled an inducible promoter system.

[0061] In some embodiments, the polynucleotide encodes a plurality of first fragments, each comprising a unique condensate-modulating protein and a unique first biomarker polypeptide. In some embodiments, each fragment of the plurality of first fragments and the second fragment is separated from the adjacent fragment by a first cleavage site or a ribosomal skipping sequence.

[0062] Nucleic acid library

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[0065] In some aspects, the present invention is directed to a nucleic acid library.

[0066] In some embodiments, the nucleic acid library comprises a plurality of polynucleotides, each of which encodes a polypeptide herein.

[0067] In some embodiments, the nucleic acid library comprises a plurality of the polynucleotide herein.

[0068] In some embodiments, the polynucleotides are viral constructs.

[0069] In some embodiments, the polynucleotides are lentiviral constructs.

[0070] In some embodiments, some or all polynucleotides of the plurality of polynucleotides comprise an inducible promoter system for controlling an expression of one or more polypeptides encoded by the polynucleotide.

[0071] In some embodiments, the first biomarker polypeptide encoded in each of the polynucleotides is the same or emits the same signal.

[0072] In some embodiments, the condensate-modulating protein encoded in each of the polynucleotides is identified by and correspond uniquely to the barcoding biomarker polypeptide encoded in the same polynucleotide.

[0073] In some embodiments, the barcoding biomarker polypeptide encoded by a first polynucleotide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide encoded by a second polynucleotide.

[0074] Method of identifying a compound for modulating aberrant protein condensates

[0075] In some aspects, the present invention is directed to a method of identifying a compound for modulating aberrant protein condensates.

[0076] In some embodiments, the method comprises forming aberrant condensates of condensate-modulating proteins tagged with first biomarker polypeptides in a plurality of population of cells and marking the cells with barcoding biomarker polypeptides by introducing the nucleic acid library herein into the cells.

[0077] In some embodiments, the method further comprises mock treating a first population of cells.

[0078] In some embodiments, the method further comprises determining the type of condensatemodulating proteins in the first population of cells based on signals from the barcoding biomarker polypeptides.

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[0081] In some embodiments, the method further comprises determining a first parameter of aberrant protein condensates in the first population of cells based on the signal of the first biomarker polypeptides.

[0082] In some embodiments, the method further comprises contacting a second population of cells with a compound of interest.

[0083] In some embodiments, the method further comprises determining a second parameter of aberrant protein condensates in the second population of cells based on the signal of the first biomarker polypeptides.

[0084] In some embodiments, the method further comprises determining the effect of the compound of interest on the aberrant protein condensates based on differences between the first parameter and the second parameter.

[0085] In some embodiments, the method further comprises the nucleic acid library is introduced into the plurality of population of cells such that more than about 95% of the cells are introduced with one or zero polynucleotide of the nucleic acid library.

[0086] In some embodiments, the method further comprises the first parameter and / or the second parameter comprises intensity, location, size, morphology, toxicity, degree, and cytotoxicity.

[0087] In some embodiments, the effect of the compound of interest on the aberrant protein condensates is determined in the form of a condensate normalization score (CNS) according to the formula:

[0088]

[0089] In some embodiments, in the formula:

[0090] Fx= average number of condensate foci in the second population of cells contacted with the compound of interest,

[0091] F Mock = average number of condensate foci in the first population of cells after the mock treatment,

[0092] lx = average integrated intensity of the barcoding biomarker polypeptide in the second population of cells contacted with the compound of interest,

[0093] I Mock = average integrated intensity of the barcoding biomarker polypeptide in the first population of cells after the mock treatment.

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[0096] In some embodiments, the nucleic acid library is introduced into the cells such that more than about 95% of the cells are introduced with one or zero polynucleotide of the nucleic acid library.

[0097] In some embodiments, the first parameter and / or the second parameter comprises intensity, location, size, morphology, toxicity, degree, and cytotoxicity.

[0098] In some embodiments, the method herein tests a plurality of compounds parallelly.

[0099] In some embodiments, the method tests about 5,000 compounds or more parallelly.

[0100] In some embodiments, the method identifies compounds that reduce or increases aberrant protein condensates of one or more condensate-forming proteins.

[0101] In some embodiments, the method further comprises validating the effect of the compound.

[0102] BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, nonlimiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0104] Fig. 1 illustrates certain aspects of protein condensation and protein homeostasis, in accordance with some embodiments.

[0105] Fig. 2 provides images of aberrant protein condensates in disease tissues, as well as in cells and in vitro, in accordance with some embodiments. (Zbinden, et. al., Developmental Cell, 2020)

[0106] Figs. 3A-3B and 4A-4B illustrate certain aspects of the non-natural polypeptides and constructs herein, in accordance with some embodiments.

[0107] Figs. 5A-5B and 6 illustrate certain aspects of the compound screening method herein, in accordance with some embodiments.

[0108] Fig. 7 illustrates certain aspects of the validation of identified compounds, in accordance with some embodiments.

[0109] Fig. 8 illustrates certain analysis aspects of the high-throughput compound screening method herein, in accordance with some embodiments.

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[0112] Figs. 9A-9B illustrate certain aspects of a non-limiting inducible MLF2 condensate reporter and chemical modulation of nuclear foci, in accordance with some embodiments. Fig.

[0113] 9A: Schematic of the doxycycline-inducible reporter system used to quantify aberrant condensate formation. In the absence of doxycycline, the reverse tetracycline transactivator (rtTA) does not activate transcription from the tetracycline response element (P -tight). Upon doxycycline addition, rtTA binds the promoter and induces expression of an MLF2-GFP fusion protein. The P2A peptide enables co-expression of a secondary fluorescent construct (e.g., tdTomato) through a ribosomal skipping mechanism during translation, resulting in production of two separate polypeptides from a single open reading frame. In the formula, CNS means condensate normalization score. The condensate normalization score is used as an empirical equation to determine the overall impact on differential protein expression relative to the effect on the condensate-specific reporter. Fx= average number of condensate foci in the tested condition; F DMSO = average number of NE condensate foci in negative control (DMSO); Ix= average integrated intensity of the secondary reporter (tdTomato) in the tested condition;

[0114] I DMSO = average integrated intensity of tdTomato in negative control (DMSO). Fig. 9B: Representative confocal images showing the number of nuclear condensates and tdTomato expression in either DMSO-treated cells (top) or cells treated with a small molecule hit (pyrithione zinc, 1.5 pM, 6 h treatment). Nuclear condensates are modulated while the secondary tdTomato reporter remains relatively unchanged. Cell nuclei are stained with DAPI. Scale bars, 5 pm.

[0115] Figs. 10A-10C illustrate certain aspects of the quantification of various drugs using the non-limiting MLF2-GFP-P2A-tdTomato system exemplified in Figs. 9A-9B, in accordance with some embodiments. Figs. 10A-10C show the quantification of the average number of condensate foci (MLF2-GFP) per cell (Fig. 10A), average integrated intensity of the secondary fluorescent construct (Fig. 10B), and the relative condensate normalization score (CNS, Fig. IOC). HeLa cells were treated with either only doxycycline (to induce condensate-biomarker expression, untreated) or doxycycline with a combination of DMSO, CHX, celastrol, or PZ. CNS-value / cell across treatment conditions normalized to the DMSO control. Asterisks indicate Bonferroni-corrected p-values determined by Mann-Whitney U testing (**: p <= 0.01; ****: p <= 0.0001).

[0116] Fig. 11 illustrate certain aspects of the identification of candidate modulators from high-content screening of a compound library with the system illustrated in Figs. 9A-9B, in

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[0119] accordance with some embodiments. Tn the experiment shown in Fig. 11, the present study used Life Chemicals Diversity Library and screened more than 10k novel chemical compounds. Each point represents a distinct test compound evaluated in a cell-based phenotypic assay. The vertical axis shows a statistically normalized (BZ-Score) activity score reflecting the compound’s effect on a cellular biomarker associated with intracellular condensates. The horizontal axis indicates relative cell viability under treatment conditions. Point shading represents the relative intensity of a co-expressed secondary fluorescent reporter (tdTomato) used as an internal expression control. Compounds meeting predefined activity and viability thresholds are highlighted (circled points). A subset of compounds exhibiting broad suppression of the reporter signal without selective biomarker modulation can be distinguished by their color profile, enabling prioritization of candidates more likely to act through specific biological mechanisms rather than nonspecific global inhibition. This multiparametric selection strategy improves identification of compounds with desired cellular activity while reducing false positives arising from general transcriptional or translational suppression.

[0120] DETAILED DESCRIPTION

[0121] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0122] In the present study, non-natural polypeptides (as well as constructs and libraries encoding the polypeptides) were developed, that would allow a large number of candidate compounds for modulating multiple different types of aberrant condensates to be screened in parallel.

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[0125] The non-natural polypeptides comprise a condensate-modulating protein, a biomarker polypeptide, a barcoding biomarker polypeptide that uniquely associates and identifies the cell containing the condensate-modulating protein , and a cleavage site separating the barcoding biomarker polypeptide from the rest of the non-natural polypeptides. When inside a cell, the cellular enzymes cleave the cleavage site (or method such as ribosomal skipping) and separates the barcoding biomarker polypeptide from the condensate-modulating protein and the biomarker polypeptide. The barcoding biomarker polypeptide is allowed to either diffuse freely in the cell or localized to predetermined locations, thereby allows the identification of the condensatemodulating protein that’s inside the cell. The biomarker polypeptide tagged condensatemodulating protein forms condensates inside the cell. As such, the strength and localizations of the biomarker polypeptide signal allow the determination of, among others, the intensity, locations, size, morphology, toxicity, degrees, and cytotoxicity of the protein aggregations and or condensation.

[0126] Since the barcoding biomarker polypeptide can be configured to give a very large number of combinations of identification signals, a very large number of unique barcoding biomarker polypeptide-condensate-modulating protein pairs can be obtained. This allows a great number of condensate-modulating proteins to be tested in a single batch of experiment.

[0127] Accordingly, in some aspects, the present invention is directed to a non-natural polypeptide, as well as constructs and libraries encoding the same.

[0128] In some aspects, the present invention is directed to a method of identifying compound for modulating aberrant protein condensates.

[0129] Definitions

[0130] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is

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[0133] not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0134] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0135] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0136] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0137] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0138] As used herein, a “biomarker polypeptide” is a polypeptide that can emit a signal, that allows the type, localization and intensity of the biomarker polypeptide to be determined or estimated.

[0139] As used herein, a “barcoding biomarker polypeptide” is a polypeptide that is able to emit a signal, or a combination of signals, which allow the identification of the barcoding biomarker polypeptide.

[0140] As used herein, a “cleavage site” is a motif, such as a polypeptide motif, that is cleaved by cellular components, such as an endogenous cellular enzyme, when inside a cell. While ribosomal skipping sequences are not normally considered cleavage sites in the art, it is

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[0143] specifically included when referring to e.g., constructs and libraries (ribosome skipping sequences are nucleic acid sequences rather than peptide sequences). The cleavage site herein can be replaced with a ribosomal skipping sequence in the nucleic acid. Ribosomal skipping sequences prevent ribosomes from creating the peptide bond with the next amino acid (such as proline), thereby resulting in the co-translational cleavage of polypeptide without the need of a cleavage site.

[0144] As used herein, the term “condensate-modulating proteins” include both condensateforming proteins and condensate dysregulating proteins.

[0145] The term “condensate-forming proteins” refers to proteins that directly form abhorrent condensates or aggregates.

[0146] The term “condensate dysregulating proteins” refers to proteins which do not directly form condensates but instead influence their formation or behavior through other mechanisms. These proteins are commonly enriched in the condensate or aggregate, and can be included in the non-natural protein herein. For instance, MLF2, a protein found in protein condensates, does not typically cause condensate formation directly. Nonetheless, MLF2 is a component of several different condensates and likely contributes to the formation of the aggregates. Similarly, many heat shock proteins typically work by dissolving protein condensates / aggregates instead of causing their formation. The barcoded system herein enables the association of individual barcodes to different labeled heat shock proteins which co-localizes with cells that have preformed condensates (for instance, cells exposed to oxidative stress).

[0147] Polypeptide

[0148] In some aspects, the present invention is directed to a polypeptide.

[0149] In some aspects, the polypeptide is a non-natural polypeptide that does not exist in nature. In some embodiments, the non-natural protein comprises: a condensate-modulating protein; a first biomarker polypeptide; a barcoding biomarker polypeptide; and a first cleavage site separating the barcoding biomarker polypeptide from the condensate-modulating protein and the first biomarker polypeptide.

[0150] In some embodiments, when inside a cell, the polypeptide is cleaved by an endogenous enzyme (or directly synthesizes as multiple fragments in the case where the cleavage site comprises a ribosomal skipping sequence), thereby producing a first fragment comprising the

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[0153] condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide.

[0154] In some embodiments, the present invention is directed to a combination of several polypeptide fragments, which comprises a first fragment comprising the condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide. As detailed elsewhere herein, the fragments can be generated directed from translation of one single polynucleotide comprising a ribosomal skipping sequence.

[0155] In some embodiments, the condensate-modulating proteins herein include condensateforming proteins, as well as condensate dysregulating proteins. Condensate dysregulating proteins are proteins which do not directly form condensates but instead influence their formation or behavior through other mechanisms. These proteins are commonly enriched in the condensate or aggregate, and can be included in the polypeptides herein. For instance, MLF2, a protein found in protein condensate, does not typically cause condensate formation directly. Nonetheless, MLF2 is a component of several different condensates and likely contribute to the formation of the aggravations. Similarly, many heat shock proteins typically work by dissolving protein condensates / aggregates instead of causing their formation. The barcoding system herein enables the association of individual barcodes to different labeled heat shock proteins which colocalizes with cells that have pre-formed condensates (for instance, cells exposed to oxidative stress).

[0156] In some embodiments, the condensate-modulating protein is a protein enriched in an aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, viral infections (including SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial viruses), spinocerebellar ataxias, developmental disorders (including Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome), primary immune-deficiency syndromes, type 2 diabetes, or cancers (including cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma among others).

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[0159] In some embodiments, the condensate-modulating protein comprises at least one selected from the group consisting of alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoVNP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP. In some embodiments, the proteins include any associated mutant variations.

[0160] In some embodiments, the biomarker polypeptides herein are fluorescent proteins.

[0161] In some embodiments, the first biomarker polypeptide comprises a first fluorescent protein.

[0162] In some embodiments, the barcoding biomarker polypeptide comprises one or more second biomarker polypeptides. In some embodiments, the one or more second biomarker polypeptides emit one or more signals different from that emitted by the first biomarker peptide. In some embodiments, the barcoding biomarker polypeptide comprises one or more fluorescent proteins emitting a different signal from the first biomarker polypeptide.

[0163] In some embodiments, the barcoding biomarker polypeptide comprises two or more second biomarkers.

[0164] In some embodiments, the two or more second biomarkers are not separated by a cleavage site.

[0165] In some embodiments, the two or more second biomarkers are separated by one or more second cleavage sites.

[0166] In some embodiments, the two or more second biomarkers are separated by one or more second cleavage sites, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

[0167] In some embodiments, the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or other membrane localization signal peptides.

[0168] In some embodiments, the polypeptide comprises a plurality of condensate-modulating proteins and a plurality of first biomarker polypeptides. In some embodiments, when in a cell, the polypeptide produces a plurality of first fragments, each comprising a unique condensate-

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[0171] modulating protein and a unique first biomarker polypeptide. As such, a plurality of condensatemodulating proteins can be studied in a single cell, by simply detecting each of the plurality of the first biomarker polypeptides

[0172] Polynucleotides

[0173] In some aspects, the present invention is directed to a polynucleotide encoding the polypeptide herein.

[0174] In some embodiments, the transcription and / or translation of the polynucleotide produces the polypeptide herein, or the first fragment and the second fragment of the polypeptide herein.

[0175] In some embodiments, the polynucleotide is a non-natural polynucleotide that does not exist in the natural.

[0176] In some embodiments, the polynucleotide comprises a vector, such as a viral vector or a plasmid.

[0177] In some embodiments, the polynucleotide comprises a lentiviral vector.

[0178] In some embodiments, the polynucleotide contains an inducible activator upstream of one or more sections of the encoded polypeptide to allow tunable expression. For example, a doxycycline inducible promoter system can be used to control the expression of some or all of the polypeptides expressed by the polynucleotide.

[0179] Cells

[0180] In some aspects, the present invention is directed to a cell, which comprises the polypeptide (or the combination of polypeptide fragments) herein, or the polynucleotide herein.

[0181] In some embodiments, the cell is a bacterial cell, a plant cell, an animal cell, a vertebrate cell, a mammalian cell, or a human cell.

[0182] In some embodiments, the cell is a cell from a cell line, or a primary cell.

[0183] Library

[0184] In some aspects, the present invention is directed to a library.

[0185] In some embodiments, the library comprises a plurality of polynucleotides that encoding the polypeptides herein.

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[0188] In some embodiments, each of the polypeptides comprises a unique combination of one condensate-modulating protein and one barcoding biomarker polypeptide.

[0189] In some embodiments, the library comprises a plurality of the polynucleotides herein. In some embodiments, each of the polynucleotides encodes a polypeptide comprising a unique combination of one condensate-modulating protein and one barcoding biomarker polypeptide.

[0190] Method of Screening Modulators of Protein Aggregation

[0191] In some aspects, the present invention is directed to a method of identifying a compound for modulating aberrant protein condensates.

[0192] In some embodiments, the method comprises forming aberrant condensates comprising condensate-modulating proteins tagged with first biomarker polypeptides in cells and marking the cells with barcoding biomarker polypeptides by introducing the library described herein into the cells.

[0193] In some embodiments, the method further comprises determining the type of condensatemodulating proteins in the cells based on signals from the barcoding biomarker polypeptides.

[0194] In some embodiments, the method further comprises determining a first parameter of aberrant protein condensates in the cells based on the signal of the first biomarker polypeptides.

[0195] In some embodiments, the method further comprises contacting the cells with a compound of interest.

[0196] In some embodiments, the method further comprises determining a second parameter of aberrant protein condensates in the cells based on the signal of the first biomarker polypeptides.

[0197] In some embodiments, the method further comprises determining the effect of the compound of interest on the aberrant protein condensates based on differences between the first parameter and the second parameter.

[0198] In some embodiments, the nucleic acid library is introduced into the cells such that more than about 90% of the cells, more than about 95% of the cells, more than about 98% of the cells, more than about 99% of the cells, more than about 98% of the cells, more than about 99% of the cells, or 100% of the cells are introduced with one or zero construct of the library. This can be achieved by, for example, introducing each lentiviral vector into cells at a low multiplicity of

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[0201] infection (such as an MOI of 0.3) to ensure that most cells receive only one viral construct or none.

[0202] In some embodiments, the method tests a plurality of compounds parallelly.

[0203] In some embodiments, the method tests about 50 compounds or more, about 100 compounds or more, about 200 compounds or more, about 500 compounds or more, about 1,000 compounds or more, about 2,000 compounds or more, about 5,000 compounds or more, about 10,000 compounds or more, about 20,000 compounds or more, about 50,000 compounds or more, about 100,000 compounds or more, about 200,000 compounds or more parallelly. For example, N (N is an integer of 1 or larger) 384-well plates can be used to test 384 x N compounds in one single batch of experiment.

[0204] In some embodiments, the method identifies compounds that reduce or increases aberrant protein condensates of one or more condensate-modulating proteins .

[0205] In some embodiments, the first parameter and / or the second parameter comprises intensity, location, size, morphology, toxicity, degree, and cytotoxicity.

[0206] In some embodiments, the method further comprises validating the effect of the compound.

[0207] In some embodiments, the analysis is carried out using an arrayed plate format and high-throughput fluorescence microscopy. In some embodiments, cells are imaged and analyzed using a downstream computational pipeline for phenotype interpretation. In some embodiments, a highly modular and semi-automated computational workflow integrates a tailored CellProfiler pipeline and custom R scripts for efficient quantification of the number, size, and phase properties of labeled condensates is used. In some embodiments, cells are grouped according to their unique combinatorial fluorescent profiles, facilitating accurate downstream analysis of condensate characteristics.

[0208] In some embodiments, the method takes into consideration that the compound of interest may affect the viability, transcription, translation, etc. of the cells, thereby affecting the condensation signal. In some embodiments, the first parameter and / or the second parameter are normalized.

[0209] In some embodiments, the effect of the compound of interest on the aberrant protein condensates is determined by the condensate normalization score (CNS), according to the formula:

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[0212]

[0213] where:

[0214] Fx= average number of condensate foci in the second population of cells contacted with the compound of interest,

[0215] F Mock = average number of condensate foci in the first population of cells after the mock treatment,

[0216] lx = average integrated intensity of the barcoding biomarker polypeptide in the second population of cells contacted with the compound of interest,

[0217] I Mock = average integrated intensity of the barcoding biomarker polypeptide in the first population of cells after the mock treatment.

[0218] In some embodiments, when the barcoding biomarker polypeptide generates more than one type of signals (e.g., comprises more than one type of fluorescence proteins), one or more of the signals can be chosen in the calculation of the CNS.

[0219] Examples

[0220] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0221] Example 1: Polypeptide

[0222] Referring to Fig. 3 A, in some embodiments, the polypeptide 100 comprises a condensatemodulating protein 111, and a first biomarker polypeptide 113.

[0223] In some embodiments, the condensate-modulating protein 111 and the first biomarker polypeptide 113 are fused together via a stable link, such as a stable polypeptide link (not shown). In some embodiments, when inside a cell, the tagged condensate-modulating protein 110 is sufficiently stable that the first biomarker polypeptide 113 is able to reliable indicate the location and amount of the condensate-modulating protein 111 with in a cell.

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[0226] In some embodiments, the non-natural polypeptide 100 further comprises a barcoding biomarker polypeptide 150. In some embodiments, the barcoding biomarker polypeptide 150 is uniquely associated with and identifies the condensate-modulating protein 111.

[0227] In some embodiments, the barcoding biomarker polypeptide 150 comprises one single second biomarker. In some embodiments, the barcoding biomarker polypeptide 150 comprises two or more second biomarkers.

[0228] It should be noted that the barcoding biomarker polypeptide 150 does not have to be upstream of the tagged condensate-modulating protein 110 as shown in Fig. 3 A. One of ordinary skill in the art would understand that the barcoding biomarker polypeptide 150 can be located anywhere, as long as it is separated from the tagged condensate-modulating protein 110 once the non-natural polypeptide 100 is introduced into the cell.

[0229] Similarly, in the tagged condensate-modulating protein 110, the order of the condensatemodulating protein 111 and a first biomarker polypeptide 113 can be reversed, as well.

[0230] In some embodiments, the non-natural polypeptide 100 further comprises a first cleavage site 130 separating the barcoding biomarker polypeptide from the condensate-modulating protein and the first biomarker polypeptide.

[0231] Referring to Figs. 3A-3B, when inside the cell, enzymes endogenous to the cell separates the first cleavage site 130 and produces a first fragment 110’ comprising the condensatemodulating protein 111’ and the first biomarker polypeptide 113’, and a second fragment comprising the barcoding biomarker polypeptide 150’.

[0232] It should be noted that, in some embodiments, the polypeptide can exist in the form of the fragments of Fig. 3B directly without going through the enzymatic cleavage step. In some embodiments, the polynucleotide encoding the polypeptide comprises a ribosomal skipping sequence between the segment that encodes fragment 150’ and the fragment 110’. When the polynucleotide is introduced in to the cell, it is translated into the first fragment 110’ and the barcoding biomarker polypeptide 150’ due to the failure of the translation machinery to form a peptide bound between the fragments.

[0233] Example 2: Barcoding biomarker polypeptide

[0234] In some embodiments, the barcoding biomarker polypeptide is uniquely associated with and identifies the condensate-modulating protein, such that users can easily identify the type of

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[0237] the condensate-modulating protein introduced into a cell by identifying the types, combinations, and / or localizations of the second biomarker(s) of the barcoding biomarker polypeptide.

[0238] In some embodiments, the barcoding biomarker polypeptide comprises one single second biomarkers. In some embodiments, the barcoding biomarker polypeptide comprises two or more second biomarkers.

[0239] For example, referring to the upper panel of Fig. 5 A, five condensate-modulating proteins (MLF2, TDP-43, Tau, a -synuclein, and Biomarker #31 (“Biomarker #31” is a random name of any condensate-modulating proteins rather than a specific protein) ) are each associated with a barcoding biomarker polypeptide, which is either a unique second biomarker polypeptide (e.g., mCherry for MLF2), or a unique combination of second biomarker polypeptides (e.g., Cy7 and TRITC for TDP-43). (Here, the second biomarker polypeptides are fluorescence proteins selected from mCherry, YFP, TRITC, Cy5 and Cy7). When the non-natural polypeptide is introduced into cells, cellular enzymes cleave the cleavage site (alternatively, if the peptide is introduced as a nucleic acid, the cleavage site can be a ribosomal skipping sequence) separates the barcoding biomarker polypeptide from the tagged condensate-modulating proteins (the condensate-modulating proteins are tagged with GFP). Under the microscope, the cells are illuminated with lights of different wavelengths to detect the signals of mCherry, YFP, TRITC, Cy5 and Cy7. If a cell shows signals of GFP, mCherry and YFP, but not others, it could be determined that the GFP signals inside the cell are Tau signals, as mCherry plus YFP is a combination for identifying Tau protein. If a cell shows signals of GFP, mCherry, YFP and Cy7, but not others, it could be determined that the GFP signals inside the cell are a -synuclein signals, as mCherry plus YFP and Cy7is a combination for identifying a -synuclein protein. Here, the mCherry, YFP, TRITC, Cy5 and Cy7 can exist in the cell as a fusion protein.

[0240] Alternatively, cleavage sites can be introduced between one or more of these second biomarker polypeptides.

[0241] In the case that a large number of condensate-modulating proteins need to be studied simultaneously, localization signals can be attached to one or more of the second biomarker polypeptides.

[0242] Referring to Fig. 4A, in some embodiments, the non-natural polypeptide 200 comprises a barcoding biomarker polypeptide 250, a first cleave site 230, a condensate-modulating protein 211, and a first biomarker polypeptide 213.

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[0245] In some embodiments, the barcoding biomarker polypeptide 250 comprises a second biomarker polypeptide-1 251, a second biomarker polypeptide-2255, and a second cleavage site 253 separating the two second biomarker polypeptides. In some embodiments, the second biomarker polypeptide-1 251 comprises a nuclear localization signal (NLS) peptide 251-1.

[0246] Referring to Fig. 4B, when inside cell, the non-natural polypeptide 200 is cleaved by endogenous cellular enzymes to form the second biomarker polypeptide-1 251’, the second biomarker polypeptide-2255’, and the tagged condensate-modulating protein 210’.

[0247] In some embodiments, the second biomarker polypeptide-1 251’ is localized mostly in the nucleus due to the presence of the nuclear localization signal peptide 251-1’, while the second biomarker polypeptide-2255’ is relatively evenly distributed within the cell. The unique distribution patterns of the two second biomarker polypeptides 251’ and 255’ allows the determination that the cell contains the condensate-modulating protein 211’, which can be visualized by the first biomarker polypeptide 213’.

[0248] Again, in some embodiments, the cleavage sites can be replaced with ribosomal skipping sequences in the polynucleotide. According to these embodiments, the polynucleotide, when translated, produces the second biomarker polypeptide-1 251’, the second biomarker polypeptide-2255’, and the tagged condensate-modulating protein 210’ directly due to the ribosomal skipping sequences.

[0249] Example 3: Design Combinatorial Lentiviral Library

[0250] Referring to Fig. 5A, an exemplary combinatorial lentiviral library for expressing the non-natural polypeptides herein is designed. Each of the lentiviral construct in the library express one condensate-modulating protein, as well as a unique barcoding biomarker polypeptide for identifying the condensate-modulating protein.

[0251] Here, the barcoding biomarker polypeptide is constructed from five second biomarker polypeptides, which 5 fluorescence proteins: mCherry, YFP, TRITC, Cy5 and Cy7. The maximal number of unique combinations these five fluorescence proteins can support is 31, as calculated by the formula:

[0252]

[0253] (n is the number of channels, which is 5 in this case).

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[0256] Accordingly, the maximal number of condensate-modulating protein supported by the second biomarker polypeptides is 31.

[0257] Each unique second biomarker polypeptide or unique combination of second biomarker polypeptides identifies one single condensate-modulating protein, which is attached with GFP.

[0258] The constructs encoding the condensate-modulating protein are cloned into lentiviral vectors, and used to infect cells at low concentrations such that each cell is only infected with one lentiviral vector, or not infected.

[0259] Example 4: Compound Screening

[0260] The cells contacted with the lentiviral vectors of Example 3 are analyzed using microscopy analysis, which determines the condensate-modulating protein present in each infected cell according to the signals of the five fluorescence proteins used as the second biomarker polypeptides, as well as the degree of aggregation, size, intensity, morphology, and cytotoxicity of the condensate-modulating protein according to the GFP signal.

[0261] Each group of cells (e.g., cells within a single well of a 384 well plate) are then treated with a candidate compound, and analyzed for a second time for changes in the degree of aggregation, size, intensity, morphology, and cytotoxicity of the condensate-modulating protein. Alternatively, the treated cells are compared with a dedicated control group of cells.

[0262] Referring to Figs. 6 and 7, the compounds found to be effective in modulating protein aggregations can be validated.

[0263] Example 7: Dual-Reporter System

[0264] A potential limitation of phenotype-based screening is the frequent identification of false positives that reduce biomarker signal by broadly impairing cellular transcription or translation rather than specifically modulating the phenotype of interest.

[0265] Referring to Figs. 9A-9B, the present study addresses this issue by providing an inducible cell-based assay platform for identifying compounds that modulate intracellular protein condensates biomarkers while distinguishing specific biological effects from nonspecific cellular suppression.

[0266] In this system, cells are engineered to express a condensate-associated protein fused to a secondary detectable reporter upon induction with an external stimulus. The construct also

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[0269] includes a 2A peptide sequence that enables coordinated expression of a second, spectrally distinct reporter from the same transcript via a ribosomal skipping mechanism. The second reporter serves as an internal control for overall transgene expression and cellular translational capacity.

[0270] Following induction, intracellular condensates are quantified by imaging and computational analysis of the first reporter signal, while the second reporter provides a reference to identify treatments that broadly reduce protein expression or cause cytotoxicity. A normalization metric (condensate normalization score, CNS) is calculated to compare condensate-specific effects relative to changes in the control reporter, allowing selective modulation of condensate biology to be distinguished from global suppression.

[0271] This dual-reporter strategy is applied in a high-content screening format to evaluate libraries of candidate agents. Compounds are prioritized based on their ability to alter condensate-associated signals while maintaining acceptable cell viability and without substantially reducing the internal control reporter.

[0272] The platform enables identification of agents that specifically modulate condensate-related cellular pathways and reduces false positives arising from general toxicity, transcriptional inhibition, or translational suppression. Additionally, this present work can be combined for the detection of multiple condensate biomarkers as described in previously in this application.

[0273] Referring to Figs. 9A-9B, in some embodiments the cell line stably designed or transiently able to express the described MLF2-P2A-tdTomato system. Alternatively, the construct / cell line may be designed such that multiple condensate biomarkers are detected simultaneously such as, Condensate / Biomarkerl-Condensate / Biomarker2-Condensate / BiomarkerX-P2A-SecondaryFluorescentReproter.

[0274] Furthermore, the secondary fluorescent reporter may be designed such that it tests the specificity of a modulator against aberrant condensates, while not impacting constitutive, nonstress-induced condensates (housekeeping condensates, e.g., PML bodies, nucleoli, etc.). Such an embodiment could be characterized through the following design: CondensateBiomarkerl-CondensateBiomarkerX-P2A-FluorescentConstitutiveCondensate.

[0275] Enumerated embodiments:

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[0278] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0279] Embodiment 1: A polypeptide, comprising:

[0280] a condensate-modulating protein;

[0281] a first biomarker polypeptide;

[0282] a barcoding biomarker polypeptide; and

[0283] a first cleavage site separating the barcoding biomarker polypeptide from the condensatemodulating protein and the first biomarker polypeptide,

[0284] wherein, when inside a cell, the polypeptide is cleaved by an endogenous enzyme, thereby producing a first fragment comprising the condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide.

[0285] Embodiment 2: The polypeptide of Embodiment 1, wherein the condensate-modulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

[0286] Embodiment 3: The polypeptide of Embodiment 2, wherein the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a viral infection, and the viral infection is selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.

[0287] Embodiment 4: The polypeptide of Embodiment 2, wherein the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a cancer, and the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.

[0288] Embodiment 5: The polypeptide of Embodiment 1, wherein the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4,

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[0291] BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TTA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoVNP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

[0292] Embodiment 6: The polypeptide of any one of Embodiments 1-5, wherein the first biomarker polypeptide comprises a first fluorescent protein.

[0293] Embodiment 7: The polypeptide of any one of Embodiments 1-6, wherein the barcoding biomarker polypeptide comprises one or more fluorescent protein emitting a different signal from the first biomarker polypeptide.

[0294] Embodiment 8: The polypeptide of any one of Embodiments 1-7, wherein the barcoding biomarker polypeptide comprises two or more second biomarkers, and wherein:

[0295] (a) the two or more second biomarkers are not separated by a cleavage site; or

[0296] (b) the two or more second biomarkers are separated by one or more second cleavage sites.

[0297] Embodiment 9: The polypeptide of Embodiment 8, wherein the two or more second biomarkers are separated by one or more second cleavage sites, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

[0298] Embodiment 10: The polypeptide of Embodiment 9, wherein the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.

[0299] Embodiment 11: The polypeptide of any one of Embodiments 1-10, which comprises a plurality of condensate-modulating proteins and a plurality of first biomarker polypeptides, wherein, when in a cell, the polypeptide produces a plurality of first fragments, each comprising a unique condensate-modulating protein and a unique first biomarker polypeptide.

[0300] Embodiment 12: A polynucleotide, which encodes:

[0301] a first fragment comprising:

[0302] a condensate-modulating protein; and

[0303] a first biomarker polypeptide; and

[0304] a second fragment comprising a barcoding biomarker polypeptide,

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[0307] wherein at least one of the following applies:

[0308] (a) the polynucleotide further encodes a first cleavage site separating the first fragment from the second fragment, and when inside a cell, a polypeptide comprising both the first fragment and the second fragment is cleaved by an endogenous enzyme, thereby producing the first fragment and the second fragment, or

[0309] (b) the polynucleotide comprises a ribosomal skipping sequence separating the first fragment from the second fragment, such that the polynucleotide is translated into the first fragment and the second fragment.

[0310] Embodiment 13: The polynucleotide of Embodiment 12, wherein the condensatemodulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

[0311] Embodiment 14: The polynucleotide of Embodiment 13, wherein the viral infection selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.

[0312] Embodiment 15: The polynucleotide of Embodiment 13, wherein the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.

[0313] Embodiment 16: The polynucleotide of any one of Embodiments 11-15, wherein the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoVNP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

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[0316] Embodiment 17: The polynucleotide of any one of Embodiments 12-16, wherein the first biomarker polypeptide comprises a first fluorescence protein.

[0317] Embodiment 18: The polynucleotide of any one of Embodiments 12-17, wherein the barcoding biomarker polypeptide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide.

[0318] Embodiment 19: The polynucleotide of any one of Embodiments 12-18, wherein the barcoding biomarker polypeptide comprises two or more second biomarkers, and wherein:

[0319] (a) the two or more second biomarkers are neither separated by a cleavage site nor belong to two or more different fragments; or

[0320] (b) the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different sequences separated by a ribosomal skipping sequence.

[0321] Embodiment 20: The polynucleotide of Embodiment 19, wherein the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different sequences separated by a ribosomal skipping sequence, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

[0322] Embodiment 21: The polynucleotide of Embodiment 20, wherein the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.

[0323] Embodiment 21: The polynucleotide of any one of Embodiments 12-21, which is a viral construct, optionally a lentiviral construct.

[0324] Embodiment 23: The polynucleotide of any one of Embodiments 12-22, wherein transcription of one or more polypeptides encoded by the polynucleotide is controlled an inducible promoter system.

[0325] Embodiment 24: The polynucleotide of any one of Embodiments 12-23, which encodes a plurality of first fragments, each comprising a unique condensate-modulating protein and a unique first biomarker polypeptide, wherein each fragment of the plurality of first fragments and the second fragment is separated from the adjacent fragment by a first cleavage site or a ribosomal skipping sequence.

[0326] Embodiment 25: A nucleic acid library, comprising:

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[0329] (a) a plurality of polynucleotides encoding a plurality of the polypeptides of any one of Embodiments 1-11, and / or

[0330] (b) a plurality of the polynucleotide of any one of Embodiments 12-24.

[0331] Embodiment 26: The nucleic acid library of Embodiment 25, wherein the polynucleotides are viral constructs, optionally lentiviral constructs.

[0332] Embodiment 27: The nucleic acid library of any one of Embodiments 25-26, wherein some or all polynucleotides of the plurality of polynucleotides comprise an inducible promoter system for controlling an expression of one or more polypeptides encoded by the polynucleotide.

[0333] Embodiment 28: The nucleic acid library of any one of Embodiments 25-27, wherein the first biomarker polypeptide encoded in each of the polynucleotides is the same or emits the same signal.

[0334] Embodiment 29: The nucleic acid library of any one of Embodiments 25-28, wherein the condensate-modulating protein encoded in each of the polynucleotides is identified by and correspond uniquely to the barcoding biomarker polypeptide encoded in the same polynucleotide.

[0335] Embodiment 30: The nucleic acid library of any one of Embodiments 25-29, wherein the barcoding biomarker polypeptide encoded by a first polynucleotide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide encoded by a second polynucleotide.

[0336] Embodiment 31 : A method of identifying a compound for modulating aberrant protein condensates, the method comprising:

[0337] forming aberrant condensates of condensate-modulating proteins tagged with first biomarker polypeptides in a plurality of population of cells and marking the cells with barcoding biomarker polypeptides by introducing the nucleic acid library of any one of Embodiments 25-30 into the cells;

[0338] mock treating a first population of cells;

[0339] determining the type of condensate-modulating proteins in the first population of cells based on signals from the barcoding biomarker polypeptides;

[0340] determining a first parameter of aberrant protein condensates in the first population of cells based on the signal of the first biomarker polypeptides;

[0341] contacting a second population of cells with a compound of interest;

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[0344] determining a second parameter of aberrant protein condensates in the second population of cells based on the signal of the first biomarker polypeptides; and

[0345] determining the effect of the compound of interest on the aberrant protein condensates based on differences between the first parameter and the second parameter,

[0346] wherein the nucleic acid library is introduced into the plurality of population of cells such that more than about 95% of the cells are introduced with one or zero polynucleotide of the nucleic acid library, and

[0347] wherein the first parameter and / or the second parameter comprises intensity, location, size, morphology, toxicity, degree, and cytotoxicity.

[0348] Embodiment 32: The method of Embodiment 31, wherein the effect of the compound of interest on the aberrant protein condensates is determined in the form of a condensate normalization score (CNS) according to the formula:

[0349]

[0350] wherein:

[0351] Fx= average number of condensate foci in the second population of cells contacted with the compound of interest,

[0352] F Mock = average number of condensate foci in the first population of cells after the mock treatment,

[0353] Ix= average integrated intensity of the barcoding biomarker polypeptide in the second population of cells contacted with the compound of interest,

[0354] I_Mock = average integrated intensity of the barcoding biomarker polypeptide in the first population of cells after the mock treatment.

[0355] Embodiment 33: The method of any one of Embodiments 31-32, which tests a plurality of compounds parallelly.

[0356] Embodiment 34: The method of Embodiment 33, which tests about 5,000 compounds or more parallelly.

[0357] Embodiment 35: The method of any one of Embodiments 31-34, which identifies compounds that reduce or increases aberrant protein condensates of one or more condensateforming proteins.

[0358] 30

[0359] 57258171.3Attorney Docket No. 047162-7546WOl(02844)

[0360] Embodiment 36: The method of Embodiment 35, further comprising validating the effect of the compound.

[0361] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

[0362] 31

[0363] 57258171.3

Claims

Attorney Docket No. 047162-7546WOl(02844)CLAIMSWhat is claimed is:

1. A polypeptide, comprising:a condensate-modulating protein;a first biomarker polypeptide;a barcoding biomarker polypeptide; anda first cleavage site separating the barcoding biomarker polypeptide from the condensatemodulating protein and the first biomarker polypeptide,wherein, when inside a cell, the polypeptide is cleaved by an endogenous enzyme, thereby producing a first fragment comprising the condensate-modulating protein and the first biomarker polypeptide, and a second fragment comprising the barcoding biomarker polypeptide.

2. The polypeptide of claim 1, wherein the condensate-modulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEBALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

3. The polypeptide of claim 2, wherein the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a viral infection, and the viral infection is selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.

4. The polypeptide of claim 2, wherein the condensate modulating protein is a protein that is enriched in a condensate or aggregate in a cancer, and the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.3257258171.3Attorney Docket No. 047162-7546WOl(02844)5. The polypeptide of claim 1, wherein the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoV NP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

6. The polypeptide of any one of claims 1-5, wherein the first biomarker polypeptide comprises a first fluorescent protein.

7. The polypeptide of any one of claims 1-6, wherein the barcoding biomarker polypeptide comprises one or more fluorescent protein emitting a different signal from the first biomarker polypeptide.

8. The polypeptide of any one of claims 1-7, wherein the barcoding biomarker polypeptide comprises two or more second biomarkers, and wherein:(a) the two or more second biomarkers are not separated by a cleavage site; or(b) the two or more second biomarkers are separated by one or more second cleavage sites.

9. The polypeptide of claim 8, wherein the two or more second biomarkers are separated by one or more second cleavage sites, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

10. The polypeptide of claim 9, wherein the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.3357258171.3Attorney Docket No. 047162-7546WOl(02844)11. The polypeptide of any one of claims 1-10, which comprises a plurality of condensatemodulating proteins and a plurality of first biomarker polypeptides, wherein, when in a cell, the polypeptide produces a plurality of first fragments, each comprising a unique condensatemodulating protein and a unique first biomarker polypeptide.

12. A polynucleotide, which encodes:a first fragment comprising:a condensate-modulating protein; anda first biomarker polypeptide; anda second fragment comprising a barcoding biomarker polypeptide,wherein at least one of the following applies:(a) the polynucleotide further encodes a first cleavage site separating the first fragment from the second fragment, and when inside a cell, a polypeptide comprising both the first fragment and the second fragment is cleaved by an endogenous enzyme, thereby producing the first fragment and the second fragment, or(b) the polynucleotide comprises a ribosomal skipping sequence separating the sequence encoding the first fragment from the sequence encoding the second fragment, such that the polynucleotide is translated into the first fragment and the second fragment.

13. The polynucleotide of claim 12, wherein the condensate-modulating protein is a protein that is enriched in a condensate or aggregate in a disease selected from Alexander disease, Alzheimer's disease, cardiomyopathy, amyotrophic lateral sclerosis (ALS), atopic dermatitis, dystonia, frontotemporal dementia (FTD), Fragile X Syndrome, Huntington disease, dementia, Parkinson's disease, cataracts a prion disease, Limb-girdle muscular dystrophy 1G, a viral infection, spinocerebellar ataxias, a developmental disorder selected from Rett Syndrome, Noonan syndrome, CEB ALID syndrome, and Kabuki syndrome, primary immune-deficiency syndromes, type 2 diabetes, and a cancer.

14. The polynucleotide of claim 13, wherein the viral infection selected from SARS-CoV-2, Ebola, Influenza A, human immunodeficiency virus, and respiratory syncytial virus.3457258171.3Attorney Docket No. 047162-7546WOl(02844)15. The polynucleotide of claim 13, wherein the cancer is selected from cervix cancer, lung cancer, prostate cancer, skin cancer, promyelocytic leukemia, central nervous system cancer, Adenoid cystic carcinoma, melanoma, Wilm’s tumor, liver cancer, and Ewing sarcoma.

16. The polynucleotide of any one of claims 11-15, wherein the condensate-modulating protein comprises at least one selected from the group consisting of the following proteins or gene products: alpha-synuclein, amyloid beta, Tau, TDP-43, FUS, G3BP1, BRD3, BRD4, BARD1, HNRNPA1, HTT, KEAP1, MLF1, MLF2, SPOP, CHCHD10, TIA1, PML, OCT4, BCOR, EWSR1, HSF1, MeCp2, UBQL2, NONO, UTX, HNRNPDL, DAXX, RBM10, RBM20, IRAK4, MYD88, BCL11A, M2-1, PRKAR1A, MLL4, HSPA1A, SHP2, SQSTM1, PTPN11, CPSF5, CPSF6, DNAJB6, SARS-CoVNP, INSR, ENL, NF2, NUPs, p53, EWS, or VCP.

17. The polynucleotide of any one of claims 12-16, wherein the first biomarker polypeptide comprises a first fluorescence protein.

18. The polynucleotide of any one of claims 12-17, wherein the barcoding biomarker polypeptide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide.

19. The polynucleotide of any one of claims 12-18, wherein the barcoding biomarker polypeptide comprises two or more second biomarkers, and wherein:(a) the two or more second biomarkers are neither separated by a cleavage site nor belong to two or more different fragments; or(b) the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different sequences separated by a ribosomal skipping sequence.

20. The polynucleotide of claim 19, wherein the two or more second biomarkers are separated by one or more second cleavage sites or are encoded by two or more different3557258171.3Attorney Docket No. 047162-7546WOl(02844)sequences separated by a ribosomal skipping sequence, and wherein one or more of the two or more second biomarkers comprise a localization signal peptide.

21. The polynucleotide of claim 20, wherein the localization signal peptide comprises at least one selected from a nuclear export signal peptide, a nuclear localization signal peptide, an endoplasmic reticulum retention signal peptide, a mitochondrial targeting peptide, a lysosomal targeting peptide, or a membrane localization signal peptide.

21. The polynucleotide of any one of claims 12-21, which is a viral construct, optionally a lentiviral construct.

23. The polynucleotide of any one of claims 12-22, wherein transcription of one or more polypeptides encoded by the polynucleotide is controlled an inducible promoter system.

24. The polynucleotide of any one of claims 12-23, which encodes a plurality of first fragments, each comprising a unique condensate-modulating protein and a unique first biomarker polypeptide, wherein each fragment of the plurality of first fragments and the second fragment is separated from the adjacent fragment by a first cleavage site or a ribosomal skipping sequence.

25. A nucleic acid library, comprising:(a) a plurality of polynucleotides encoding a plurality of the polypeptides of any one of claims 1-11, and / or(b) a plurality of the polynucleotide of any one of claims 12-24.

26. The nucleic acid library of claim 25, wherein the polynucleotides are viral constructs, optionally lentiviral constructs.

27. The nucleic acid library of any one of claims 25-26, wherein some or all polynucleotides of the plurality of polynucleotides comprise an inducible promoter system for controlling an expression of one or more polypeptides encoded by the polynucleotide.3657258171.3Attorney Docket No. 047162-7546WOl(02844)28. The nucleic acid library of any one of claims 25-27, wherein the first biomarker polypeptide encoded in each of the polynucleotides is the same or emits the same signal.

29. The nucleic acid library of any one of claims 25-28, wherein the condensate-modulating protein encoded in each of the polynucleotides is identified by and correspond uniquely to the barcoding biomarker polypeptide encoded in the same polynucleotide.

30. The nucleic acid library of any one of claims 25-29, wherein the barcoding biomarker polypeptide encoded by a first polynucleotide comprises one or more fluorescence protein emitting a different signal from the first biomarker polypeptide encoded by a second polynucleotide.

31. A method of identifying a compound for modulating aberrant protein condensates, the method comprising:forming aberrant condensates of condensate-modulating proteins tagged with first biomarker polypeptides in a plurality of population of cells and marking the cells with barcoding biomarker polypeptides by introducing the nucleic acid library of any one of claims 25-30 into the cells;mock treating a first population of cells;determining the type of condensate-modulating proteins in the first population of cells based on signals from the barcoding biomarker polypeptides;determining a first parameter of aberrant protein condensates in the first population of cells based on the signal of the first biomarker polypeptides;contacting a second population of cells with a compound of interest;determining a second parameter of aberrant protein condensates in the second population of cells based on the signal of the first biomarker polypeptides; anddetermining the effect of the compound of interest on the aberrant protein condensates based on differences between the first parameter and the second parameter,wherein the nucleic acid library is introduced into the plurality of population of cells such that more than about 95% of the cells are introduced with one or zero polynucleotide of the nucleic acid library, and3757258171.3Attorney Docket No. 047162-7546WOl(02844)wherein the first parameter and / or the second parameter comprises intensity, location, size, morphology, toxicity, degree, and cytotoxicity.

32. The method of claim 31, wherein the effect of the compound of interest on the aberrant protein condensates is determined in the form of a condensate normalization score (CNS) according to the formula:F* \ / VCNS - ) •Mock ' ' I Mock 'wherein:Fx= average number of condensate foci in the second population of cells contacted with the compound of interest,F Mock = average number of condensate foci in the first population of cells after the mock treatment,Ix= average integrated intensity of the barcoding biomarker polypeptide in the second population of cells contacted with the compound of interest,I Mock = average integrated intensity of the barcoding biomarker polypeptide in the first population of cells after the mock treatment.

33. The method of any one of claims 31-32, which tests a plurality of compounds parallelly.

34. The method of claim 33, which tests about 5,000 compounds or more parallelly.

35. The method of any one of claims 31-34, which identifies compounds that reduce or increases aberrant protein condensates of one or more condensate-forming proteins.

36. The method of claim 35, further comprising validating the effect of the compound.3857258171.3

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