Variants of phase separation-reversing polypeptides and their applications

Abstract

The present disclosure provides a variant of a phase separation reversing polypeptide. Also provided is a method of treating phase separation-related diseases, such as phase separation-related visual disorders, age-related macular degeneration, and neurodegenerative diseases, using the variant of the phase separation reversing polypeptide provided herein.

Description

Technical Field

[0001] The present invention generally relates to methods for enhancing the phase separation reversal ability of phase separation-reversing polypeptides. Specifically, the present invention relates to variants of phase separation-reversing polypeptides and methods for treating phase separation-related diseases using the same. Background Art

[0002] "Phase separation", in cell biology, refers to a special state in which biomacromolecules aggregate within a cell, which is a phenomenon where different components within a cell collide and fuse to form droplets, with some components being enclosed within the droplets and some being blocked outside the droplets, and it is ubiquitous in cells. Phase separation has become an emerging field of biological research in recent years. Existing research has shown that it is ubiquitous in cells and may be closely related to genome assembly and transcriptional regulation. The dysregulation of phase separation may be the cause of some diseases (such as neurodegenerative / muscular degenerative diseases). Scientists in related fields have also begun to re-examine related diseases from the perspective of phase separation and aim to treat related diseases by interfering with abnormal "phase separation".

[0003] Currently, there are few drugs in the prior art that can effectively reverse phase separation. Therefore, there is an urgent need for drug molecules that can effectively reverse phase separation, or even have enhanced phase separation reversal ability. Summary of the Invention

[0004] The present invention provides a variant of a phase separation-reversing polypeptide and methods for treating phase separation-related diseases, such as phase separation-related visual disorders, age-related macular degeneration, and neurodegenerative diseases, using the same.

[0005] On the one hand, the present invention provides a variant of a phase separation-reversing polypeptide, wherein the amino acid sequence of the variant is the amino acid sequence after incorporating n cysteine residues based on the phase separation-reversing polypeptide, and the variant has comparable or enhanced phase separation reversal ability compared to the phase separation-reversing polypeptide; where n is an integer greater than or equal to 0.

[0006] In certain embodiments, the incorporation includes insertion and / or substitution.

[0007] In certain embodiments, the n cysteine residues are consecutive or non-consecutive.

[0008] In certain embodiments, the n cysteine residues are located at the N-terminus and / or C-terminus of the phase separation-reversing polypeptide.

[0009] In certain embodiments, the phase separation-reversing polypeptide comprises a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment has a sequence selected from the group consisting of:

[0010] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 (SEQ ID NO: 47),

[0011] X 13 Z2X 14 X 15 X 16 Y4X 17 X 18 X 19 Z3X 20 X 21 X 22 Y5X 23 X 24 X 25 (SEQ ID NO: 44),

[0012] X 26 X 27 X 28 Z4X 29 X 30 X 31 X 32 X 33 Z5X 34 X 35 (SEQ ID NO: 45), and

[0013] X 36 X 37 Y6X 38 Y7X 39 Z6X 40 (SEQ ID NO: 46);

[0014] wherein X1 - X 40 are independently selected from the group consisting of Asp, Glu, Lys, and Arg; Y1 - Y7 are independently selected from the group consisting of Thr, Gln, and Ser; and Z1 - Z6 are independently selected from the group consisting of Val, Phe, Leu, and Ile.

[0015] In certain embodiments, the hydrophilic segment has the following sequence:

[0016] Y1X1PY2X3X4Y3X5X6X7VX8X9PX 10 X 11 X 12 (SEQ ID NO: 43),

[0017] wherein X1 - X 12Independently selected from the group consisting of Asp, Glu, Lys, and Arg, and Y1 - Y3 are independently selected from the group consisting of Thr, Gln, and Ser.

[0018] In certain embodiments, the hydrophilic segment has a sequence selected from the group consisting of:

[0019] TX1PQX1X1SX1X1X1VX1X1PX1X1R (SEQ ID NO: 11),

[0020] X1LX1X l X1SX1X1X1VX l X1X1QX1X l X1 (SEQ ID NO: 12),

[0021] X1X1X1VX1X1X1X1X1VX1X1 (SEQ ID NO: 13), and

[0022] X1X1SX1QX1LX1 (SEQ ID NO: 14);

[0023] wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, or His.

[0024] In certain embodiments, the hydrophilic segment has a sequence selected from the group consisting of:

[0025]

[0026] KEEVDEDRDVDE (SEQ ID NO: 20); and

[0027]

[0028] In certain embodiments, the hydrophobic segment has a sequence selected from the group consisting of:

[0029]

[0030] PPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 24); and

[0031]

[0032] In certain embodiments, the phase separation reversing polypeptide comprises a sequence selected from the group consisting of:

[0033]

[0034] SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37); and

[0035]

[0036] In certain embodiments, the variant further comprises a cell-penetrating peptide.

[0037] In certain embodiments, the cell-penetrating peptide comprises a sequence selected from the group consisting of:

[0038]

[0039] In certain embodiments, the cell-penetrating peptide is at the N-terminus or C-terminus of the variant.

[0040] In certain embodiments, the variant further comprises the insertion and / or replacement of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0.

[0041] In certain embodiments, the variant further comprises a his-tag.

[0042] In certain embodiments, the his-tag is linked to the variant via a linker.

[0043] In certain embodiments, the linker comprises a sequence selected from the group consisting of: SGRPVL (SEQ ID NO: 28), GAPGSAGSAAGGSG (SEQ ID NO: 29), ENLVFQG (SEQ ID NO: 30), and (GSG)n; where n is an integer greater than 0, for example 1.

[0044] In certain embodiments, the variant further comprises the insertion and / or replacement of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0.

[0045] In certain embodiments, the variant sequentially comprises a phase separation reversal polypeptide, optionally a cell-penetrating peptide, optionally a linker, and optionally a his-tag from the N-terminus to the C-terminus, where the n cysteine residues are at the N-terminus or C-terminus of the variant.

[0046] In certain embodiments, the variant sequentially comprises a phase separation reversal polypeptide, a cell-penetrating peptide, a linker, and a his-tag from the N-terminus to the C-terminus, where the n cysteine residues are at the N-terminus or C-terminus of the variant; where,

[0047] The phase separation reversal polypeptide comprises a sequence selected from the group consisting of:

[0048]

[0049]

[0050] SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37), and

[0051]

[0052] The cell-penetrating peptide comprises a sequence selected from the group consisting of:

[0053] GGRKKRRQRRR (SEQ ID NO: 26), and

[0054] RQIKIWFQNRRMKWKKK (SEQ ID NO: 27); and

[0055] The linker comprises a sequence selected from the group consisting of:

[0056] SGRPVL (SEQ ID NO: 28),

[0057] GAPGSAGSAAGGSG (SEQ ID NO: 29),

[0058] ENLVFQG (SEQ ID NO: 30), and

[0059] GSG.

[0060] In certain embodiments, the variant has a sequence selected from the group consisting of:

[0061]

[0062]

[0063] CCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 53); and

[0064]

[0065] On the other hand, provided herein is also a method for improving the ability of a phase separation-reversing polypeptide to reverse phase separation, wherein the method comprises incorporating n cysteine residues into the phase separation-reversing polypeptide; wherein n is an integer greater than or equal to 0.

[0066] In certain embodiments, the incorporation comprises insertion and / or substitution.

[0067] In some embodiments, the n cysteine residues are consecutive or non-consecutive.

[0068] In some embodiments, the n cysteine residues are at the N-terminus and / or C-terminus of the phase separation reversing polypeptide.

[0069] In some embodiments, the phase separation reversing polypeptide comprises a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment has a sequence selected from the group consisting of:

[0070] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 (SEQ ID NO: 47),

[0071] X 13 Z2X 14 X 15 X 16 Y4X 17 X 18 X 19 Z3X 20 X 21 X 22 Y5X 23 X 24 X 25 (SEQ ID NO: 44),

[0072] X 26 X 27 X 28 Z4X 29 X 30 X 31 X 32 X 33 Z5X 34 X 35 (SEQ ID NO: 45), and

[0073] X 36 X 37 Y6X 38 Y7X 39 Z6X 40 (SEQ ID NO: 46);

[0074] wherein X1 - X 40 are independently selected from the group consisting of Asp, Glu, Lys, and Arg; Y1 - Y7 are independently selected from the group consisting of Thr, Gln, and Ser; and Z1 - Z6 are independently selected from the group consisting of Val, Phe, Leu, and Ile.

[0075] In certain embodiments, the hydrophilic segment has the following sequence:

[0076] Y1X1PY2X3X4Y3X5X6X7VX8X9PX 10 X 11 X 12 (SEQ ID NO: 43),

[0077] wherein X1 - X 12 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, and Y1 - Y3 are independently selected from the group consisting of Thr, Gln, and Ser.

[0078] In certain embodiments, the hydrophilic segment has a sequence selected from the group consisting of:

[0079] TX1PQX1X1SX1X1X1VX1X1PX1X1R (SEQ ID NO: 11),

[0080] X1LX1X1X1SX1X1X1VX1X1X1QX1X1X1 (SEQ ID NO: 12),

[0081] X1X1X1VX1X1X1X1X1VX1X1 (SEQ ID NO: 13), and

[0082] X1X1SX1QX1LX1 (SEQ ID NO: 14);

[0083] wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, or His.

[0084] In certain embodiments, the hydrophilic segment has a sequence selected from the group consisting of:

[0085]

[0086]

[0087] KEEVDEDRDVDE (SEQ ID NO: 20); and

[0088]

[0089] In certain embodiments, the hydrophobic segment has a sequence selected from the group consisting of:

[0090]

[0091] PPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 24); and

[0092]

[0093] In certain embodiments, the phase separation reversal polypeptide comprises a sequence selected from the group consisting of:

[0094]

[0095] SNRLMMLRMHNLHGTKPPP SEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37); and

[0096]

[0097] On the other hand, the present disclosure also provides a method for treating a subject in need thereof suffering from a phase separation-related disease, which comprises administering to the subject in need thereof a therapeutically effective amount of the variant described herein.

[0098] On the other hand, the present disclosure also provides a method for reversing, inhibiting, alleviating and / or preventing abnormal protein aggregation or abnormal metabolite generation in cells, which comprises introducing the variant described herein into the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. By referring to one or more of these drawings, in conjunction with the detailed description of the specific embodiments presented herein, the present disclosure can be better understood.

[0100] Figure 1 and Figure 2 show a comparison of the ability of different polypeptides to depolymerize proteins in in vitro experiments.

[0101] Figure 3 and Figure 4 show a comparison of the ability of different polypeptide variants to depolymerize the protein FUS525 in cell experiments. DETAILED DESCRIPTION

[0102] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus can of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, as the scope of the present disclosure will be defined only by the appended claims.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0104] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that this disclosure is not entitled to antedate such publication by virtue of prior disclosure. In addition, the provided publication dates may be different from the actual publication dates, which may need to be independently confirmed.

[0105] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of the several other embodiments without departing from the scope or spirit of this disclosure. Any of the recited methods can be performed in the recited order of events or in any other order that is logically possible.

[0106] I. Definitions

[0107] It should be understood that the foregoing general description and the following detailed description are both exemplary and explanatory and do not limit the claimed invention. In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this disclosure, unless clearly indicated to refer to only the alternative or the alternatives are mutually exclusive, the term "or" is used to mean "and / or". As used herein, "another" can mean at least a second or more. In addition, the use of the term "including" and other forms such as "includes" and "included" is not limiting. In addition, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components that include one unit and elements and components that include more than one subunit. In addition, the use of the term "portion" can include a portion of a portion or the whole portion.

[0108] As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents.

[0109] As used herein, the term "variant" refers to a polypeptide that contains one or more amino acid residue alterations relative to the wild-type polypeptide, i.e., substitution of some of the amino acid residues therein, insertion of new amino acid residues, and / or deletion of some of the amino acid residues therein, and still retains its activity, or even has higher activity.

[0110] As used herein, the term "administer" means to provide an agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0111] As used herein, the term "amino acid" refers to an organic compound containing an amino (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain unique to each amino acid. Amino acid names are also represented in this disclosure by standard single-letter or three-letter codes, and the common amino acids are shown in the following table.

[0112]

[0113]

[0114] As used herein, the term "effective amount" or "therapeutically effective amount" means the amount of an agent sufficient to prevent, treat, alleviate, and / or improve the symptoms and / or underlying causes of any disorder or disease, or the amount of an agent sufficient to produce a desired effect on cells. In one embodiment, a "therapeutically effective amount" means the amount sufficient to reduce or eliminate the symptoms of a disease. In another embodiment, a therapeutically effective amount is the amount sufficient to overcome the disease itself. In certain embodiments, the therapeutically effective amount of the variants provided herein in the pharmaceutical formulations provided herein is relatively low in concentration in a liquid pharmaceutical composition or other agent, such as at least 10 -9 M, at least 0.5 to 1×10 -8 M, at least 0.5 to 1×10 -7 M, at least 0.5 to 1×10 -6 M, at least 0.5 to 1×10 -5 M, at least 0.5 to 1×10 -4 M, at least 0.5 to 1×10 -3 M, at least 0.5 to 1×10 -2 M, at least 0.5 to 1×10 -1 M, or at least 0.5 to 1M or any concentration falling within the range between these values (e.g., 10 -9 M to 1M), and such visual impairments can be reversed by application once, twice, three times, or multiple times a day, and the effect is rapid.

[0115] The term "host cell" means a cell that has been transformed with a nucleic acid sequence or is capable of being transformed and thereby expressing a protein of interest. The term includes the progeny of a parent cell, whether or not the progeny are identical in morphology or genetic constitution to the original parent cell, so long as the gene of interest is present.

[0116] The term "engineered cell" means a cell that has been subjected to manipulation such that its genetic, epigenetic, and / or phenotypic identity has been altered relative to a suitable reference cell (such as an otherwise identical cell that has not been so manipulated). In some embodiments, the manipulation is or includes genetic manipulation. In some embodiments, the genetic manipulation is or includes one or more of the following: (i) introduction of a nucleic acid that was not present in the cell prior to the manipulation (i.e., introduction of a heterologous nucleic acid); (ii) removal of a nucleic acid or a portion thereof that was present in the cell prior to the manipulation; and / or (iii) alteration (e.g., by sequence substitution) of a nucleic acid or a portion thereof that was present in the cell prior to the manipulation. In some embodiments, an engineered cell is a cell that has been manipulated such that it contains and / or expresses an altered amount and / or in an altered temporal pattern of a particular agent of interest (e.g., a variant, polynucleotide, and / or a particular form thereof herein) relative to such a suitable reference cell. One of ordinary skill in the art will understand that in some embodiments, the "engineered cells" referred to herein encompass a particular cell to which the manipulation has been applied and also encompass any progeny of such a cell.

[0117] As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly recited sequence. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0118] "Percent sequence identity (%)" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered as identical residues. Publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., Journal of Molecular Biology (J. Mol. Biol.), 215: 403-410 (1990); Stephen F. et al., Nucleic Acids Research, 25: 3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins D.G. et al., Methods In Enzymology, 266: 383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, UK), 23(21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software can be used to perform the alignment to determine the percent amino acid (or nucleic acid) sequence identity. A person skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters according to the needs of the alignment, for example, by selecting a suitable algorithm.

[0119] As used herein, the term "non-conservative amino acid" refers to an amino acid residue in a polypeptide that, when mutated (e.g., substituted, deleted), does not affect the pre-mutation function of the polypeptide (e.g., the phase separation reversal function).

[0120] The term "polypeptide" or "protein" refers to a chain of at least two amino acids linked to each other by peptide bonds. Polypeptides and proteins can include moieties other than amino acids (e.g., they can be glycosylated) and / or can be otherwise processed or modified. A person of ordinary skill in the art will understand that a "polypeptide" or "protein" can be a full-length polypeptide chain produced by a cell (with or without a signal sequence) or can be a functional portion thereof. A person of ordinary skill in the art will further understand that a protein can sometimes include more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated in other ways. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers.

[0121] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffering solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0122] As used herein, the term "protein aggregate" refers to the aggregation of proteins that occurs intracellularly or extracellularly. In some embodiments, the protein is an intrinsically disordered protein or a misfolded protein. In some embodiments, aggregation occurs when the concentration of the protein exceeds the solubility of the protein. In some embodiments, the concentration of the protein exceeds the thermodynamic solubility, but the protein is maintained in a metastable liquid-like state by buffering of heterotypic interactions. Disorder of the metastable form of the protein leads to loss of protein solubility and results in protein aggregation.

[0123] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include pre-natal and post-natal forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a human who presents to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". The subject can have or be predisposed to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder. For example, a subject may have or be at risk of developing cataracts. Subjects at risk of developing cataracts include, but are not limited to, subjects having a mutation associated with cataracts, subjects having a family history of cataracts, subjects exposed to radiation, and diabetic patients, among others.

[0124] As used herein, "treating" a condition includes preventing or alleviating the condition, slowing the rate of onset or development of the condition, reducing the risk of contracting the condition, preventing or delaying the development of symptoms associated with the condition, alleviating or ending symptoms associated with the condition, producing a complete or partial regression of the condition, curing the condition, or some combination thereof. It should be understood that "treating" does not require 100% elimination or reversal of the condition. By way of example, for the "treatment" of visual impairment, it is not required to 100% eliminate or reverse the visual impairment; in certain embodiments, compared to the levels observed in the absence of the pharmaceutical compositions or methods provided herein (e.g., in biologically matched control subjects or samples not exposed to the pharmaceutical compositions or methods provided herein), "treating" visual impairment by the methods provided herein reduces, inhibits, prevents, and / or reverses lens dysfunction (e.g., nuclear opacity (N), cortical opacity (C), posterior subcapsular opacity (P), and nuclear color (NC)) of the lens by, for example, at least about 5%, at least about 10%, or at least about 20%.

[0125] As used herein, a "vector" refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit its replication in a host cell (such as an origin of replication). A vector may also include one or more therapeutic genes and / or selectable marker genes, as well as other genetic elements known in the art. A vector may transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those that are native to the cell. A vector optionally includes materials that facilitate entry of the nucleic acid into the cell, such as viral particles, liposomes, protein coatings, and the like.

[0126] II. Variants of Phase Separation-Reversing Polypeptides

[0127] Phase separation (also known as phase transition) is a concept originally derived from physical chemistry. A phase refers to a part of a system that has completely uniform physical and chemical properties. When two phases are mixed, such as when oil is dropped into water, a two-phase separation phenomenon will occur. For cells with complex components, certain protein and nucleic acid molecules can combine through multivalent interactions, thereby spontaneously forming another phase with physical and chemical properties different from the original environment - this phenomenon is called "intracellular and biological phase separation".

[0128] In 2009, Brangwynne and Hyman first introduced the concept of "phase separation" into the assembly of biological structures to explain the liquid-like behavior of P granules when studying P granules in Caenorhabditis elegans (see Brangwynne, Clifford P. et al., "Germline P granules are liquid droplets that localize by controlled dissolution / condensation" Science 324.5935 (2009): 1729-1732). Recent studies have shown that phase separation plays an important role in the assembly of membraneless organelles, signaling complexes, the cytoskeleton, etc., and phase separation has also been observed in membraneless organelles, including stress granules and the formation of the structural nucleolus. Changes in physiologically relevant conditions can also alter the homeostasis formed by phase separation. In cells, phase separation may be caused by, for example, a decrease in pH induced by starvation, changes in viscosity, or an increase in the concentration of calcium ions and other polyvalent cations, changes in the expression level of the protein itself, and phosphorylation modifications. For non-isothermal organisms such as yeast and nematodes, temperature changes may also trigger intracellular protein phase separation. In other words, phase separation is ubiquitous in cells.

[0129] Abnormal phase separation of specific proteins may form more stable but difficult-to-reverse structures and may be the cause of specific diseases. There is a large amount of evidence indicating a link between membraneless structures formed by phase separation and diseases.

[0130] On the one hand, the present disclosure provides a variant of a phase separation-reversing polypeptide, wherein the amino acid sequence of the variant is the amino acid sequence after incorporating n cysteine residues on the basis of the phase separation-reversing polypeptide, and the variant has a comparable or enhanced phase separation-reversing ability compared to the phase separation-reversing polypeptide; wherein n is an integer greater than or equal to 0. The terms "phase separation reversal" and "depolymerization" can be used interchangeably.

[0131] In some embodiments, n is an integer between 1-50, 10-40, or 15-30. In some embodiments, n is 1, 2, 3, 4, 5, 6, or 7, etc. In a preferred embodiment, n is 1 or 2.

[0132] In some embodiments, the incorporation includes inserting n cysteines on the basis of the phase separation-reversing polypeptide. For example, it can be inserted at the N-terminus, C-terminus, and / or non-terminal positions of the phase separation-reversing polypeptide.

[0133] In some embodiments, the n cysteine residues are at the N-terminus and / or C-terminus of the phase separation reversing polypeptide. In some embodiments, the n cysteine residues are at the N-terminus of the phase separation reversing polypeptide.

[0134] In some embodiments, the incorporation includes replacing some amino acid residues (such as non-conserved amino acids) in the phase separation reversing polypeptide with n cysteine residues. For example, one or more consecutive or non-consecutive non-conserved amino acids in the phase separation reversing polypeptide are replaced with n cysteine residues.

[0135] In some embodiments, the incorporation includes inserting cysteine residues and replacing some amino acid residues in the phase separation reversing polypeptide with cysteine residues. Compared with the phase separation reversing polypeptide, the variant after insertion and replacement incorporates n cysteine residues.

[0136] It should be understood that the incorporated n cysteine residues can be consecutive or non-consecutive. For example, when n>1, 1 newly inserted cysteine residue is located at the N-terminus of the phase separation reversing polypeptide, and n-1 newly inserted cysteine residues are located at the C-terminus of the phase separation reversing polypeptide; or, when n>2, 1 newly inserted cysteine residue is located at the N-terminus of the phase separation reversing polypeptide, 1 newly inserted cysteine residue is located at the C-terminus of the phase separation reversing polypeptide, and n-2 consecutive cysteine residues replace one or more consecutive non-conserved amino acids in the phase separation reversing polypeptide.

[0137] In some embodiments, the phase separation reversing polypeptide has one or more of the following characteristics: 1) having a charge of greater than or equal to 30%; 2) having 14%-26% hydrophobic amino acids and greater than or equal to 10% polar amino acids; and 3) having a length of 20-100 amino acids. It should be understood that charged amino acids can be replaced with each other, such as Glu (E) mutated to Asp (D), or Lys (K) or Arg (R), etc.

[0138] In some embodiments, the phase separation reversing polypeptide comprises a charged amphiphilic (Champ) region having a length of 40-100 amino acid residues, wherein in the Champ region: (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys, and at least 80% of the charged amino acid residues are at the N-terminus or C-terminus of the Champ region; (b) 14%-26% of the amino acid residues are hydrophobic amino acid residues selected from Phe, Cys, Leu, Val, and Ile; and (c) at least 10% of the amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, Cys, and His.

[0139] In some embodiments, the phase separation reversing polypeptide comprises a charged amphiphilic (Champ) region having a length of 40-100 amino acid residues, wherein in the Champ region: (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys, and at least 80% of the charged amino acid residues are at the N-terminus or C-terminus of the Champ region; (b) 14%-26% of the amino acid residues are hydrophobic amino acid residues selected from Phe, Leu, Val, Ala, Met, His, Trp, and Ile; and (c) at least 10% of the amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, and Cys.

[0140] In some embodiments, the phase separation reversing polypeptide comprises a hydrophilic segment and a hydrophobic segment. The hydrophilic segment has a length of 10-20 (e.g., 11-19, 12-18, 13-17, 14-16, or 15) amino acid residues, and among these amino acid residues, at least 50% (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%) are Asp, Glu, Lys, and / or Arg. The hydrophobic segment has a length of 10-20 amino acid residues, and among these amino acid residues, at least 30% (e.g., at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%) are Tyr, Phe, Trp, Leu, Ile, Val, Met, Pro, Ala, and / or Cys. The hydrophilic segment is at the N-terminus and the hydrophobic segment is at the C-terminus, or vice versa, and the length of the polypeptide is 20-60 (e.g., 25-55, 30-50, 35-45, or 40) amino acid residues.

[0141] In some embodiments, the hydrophilic segment has a sequence selected from the group consisting of: Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX10X 11 X 12 (SEQ ID NO: 47), X 13 Z2X 14 X 15 X 16 Y4X 17 X 18 X 19 Z3X 20 X 21 X 22 Y5X 23 X 24 X25 (SEQ ID NO: 44), X 26 X 27 X 28 Z4X 29 X 30 X 31 X 32 X 33 Z5X 34 X 35 (SEQ ID NO: 45), and X 36 X 37 Y6X 38 Y7X 39 Z6X 40 (SEQ ID NO: 46); wherein X1 - X 40 are independently selected from the group consisting of Asp, Glu, Lys, and Arg; Y1 - Y7 are independently selected from the group consisting of Thr, Gln, and Ser; and Z1 - Z6 are independently selected from the group consisting of Val, Phe, Leu, and Ile.

[0142] In some embodiments, the hydrophilic segment has the following sequence: Y1X1PY2X3X4Y3X5X6X7VX8X9PX 10 X11X 12 (SEQ ID NO: 43), wherein X1 - X 12 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, and Y1 - Y3 are independently selected from the group consisting of Thr, Gln, and Ser.

[0143] In some embodiments, the length of the hydrophilic segment is 15 - 20 amino acid residues, and among the amino acid residues, up to 55% are Asp, Glu, Lys, and / or Arg, wherein the length of the hydrophobic segment is 10 - 12 amino acid residues, and among the amino acid residues, at least 35% are Tyr, Phe, Leu, Val, Ala, Ile, Met, Trp, or a combination thereof, and wherein the length of the phase separation reversing polypeptide is 21 - 55 amino acid residues.

[0144] In some embodiments, the phase separation reversing polypeptide comprises the following sequence: Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 Y4Y5Y6PX 13 Z2Z3PX 14 X 15 Y7GY8Z4Y9X 16 Y 10 Y 11 Z5Y12 Y 13 X 17 Z6X 18 (SEQ ID NO: 48), wherein X1-X 18 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, Y1-Y 13 are independently selected from the group consisting of Thr, Gln, Asn, Tyr, and Ser, and Z1-Z6 are independently selected from the group consisting of Val, Phe, and Leu.

[0145] In some embodiments, the phase separation reversal polypeptide comprises the following sequence: TX1PQX1X1SX1X1X1VX1X1PX1X1RQQTPX1VVPDDSGTFYDQTVSNDLX1 (SEQ ID NO: 31); wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, or His.

[0146] In some embodiments, the hydrophilic segment has a sequence selected from the group consisting of: TX1PQX1X1SX1X l X1VX1X1PX1X l R (SEQ ID NO: 11); X1LX l X1X1SX1X1X1VX1X1X1QX1X1X1 (SEQ ID NO: 12); X1X1X1VX1X1X1X1X1VX1X1 (SEQ ID NO: 13); and X1X1SX1QX1LX1 (SEQ ID NO: 14), wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, or His.

[0147] In some embodiments, the hydrophilic segment has a sequence selected from the group consisting of: TEPQEESEEEVEEPEER (SEQ ID NO: 15); TDPQDDSDDDVDDPDDR (SEQ ID NO: 16); TKPQKKSKKKVKKPKKR (SEQ ID NO: 17); TRPQRRSRRRVRRPRRR (SEQ ID NO: 18); ELDEESEDEVEEEQEDR (SEQ ID NO: 19); KEEVDEDRDVDE (SEQ ID NO: 20); and EKSEQDLE (SEQ ID NO: 21), or a sequence having at least 90% identity thereto or a sequence having 1, 2, 3, 4, or 5 amino acid residue differences therefrom.

[0148] In some embodiments, the hydrophobic segment has a sequence selected from the following: TFYDQTVSNDL (SEQ ID NO: 22); ANSAYYDAHPVTNGI (SEQ ID NO: 23); PPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 24); and EGEVAEEPNSRP (SEQ ID NO: 25), or a sequence having at least 90% identity thereto or a sequence having 1, 2, 3, 4 or 5 amino acid residue differences therefrom.

[0149] In some embodiments, the phase separation reversal polypeptide comprises a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1), TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2), TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3), TRPQRRSRRRVRRPRRRQQTPRVVPDDSGTFYDQTVSNDLR (SEQ ID NO: 4), ELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIE (SEQ ID NO: 8), KEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 9), EGEVAEEPNSRPQEKSEQDLE (SEQ ID NO: 10), PMAEEEEEEEDEEEEEEEEEEEDEGPAPPSLYPTVQARPG (SEQ ID NO: 35), RSSRRRRRRRRRKQRKVKRESRERNAERMESILQALEDIQ (SEQ ID NO: 38), REKKEKKTKRRKKGEGDGGQKQVEQKSSATLLLTWGLEDV (SEQ ID NO: 42), RKKKKKKTKRRKKGKGDGGQKQVKQKSSATLLLTWGLKDV (SEQ ID NO: 5), PCPGAPCCSLVATGSRVPFSGLKEEEEEDGEDDEEEEEEG (SEQ ID NO: 36), SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37), and LPDVNERIEQFSQEHSVKKKKKKDKHSKKAKKEKKKKSKK (SEQ ID NO: 39); or a sequence having at least 90% identity thereto or a sequence having 1, 2, 3, 4, or 5 amino acid residue differences therefrom.

[0150] In some embodiments, the phase separation reversal polypeptide comprises a hydrophilic segment and a hydrophobic segment. The hydrophilic segment has a length of 10-17 (e.g., 11-16, 12-15, or 13-14) amino acid residues, and at least 60% (e.g., at least 65%, at least 70%, at least 75%, or at least 80%) of these amino acid residues are Asp, Glu, Lys, or Arg. The hydrophobic segment has a length of 10-12 amino acid residues, and at least 35% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%) of these amino acid residues are Tyr, Phe, Leu, or Val. The hydrophilic segment is at the N-terminus and the hydrophobic segment is at the C-terminus, or vice versa. The phase separation reversal polypeptide has a length of 20-29 (e.g., 21-28, 22-27, 23-26, or 24-25) amino acid residues, and the phase separation reversal polypeptide is capable of reversing phase separation.

[0151] In some embodiments, the hydrophilic segment has the following sequence: TX1PQX1X1SX1X1X1VX1X1PX1X1R (SEQ ID NO: 11), where each X1 is Asp, Glu, or Lys.

[0152] In some embodiments, the hydrophobic segment has the following sequence: TFYDQTVSNDL (SEQ ID NO: 22), or a sequence having at least 90% identity thereto or a sequence having 1, 2, 3, 4, or 5 amino acid residue differences therefrom.

[0153] In some embodiments, the phase separation reversal polypeptide comprises the following sequence: TX1PQX1X1SX1X1X1VX1X1PX1X1RQQTPX1VVPDDSGTFYDQTVSNDLX1, where each X1 is Asp, Glu, or Lys.

[0154] It should be understood that hydrophilic amino acids other than Asp, Glu, and Lys (such as Arg, Asn, Gln, His, Ser, and Thr) can also be used as X1 in the above sequences, and similar technical effects can be expected.

[0155] In some embodiments, the phase separation reversal polypeptide comprises a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1), TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2), and TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3).

[0156] The variants provided herein can be identified using the methods described in PCT / CN2021 / 111683, the disclosure of which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the variant further comprises a cell-penetrating peptide (CPP). In some embodiments, the cell-penetrating peptide comprises a sequence selected from the group consisting of: GGRKKRRQRRR (SEQ ID NO: 26) and RQIKIWFQNRRMKWKKK (SEQ ID NO: 27). Other available CPPs that can assist the phase separation reversal polypeptide or its variant provided herein to enter cells are also within the scope of this disclosure. The CPP can be at the N-terminus or C-terminus of the variant provided herein.

[0158] In some embodiments, the variant further comprises the insertion and / or replacement of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0.

[0159] In some embodiments, n is an integer between 1-50, 10-40, or 15-30. In some embodiments, n is 1, 2, 3, 4, 5, 6, or 7, etc.

[0160] In some embodiments, the n cysteine residues are consecutive. In some embodiments, the n cysteine residues are non-consecutive.

[0161] In some embodiments, the variant further comprises a his tag. In some embodiments, the his tag is linked to the variant via a linker.

[0162] In some embodiments, the linker comprises a sequence selected from the group consisting of: SGRPVL (SEQ ID NO: 28), GAPGSAGSAAGGSG (SEQ ID NO: 29), ENLVFQG (SEQ ID NO: 30), and (GSG)n, where n is an integer greater than 0, such as 1.

[0163] The His tag used in the present disclosure can be used to purify polypeptides. In some embodiments, the His tag used in the present disclosure can increase the half-life of the variants provided herein.

[0164] In some embodiments, the variant further comprises an insertion and / or substitution of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0. For example, n can be an integer between 1-50, 10-40, or 15-30. In some embodiments, n is 1, 2, 3, 4, 5, 6, or 7, etc. In some embodiments, n is 1 or 2.

[0165] In some embodiments, the variant sequentially contains a phase separation reversal polypeptide, optionally a cell-penetrating peptide, optionally a linker, and optionally a His tag from the N-terminus to the C-terminus, where the n cysteine residues are at the N-terminus or C-terminus of the variant.

[0166] In some embodiments, the variant sequentially contains a phase separation reversal polypeptide, a cell-penetrating peptide, a linker, and a his-tag from the N-terminus to the C-terminus, wherein the n cysteine residues are at the N-terminus or C-terminus of the variant; the phase separation reversal polypeptide comprises a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1), TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2), TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3), TRPQRRSRRRVRRPRRRQQTPRVVPDDSGTFYDQTVSNDLR (SEQ ID NO: 4), ELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIE (SEQ ID NO: 8), KEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 9), EGEVAEEPNSRPQEKSEQDLE (SEQ ID NO: 10), PMAEEEEEEEDEEEEEEEEEEEDEGPAPPSLYPTVQARPG (SEQ ID NO: 35), RSSRRRRRRRRRKQRKVKRESRERNAERMESILQALEDIQ (SEQ ID NO: 38), REKKEKKTKRRKKGEGDGGQKQVEQKSSATLLLTWGLEDV (SEQ ID NO: 42), RKKKKKKTKRRKKGKGDGGQKQVKQKSSATLLLTWGLKDV (SEQ ID NO: 5), PCPGAPCCSLVATGSRVPFSGLKEEEEEDGEDDEEEEEEG (SEQ ID NO: 36), SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37), and LPDVNERIEQFSQEHSVKKKKKKDKHSKKAKKEKKKKSKK (SEQ ID NO: 39); the cell-penetrating peptide comprises a sequence selected from the group consisting of: GGRKKRRQRRR (SEQ ID NO: 26), RQIKIWFQNRRMKWKKK (SEQ ID NO: 27);and the linker comprises a sequence selected from the group consisting of: SGRPVL (SEQ ID NO: 28), GAPGSAGSAAGGSG (SEQ ID NO: 29), ENLVFQG (SEQ ID NO: 30), and GSG.;

[0167] In some embodiments, the variant has a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 6), CTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 7), TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLDGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 32), CTDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLDGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 33), TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 34), CTEPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 40), CTEPQEESEEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRHHHHHHHH (SEQ ID NO: 41), TEPQEESEEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRHHHHHHHHC (SEQ ID NO: 49), CRQIKIWFQNRRMKWKKKTKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKSSGRPVLHHHHHHH (SEQ ID NO: 50); RQIKIWFQNRRMKWKKKTKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKSSGRPVLHHHHHHHC (SEQ ID NO: 51); CCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 52); CCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 53);and SCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 54).;

[0168] Polynucleotides, Vectors, Host Cells, Engineered Cells

[0169] The variants provided herein can be produced by culturing a host cell (e.g., a eukaryotic or prokaryotic cell) containing the polynucleotide provided herein under conditions that permit expression of the polynucleotide provided herein. The polynucleotides provided herein can be constructed using a cell-free protein synthesis (CFPS) system. The polynucleotides provided herein can be constructed using recombinant techniques. To this end, DNA encoding the phase separation reverse transcription polypeptide provided herein and DNA encoding a CPP, linker, and / or his tag can be obtained and operably linked to permit transcription and expression in a host cell to produce the variant.

[0170] To produce the variants provided herein, host cells transformed with an expression vector can be cultured in a variety of media. Commercially available bacterial growth media (such as Terrific Broth, LB Broth, LB Agar, M9 Minimal Medium, MagiaMedia Medium, and ImMedia Medium (ThermoFisher)) are suitable for culturing bacterial host cells. Commercially available media (such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma)) are suitable for culturing eukaryotic host cells. Any of these media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds whose final concentrations are typically in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. The culture conditions (such as temperature, pH, etc.) are those previously used with the selected host cell for expression and will be apparent to one of ordinary skill in the art.

[0171] Variants provided herein prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.

[0172] Depending on the protein to be recovered, other techniques for protein purification are also available, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE TM chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0173] In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is single-stranded DNA or double-stranded DNA.

[0174] On the other hand, the present disclosure provides a host cell comprising the vector provided herein. The host cell is a prokaryotic cell or a eukaryotic cell. Host cells transformed with the above expression or cloning vectors can be cultured in a conventional nutrient medium, which is modified as needed to induce the promoter, select transformants, or amplify the cloning vector.

[0175] In some embodiments, the vector is a viral vector.

[0176] In some embodiments, the vector further comprises additional elements that promote polypeptide expression, such as promoters, enhancers, polyA regions, etc. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector further comprises ITR sequences.

[0177] In some embodiments, the AAV has a serotype selected from AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhl0, AAV11, and AAV12.

[0178] On the other hand, the present disclosure provides an engineered cell comprising the variant, polynucleotide, and / or vector provided herein.

[0179] Pharmaceutical Compositions

[0180] On the other hand, the present disclosure provides a pharmaceutical composition for treating a phase separation disease in a subject in need thereof, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a variant provided herein, a polynucleotide encoding the phase separation reversing polypeptide, a vector comprising the polynucleotide, a host cell comprising the vector, and / or an engineered cell comprising the variant, the polynucleotide, and / or the vector.

[0181] As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable excipient, binder, carrier, and / or diluent for directly or indirectly delivering a variant of the phase separation reversing polypeptide provided herein, a polynucleotide encoding the variant, and / or a vector comprising the polynucleotide to a diseased tissue or near it. The pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, the pharmaceutically acceptable carrier can be water. In addition, saline solutions and aqueous solutions of glucose, glycerol, etc. can also be used as pharmaceutically acceptable carriers in liquid form, and this form of carrier is commonly used in injection solutions.

[0182] The pharmaceutical composition can have different forms depending on the disease being treated. For example, when the pharmaceutical composition is an ophthalmic pharmaceutical composition, it can be in the form of eye drops, and the eye drops can be prepared using aqueous solutions and diluents, including but not limited to distilled water, physiological saline, etc. The pharmaceutical composition can contain various additives as needed. These additives can include but are not limited to additional ingredients, additives, or carriers suitable for contacting the diseased tissue or its surroundings without excessive toxicity, incompatibility, emollients, instability, irritation, isotonicity regulators, allergic reactions, etc. Additives (such as solvents, bases, solubilizing aids, suspending agents, thickening agents, emulsifying agents, stabilizers, buffers, pH regulators, flavoring agents, chelating agents, preservatives, flavoring agents, coloring agents, excipients, binders, lubricants, surfactants, absorption promoters, dispersants, preservatives, solubilizing agents, etc.) can be added to the formulation as appropriate.

[0183] Kits

[0184] On the other hand, the present disclosure provides a kit for treating a phase separation-related disease (e.g., cataract), the kit comprising a therapeutically effective amount of a formulation of a variant provided herein, a polynucleotide provided herein, and / or a vector provided herein, a pharmaceutically acceptable carrier, and instructions for administering the formulation such that the administration treats the phase separation-related disease.

[0185] In some embodiments, the kit comprises one or more containers that contain one or more of the variants provided herein, the polynucleotides provided herein, and / or the vectors provided herein. The variants provided herein, the polynucleotides provided herein, and / or the vectors provided herein may be present in the container as a formulated pharmaceutical composition or, alternatively, may be unformulated. In such embodiments, the kit may include the variants provided herein, the polynucleotides provided herein, and / or the vectors provided herein that are unformulated in a container, separate from the pharmaceutically acceptable carrier present in another container. Prior to use, the variants provided herein, the polynucleotides provided herein, and / or the vectors provided herein are diluted or otherwise mixed with a pharmaceutically acceptable carrier.

[0186] In some embodiments, the kit provided herein further comprises instructions that describe a method of administering the pharmaceutical composition in a manner related to one or more symptoms associated with a disease associated with phase separation (e.g., cataract). In some embodiments, the instructions further describe a procedure for mixing the variants provided herein, the polynucleotides provided herein, and / or the vectors provided herein included in the kit with a pharmaceutically acceptable carrier.

[0187] III. Methods for Enhancing the Phase Separation-Reversing Ability of Phase Separation-Reversing Polypeptides

[0188] The present disclosure provides a method for enhancing the phase separation reversal ability of a phase separation reversing polypeptide, wherein the method comprises incorporating n cysteine residues into the phase separation reversing polypeptide; wherein n is an integer greater than or equal to 0.

[0189] In some embodiments, n is an integer between 1-50, 10-40, or 15-30. In some embodiments, n is 1, 2, 3, 4, 5, 6, or 7, etc. In some embodiments, n is 1 or 2.

[0190] In some embodiments, the incorporation comprises inserting n cysteines on the basis of the phase separation reversing polypeptide. For example, it can be inserted at the N-terminus, C-terminus, and / or non-terminal positions of the phase separation reversing polypeptide. In some embodiments, the incorporation comprises inserting 1 or 2 cysteines at the N-terminus of the phase separation reversing polypeptide.

[0191] In some embodiments, the n cysteine residues are at the N-terminus and / or C-terminus of the phase separation reversing polypeptide. In some embodiments, the n cysteine residues are at the N-terminus of the phase separation reversing polypeptide.

[0192] In some embodiments, the incorporation includes replacing some amino acid residues (e.g., non-conserved amino acids) in the phase separation reversing polypeptide with n cysteines. For example, one or more consecutive or non-consecutive non-conserved amino acids in the phase separation reversing polypeptide are replaced with n cysteine residues.

[0193] In some embodiments, the incorporation simultaneously includes the insertion and replacement of cysteine residues.

[0194] For the relevant definitions and preferred limitations of the phase separation reversing polypeptide in this regard, see the relevant content in "II. Variants of Phase Separation Reversing Polypeptides". Although the present disclosure verifies that incorporating cysteine residues based on one phase separation reversing polypeptide can improve its phase separation reversing ability, it should be understood that incorporating cysteine residues in a similar manner based on other phase separation reversing polypeptides described in "II. Variants of Phase Separation Reversing Polypeptides" can also improve the phase separation reversing ability of the original phase separation reversing polypeptide. Moreover, the amino acid sequence of the original phase separation reversing polypeptide verified in the present disclosure contains the cell-penetrating peptide GGRKKRRQRRR for penetrating cell membranes, GSG as a linker, and His-tag for polypeptide purification and / or extending the half-life. Without wishing to be bound by theory, it should be understood that the functions of the cell-penetrating peptide, linker, and His-tag are independent of the phase separation reversing function of the phase separation reversing polypeptide. Therefore, whether to include the cell-penetrating peptide, linker, and / or His-tag and what kind of cell-penetrating peptide, linker, and / or His-tag to include will not affect the phase separation reversing function of the polypeptide, nor will it be related to the effect on the phase separation reversing ability of the phase separation reversing polypeptide after incorporating cysteine.

[0195] IV. Methods for Treating Phase Separation-Related Diseases

[0196] On the other hand, the present disclosure provides a method for treating a subject in need thereof for a phase separation-related disease, the method comprising administering to the subject in need thereof a therapeutically effective amount of a variant of a phase separation-reversing polypeptide, a polynucleotide encoding the phase separation-reversing polypeptide, a vector comprising the polynucleotide, a host cell comprising the vector, an engineered cell comprising the variant, the polynucleotide, and / or the vector, or a pharmaceutical composition comprising the variant, the polynucleotide, the vector, and / or the engineered cell. In certain embodiments, the phase separation-related diseases are selected from the group consisting of phase separation-related visual impairments (e.g., cataracts), age-related macular degeneration, and neurodegenerative diseases (e.g., frontotemporal dementia, amyotrophic lateral sclerosis, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease, Lewy body disease, motor neuron disease, frontotemporal lobar degeneration, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease, argentophilic grain disease, Alzheimer's disease, Kii Peninsula Parsons / dementia syndrome, progressive supranuclear palsy, Pick's disease, and prion disease).

[0197] On the other hand, the present disclosure provides a method for reversing, inhibiting, reducing, and / or preventing abnormal protein aggregation or abnormal metabolite generation in a cell, the method comprising introducing into the cell a variant of a phase separation-reversing polypeptide, a polynucleotide encoding the variant, and / or a vector comprising the polynucleotide.

[0198] On the other hand, the present disclosure provides a method for dissolving protein aggregates (e.g., crystalline aggregates (e.g., γD-crystallin aggregates), stress granule protein aggregates (G3BP1 protein aggregates)) and / or preventing the formation of crystalline protein aggregates (e.g., crystalline aggregates (e.g., γD-crystallin aggregates), stress granule protein aggregates (G3BP1 protein aggregates)), the method comprising contacting the protein aggregates with a variant of a phase separation-reversing polypeptide.

[0199] On the other hand, the present disclosure provides a method for dissolving protein aggregates (e.g., crystalline aggregates (e.g., γD-crystallin aggregates), stress granule protein aggregates (G3BP1 protein aggregates)) and / or preventing the formation of crystalline protein aggregates (e.g., crystalline aggregates (e.g., γD-crystallin aggregates), stress granule protein aggregates (G3BP1 protein aggregates)) in a cell, the method comprising introducing into the cell a variant of a phase separation-reversing polypeptide. As used herein, the term "G3BP1" refers to an adjustable switch that triggers phase separation to assemble stress granules, e.g., triggering RNA-dependent liquid-liquid phase separation (LLPS) in response to an increase in the intracellular free RNA concentration.

[0200] Exemplary embodiments of the present application include, but are not limited to, the following:

[0201] Item 1. A variant of a phase separation reversing polypeptide, wherein the amino acid sequence of the variant is the amino acid sequence after incorporating n cysteine residues on the basis of the phase separation reversing polypeptide, and the variant has equivalent or enhanced phase separation reversing ability compared with the phase separation reversing polypeptide; wherein n is an integer greater than or equal to 0 (for example, n is 1 or 2).

[0202] Item 2. The variant according to Item 1, wherein the incorporation includes insertion and / or substitution.

[0203] Item 3. The variant according to Item 1 or 2, wherein the n cysteine residues are consecutive or non - consecutive.

[0204] Item 4. The variant according to any one of the foregoing items, wherein the n cysteine residues are at the N - terminus and / or C - terminus of the phase separation reversing polypeptide (preferably, the n cysteine residues are at the N - terminus of the phase separation reversing polypeptide).

[0205] Item 5. The variant according to any one of the foregoing items, wherein the phase separation reversing polypeptide comprises a charged amphiphilic (Champ) region having a length of 40 - 100 amino acid residues, wherein in the Champ region,

[0206] (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys,

[0207] wherein at least 80% of the charged amino acid residues are at the N - terminus or C - terminus of the Champ region,

[0208] (b) 14% - 26% of the amino acid residues are hydrophobic amino acid residues selected from Phe, Cys, Leu, Val, and Ile, and

[0209] (c) at least 10% of the amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, Cys, and His;

[0210] Or, in the Champ region,

[0211] (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys,

[0212] wherein at least 80% of the charged amino acid residues are at the N - terminus or C - terminus of the Champ region,

[0213] (b) 14% - 26% of said amino acid residues are hydrophobic amino acid residues selected from Phe, Leu, Val, Ala, Met, His, Trp, and Ile, and

[0214] (c) At least 10% of said amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, and Cys.

[0215] Item 6. The variant according to any one of the preceding items, wherein said phase separation reversing polypeptide comprises a hydrophilic segment and a hydrophobic segment,

[0216] wherein the length of said hydrophilic segment is 10 - 20 amino acid residues, and among said amino acid residues, at least 50% are Asp, Glu, Lys, and / or Arg,

[0217] wherein the length of said hydrophobic segment is 10 - 20 amino acid residues, and among said amino acid residues, at least 50% are Tyr, Phe, Trp, Leu, Ile, Val, Met, Pro, Ala, and / or Cys,

[0218] wherein said hydrophilic segment is at the N - terminus and said hydrophobic segment is at the C - terminus, or vice versa,

[0219] wherein the length of said phase separation reversing polypeptide is 20 - 60 amino acid residues.

[0220] Item 7. The variant according to Item 6, wherein said hydrophilic segment has a sequence selected from the group consisting of:

[0221] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 (SEQ ID NO: 47), X 13 Z2X 14 X 15 X 16 Y4X 17 X 18 X 19 Z3X20X 21 X 22 Y5X 23 X 24 X 25 (SEQ ID NO: 44), X 26 X 27 X 28 Z4X 29 X 30 X 31 X 32 X 33 Z5X34 X 35 (SEQ ID NO: 45), and X 36 X 37 Y6X 38 Y7X 39 Z6X 40 (SEQ ID NO: 46);

[0222] wherein X1 - X 40 are independently selected from the group consisting of Asp, Glu, Lys, and Arg; Y1 - Y7 are independently selected from the group consisting of Thr, Gln, and Ser; and Z1 - Z6 are independently selected from the group consisting of Val, Phe, Leu, and Ile.

[0223] Item 8. The variant according to item 6 or 7, wherein the hydrophilic segment has the following sequence:

[0224] Y1X1PY2X3X4Y3X5X6X7VX8X9PX 10 X 11 X 12 (SEQ ID NO: 43), wherein X1 - X 12 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, and Y1 - Y3 are independently selected from the group consisting of Thr, Gln, and Ser.

[0225] Item 9. The variant according to any one of items 6 - 8, wherein the hydrophilic segment has a sequence selected from the group consisting of: TX1PQX1X1SX1X1X1VX1X1PX1X1R (SEQ ID NO: 11), X1LX1X1X1SX1X1X1VX1X1X1QX1X1X1 (SEQ ID NO: 12), X1X1X1VX1X1X1X1X1VX1X1 (SEQ ID NO: 13), and X1X1SX1QX1LX1 (SEQ ID NO: 14); wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, His, or a combination thereof.

[0226] Item 10. The variant according to item 6, wherein the hydrophilic segment has a length of 15 - 20 amino acid residues, and among the amino acid residues, up to 55% are Asp, Glu, Lys, and / or Arg,

[0227] wherein the hydrophobic segment has a length of 10 - 12 amino acid residues, and among the amino acid residues, at least 35% are Tyr, Phe, Leu, Val, Ala, Ile, Met, Trp, or a combination thereof, and

[0228] The length of the phase separation reversing polypeptide is 21-55 amino acid residues.

[0229] Item 11. The variant according to item 10, wherein the phase separation reversing polypeptide comprises the following sequence:

[0230] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 Y4Y5Y6PX 13 Z2Z3PX 14 X 15 Y7GY8Z4Y9X 16 Y 10 Y 11 Z5Y 12 Y13X 17 Z6X 18 (SEQ ID NO: 48),

[0231] wherein X1-X 18 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, Y1-Y 13 are independently selected from the group consisting of Thr, Gln, Asn, Tyr, and Ser, and Z1-Z6 are independently selected from the group consisting of Val, Phe, and Leu.

[0232] Item 12. The variant according to item 10 or 11, wherein the phase separation reversing polypeptide comprises the following sequence:

[0233] TX1PQX1X1SX1X1X1VX1X1PX1X1RQQTPX1VVPDDSGTFYDQTVSNDLX1(SEQ ID NO: 31);

[0234] wherein each X1 is respectively Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, or His.

[0235] Item 13. The variant according to any one of Items 6 - 12, wherein the hydrophilic segment has a sequence selected from the group consisting of: TEPQEESEEEVEEPEER (SEQ ID NO: 15); TDPQDDSDDDVDDPDDR (SEQ ID NO: 16); TKPQKKSKKKVKKPKKR (SEQ ID NO: 17); TRPQRRSRRRVRRPRRR (SEQ ID NO: 18); ELDEESEDEVEEEQEDR (SEQ ID NO: 19); KEEVDEDRDVDE (SEQ ID NO: 20); and EKSEQDLE (SEQ ID NO: 21).

[0236] Item 14. The variant according to any one of Items 6 - 13, wherein the hydrophobic segment has a sequence selected from the group consisting of: TFYDQTVSNDL (SEQ ID NO: 22); ANSAYYDAHPVTNGI (SEQ ID NO: 23); PPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 24); and EGEVAEEPNSRP (SEQ ID NO: 25).

[0237] Item 15. The variant according to any one of claims 6-14, wherein the phase separation reversing polypeptide comprises a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1); TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2); TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3); TRPQRRSRRRVRRPRRRQQTPRVVPDDSGTFYDQTVSNDLR (SEQ ID NO: 4); ELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIE (SEQ ID NO: 8); KEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 9); EGEVAEEPNSRPQEKSEQDLE (SEQ ID NO: 10); PMAEEEEEEEDEEEEEEEEEEEDEGPAPPSLYPTVQARPG (SEQ ID NO: 35); RSSRRRRRRRRKQRKVKRESRERNAERMESILQALEDIQ (SEQ ID NO: 38); REKKEKKTKRRKKGEGDGGQKQVEQKSSATLLLTWGLEDV (SEQ ID NO: 42); RKKKKKKTKRRKKGKGDGGQKQVKQKSSATLLLTWGLKDV (SEQ ID NO: 5); PCPGAPCCSLVATGSRVPFSGLKEEEEEDGEDDEEEEEEG (SEQ ID NO: 36); SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37); and LPDVNERIEQFSQEHSVKKKKKKDKHSKKAKKEKKKKSKK (SEQ ID NO: 39).

[0238] Item 16. The variant according to any one of the preceding items, wherein the variant further comprises a cell-penetrating peptide.

[0239] Item 17. The variant according to item 16, wherein the cell-penetrating peptide comprises a sequence selected from the group consisting of: GGRKKRRQRRR (SEQ ID NO: 26); RQIKIWFQNRRMKWKKK (SEQ ID NO: 27).

[0240] Item 18. The variant according to item 16 or 17, wherein the cell-penetrating peptide is at the N-terminus or C-terminus of the variant.

[0241] Item 19. The variant according to item 18, wherein the variant further comprises an insertion and / or substitution of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0.

[0242] Item 20. The variant according to item 19, wherein the n cysteine residues are consecutive or non-consecutive.

[0243] Item 21. The variant according to any one of the preceding items, wherein the variant further comprises a his-tag.

[0244] Item 22. The variant according to item 21, wherein the his-tag is linked to the variant via a linker.

[0245] Item 23. The variant according to item 22, wherein the linker comprises a sequence selected from the group consisting of:

[0246] SGRPVL (SEQ ID NO: 28);

[0247] GAPGSAGSAAGGSG (SEQ ID NO: 29);

[0248] ENLVFQG (SEQ ID NO: 30); and

[0249] (GSG)n, where n is an integer greater than 0, for example 1.

[0250] Item 24. The variant according to any one of items 21 - 23, wherein the variant further comprises an insertion and / or substitution of n cysteine residues at its N-terminus or C-terminus; where n is an integer greater than or equal to 0.

[0251] Item 25. The variant according to any one of the preceding items, wherein the variant sequentially contains a phase separation reversal polypeptide, optionally a cell-penetrating peptide, optionally a linker, and optionally a his-tag from the N-terminus to the C-terminus, where the n cysteine residues are at the N-terminus or C-terminus of the variant; wherein,

[0252] the phase separation reversal polypeptide comprises a sequence selected from the group consisting of:

[0253] TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1), TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2), TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3), TRPQRRSRRRVRRPRRRQQTPRVVPDDSGTFYDQTVSNDLR (SEQ ID NO: 4), ELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIE (SEQ ID NO: 8), KEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 9), EGEVAEEPNSRPQEKSEQDLE (SEQ ID NO: 10), PMAEEEEEEEDEEEEEEEEEEEDEGPAPPSLYPTVQARPG (SEQ ID NO: 35), RSSRRRRRRRRRKQRKVKRESRERNAERMESILQALEDIQ (SEQ ID NO: 38), REKKEKKTKRRKKGEGDGGQKQVEQKSSATLLLTWGLEDV (SEQ ID NO: 42), RKKKKKKTKRRKKGKGDGGQKQVKQKSSATLLLTWGLKDV (SEQ ID NO: 5), PCPGAPCCSLVATGSRVPFSGLKEEEEEDGEDDEEEEEEG (SEQ ID NO: 36), SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37), and LPDVNERIEQFSQEHSVKKKKKKDKHSKKAKKEKKKKSKK (SEQ ID NO: 39);

[0254] The cell-penetrating peptide comprises a sequence selected from the group consisting of: GGRKKRRQRRR (SEQ ID NO: 26), RQIKIWFQNRRMKWKKK (SEQ ID NO: 27); and

[0255] The linker comprises a sequence selected from the group consisting of: SGRPVL (SEQ ID NO: 28), GAPGSAGSAAGGSG (SEQ ID NO: 29), ENLVFQG (SEQ ID NO: 30), and GSG.

[0256] Item 26. The variant according to item 25, wherein the variant has a sequence selected from the group consisting of:

[0257] TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 6); CTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 7); TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLDGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 32); CTDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLDGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 33); TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 34); CTKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 40); CTEPQEESEEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRHHHHHHHH (SEQ ID NO: 41); TEPQEESEEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRHHHHHHHHC (SEQ ID NO: 49); CRQIKIWFQNRRMKWKKKTKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKSSGRPVLHHHHHHH (SEQ ID NO: 50); RQIKIWFQNRRMKWKKKTKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLKSSGRPVLHHHHHHHC (SEQ ID NO: 51); CCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ IDNO: 52); CCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 53);and SCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 54);

[0258] Item 27. A polynucleotide encoding a variant according to any one of Items 1 - 26.

[0259] Item 28. The polynucleotide according to Item 27, which is DNA or RNA.

[0260] Item 29. A vector comprising the polynucleotide according to Item 27 or 28.

[0261] Item 30. The vector according to Item 29, which is a viral vector, such as a lentiviral vector, a retroviral vector or an AAV vector (such as an AAV2 vector).

[0262] Item 31. A host cell comprising the vector according to Item 29 or 30.

[0263] Item 32. An engineered cell comprising a variant according to any one of Items 1 - 26, a polynucleotide according to Item 27 or 28, and / or a vector according to Item 29 or 30.

[0264] Item 33. A pharmaceutical composition comprising a variant according to any one of Items 1 - 26, a polynucleotide according to Item 27 or 28, a vector according to Item 29 or 30, a host cell according to Item 31, or an engineered cell according to Item 32, and a pharmaceutically acceptable carrier.

[0265] Item 34. A kit comprising a variant according to any one of Items 1 - 26, a polynucleotide according to Item 27 or 28, a vector according to Item 29 or 30, a formulation of a pharmaceutically acceptable carrier, and instructions for administering the formulation such that the administration treats the phase separation-related disease.

[0266] Item 35. A method for enhancing the phase separation reversal ability of a phase separation-reversing polypeptide, wherein the method comprises incorporating n cysteine residues into the phase separation-reversing polypeptide; where n is an integer greater than or equal to 0.

[0267] Item 36. The method according to Item 35, wherein the incorporation comprises insertion and / or replacement.

[0268] Item 37. The method according to Item 35 or 36, wherein the n cysteine residues are consecutive or non-consecutive.

[0269] Item 38. The method according to any one of Items 35 - 37, wherein the n cysteine residues are at the N-terminus and / or C-terminus of the phase separation reversing polypeptide.

[0270] Item 39. The method according to any one of Items 35 - 38, wherein the phase separation reversing polypeptide comprises a charged amphiphilic (Champ) region having a length of 40 - 100 amino acid residues, wherein in the Champ region,

[0271] (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys,

[0272] wherein at least 80% of the charged amino acid residues are at the N-terminus or C-terminus of the Champ region,

[0273] (b) 14% - 26% of the amino acid residues are hydrophobic amino acid residues selected from Phe, Cys, Leu, Val, and Ile, and

[0274] (c) at least 10% of the amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, Cys, and His;

[0275] Alternatively, in the Champ region,

[0276] (a) at least 30% of the amino acid residues are charged amino acid residues selected from Asp, Glu, Arg, and Lys,

[0277] wherein at least 80% of the charged amino acid residues are at the N-terminus or C-terminus of the Champ region,

[0278] (b) 14% - 26% of the amino acid residues are hydrophobic amino acid residues selected from Phe, Leu, Val, Ala, Met, His, Trp, and Ile, and

[0279] (c) at least 10% of the amino acid residues are polar amino acid residues selected from Ser, Asn, Thr, Gln, Tyr, and Cys.

[0280] Item 40. The method according to any one of Items 35 - 39, wherein the phase separation reversing polypeptide comprises a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment has a length of 10 - 20 amino acid residues, and among the amino acid residues, at least 50% are Asp, Glu, Lys, and / or Arg; wherein the hydrophobic segment has a length of 10 - 20 amino acid residues, and among the amino acid residues, at least 50% are Tyr, Phe, Trp, Leu, Ile, Val, Met, Pro, Ala, and / or Cys, wherein the hydrophilic segment is at the N-terminus and the hydrophobic segment is at the C-terminus, or vice versa, and the length of the phase separation reversing polypeptide is 20 - 60 amino acid residues.

[0281] Item 41. The method according to Item 40, wherein the hydrophilic segment has a sequence selected from the group consisting of:

[0282] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 (SEQ ID NO: 47), X 13 Z2X 14 X 15 X 16 Y4X 17 X 18 X 19 Z3X20X 21 X 22 Y5X 23 X 24 X 25 (SEQ ID NO: 44), X 26 X 27 X 28 Z4X 29 X 30 X 31 X 32 X 33 Z5X 34 X 35 (SEQ ID NO: 45), and X 36 X 37 Y6X 38 Y7X 39 Z6X 40 (SEQ ID NO: 46);

[0283] wherein X1 - X 40 are independently selected from the group consisting of Asp, Glu, Lys, and Arg; Y1 - Y7 are independently selected from the group consisting of Thr, Gln, and Ser; and Z1 - Z6 are independently selected from the group consisting of Val, Phe, Leu, and Ile.

[0284] Item 42. The method according to Item 40 or 41, wherein the hydrophilic segment has the following sequence:

[0285] Y1X1PY2X3X4Y3X5X6X7VX8X9PX 10 X 11 X 12 (SEQ ID NO: 43),

[0286] wherein X1 - X 12 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, and Y1 - Y3 are independently selected from the group consisting of Thr, Gln, and Ser.

[0287] Item 43. The method according to any one of Items 40 - 42, wherein the hydrophilic segment has a sequence selected from the group consisting of:

[0288] TX1PQX1X1SX1X1X1VX1X1PX1X1R (SEQ ID NO: 11), X1LX1X1X1SX1X1X1VX1X1X1QX1X1X1 (SEQ ID NO: 12), X1X1X1VX1X1X1X1X1VX1X1 (SEQ ID NO: 13), and X1X1SX1QX1LX1 (SEQ ID NO: 14);

[0289] wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg, His, or a combination thereof.

[0290] Item 44. The method according to any one of Items 36 - 39, wherein the hydrophilic segment has a sequence selected from the group consisting of:

[0291] TEPQEESEEEVEEPEER (SEQ ID NO: 15); TDPQDDSDDDVDDPDDR (SEQ ID NO: 16); TKPQKKSKKKVKKPKKR (SEQ ID NO: 17); TRPQRRSRRRVRRPRRR (SEQ ID NO: 18); ELDEESEDEVEEEQEDR (SEQ ID NO: 19); KEEVDEDRDVDE (SEQ ID NO: 20); and EKSEQDLE (SEQ ID NO: 21).

[0292] Item 45. The method according to any one of Items 36-44, wherein the hydrophobic segment has a sequence selected from the group consisting of: TFYDQTVSNDL (SEQ ID NO: 22); ANSAYYDAHPVTNGI (SEQ ID NO: 23); PPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 24); and EGEVAEEPNSRP (SEQ ID NO: 25).

[0293] Item 46. The method according to Item 36, wherein the hydrophilic segment has a length of 15-20 amino acid residues, among which up to 55% are Asp, Glu, Lys, and / or Arg, wherein the hydrophobic segment has a length of 10-12 amino acid residues, among which at least 35% are Tyr, Phe, Leu, Val, Ala, Ile, Met, Trp, or a combination thereof, and wherein the length of the phase separation reversing polypeptide is 21-55 amino acid residues.

[0294] Item 47. The method according to Item 46, wherein the phase separation reversing polypeptide comprises the following sequence:

[0295] Y1X1PY2X3X4Y3X5X6X7Z1X8X9PX 10 X 11 X 12 Y4Y5Y6PX 13 Z2Z3PX 14 X 15 Y7GY8Z4Y9X 16 Y 10 Y 11 Z5Y 12 Y13X 17 Z6X 18 (SEQ ID NO: 48), wherein X1-X 18 are independently selected from the group consisting of Asp, Glu, Lys, and Arg, Y1-Y 13 are independently selected from the group consisting of Thr, Gln, Asn, Tyr, and Ser, and Z1-Z6 are independently selected from the group consisting of Val, Phe, and Leu.

[0296] Item 48. The method according to Item 46 or 47, wherein the phase separation reversing polypeptide comprises the following sequence: TX1PQX1X1SX1X1X1VX1X1PX1X1RQQTPX1VVPDDSGTFYDQTVSNDLX1 (SEQ ID NO: 31); wherein each X1 is independently Asp, Glu, Lys, Ser, Thr, Cys, Asn, Gln, Arg or His.

[0297] Item 49. The method according to any one of Items 35-48, wherein the phase separation reversing polypeptide comprises a sequence selected from the group consisting of: TEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLE (SEQ ID NO: 1); TDPQDDSDDDVDDPDDRQQTPDVVPDDSGTFYDQTVSNDLD (SEQ ID NO: 2); TKPQKKSKKKVKKPKKRQQTPKVVPDDSGTFYDQTVSNDLK (SEQ ID NO: 3); TRPQRRSRRRVRRPRRRQQTPRVVPDDSGTFYDQTVSNDLR (SEQ ID NO: 4); ELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIE (SEQ ID NO: 8); KEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPV (SEQ ID NO: 9); EGEVAEEPNSRPQEKSEQDLE (SEQ ID NO: 10); PMAEEEEEEEDEEEEEEEEEEEDEGPAPPSLYPTVQARPG (SEQ ID NO: 35); RSSRRRRRRRRRKQRKVKRESRERNAERMESILQALEDIQ (SEQ ID NO: 38); REKKEKKTKRRKKGEGDGGQKQVEQKSSATLLLTWGLEDV (SEQ ID NO: 42); RKKKKKKTKRRKKGKGDGGQKQVKQKSSATLLLTWGLKDV (SEQ ID NO: 5); PCPGAPCCSLVATGSRVPFSGLKEEEEEDGEDDEEEEEEG (SEQ ID NO: 36); SNRLMMLRMHNLHGTKPPPSEGSDEEEEEEDEEDEEERKP (SEQ ID NO: 37); and LPDVNERIEQFSQEHSVKKKKKKDKHSKKAKKEKKKKSKK (SEQ ID NO: 39).

[0298] Item 50. A method for treating a phase separation-related disease in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a variant according to any one of Items 1-26, a polynucleotide according to Item 27 or 28, a vector according to Item 29 or 30, a host cell according to Item 31, an engineered cell according to Item 32, and / or a pharmaceutical composition according to Item 33.

[0299] Item 51. The method according to Item 50, wherein the phase separation-related disease is selected from the group consisting of phase separation-related visual impairment, age-related macular degeneration, and neurodegenerative diseases.

[0300] Item 52. The method according to Item 51, wherein the neurodegenerative disease is selected from the group consisting of frontotemporal dementia, amyotrophic lateral sclerosis, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease, Lewy body disease, motor neuron disease, frontotemporal lobar degeneration, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease, argentophilic grain disease, Alzheimer's disease, Parsons / dementia syndrome of the Kii Peninsula, progressive supranuclear palsy, Pick's disease, and prion disease.

[0301] Item 53. A method for reversing, inhibiting, reducing, and / or preventing abnormal protein aggregation or abnormal metabolite generation in a cell, comprising introducing into the cell a variant according to any one of Items 1-26, a polynucleotide according to Item 27 or 28, or a vector according to Item 29 or 30.

[0302] The following examples are provided to better illustrate the claimed invention and should not be construed in any way as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention but merely to illustrate specific embodiments falling within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive faculty and without departing from the scope of the invention. It should be understood that many variations can be made in the procedures described herein while still remaining within the scope of the invention. The intention of the inventors of the present invention is that such variations are all included within the scope of the invention.

[0303] V. Examples

[0304] Amino acid sequence of variant 2016#:

[0305]

[0306] Amino acid sequence of variant 2017#:

[0307]

[0308] Amino acid sequence of Variant 2019#:

[0309]

[0310] Amino acid sequence of Variant 2020#:

[0311]

[0312] Amino acid sequence of Variant 2021#:

[0313]

[0314] Amino acid sequence of phase separation reversal polypeptide 0009#:

[0315]

[0316] Those skilled in the art can understand that the methionine (M) at the N-terminus of the above-listed amino acid sequences is encoded by the start codon, but will be cleaved by enzymes during protein maturation. In some embodiments, the above amino acid sequences may not include the methionine (M) at the N-terminus. However, the codon encoding methionine (M) is necessary during translation. Therefore, in embodiments regarding transfection of plasmids in cell experiments, the coding sequence of the phase separation reversal polypeptide includes the codon encoding methionine (M).

[0317] Example 1: Determination of in vitro depolymerization ability

[0318] 1.1 In vitro cell-free protein synthesis system

[0319] An in vitro cell-free protein synthesis system was used. After separately expressing the phase separation protein and the phase separation reversal protein or its variants in the system, they were mixed.

[0320] Experimental group: Various variants of phase separation reversal polypeptide 0009#

[0321] Blank control group: Phase separation reversal polypeptide 0009#.

[0322] Negative control group (NC): No phase separation reversal polypeptide was added to the reaction system.

[0323] 1.2 Determination of polypeptide activity intensity by fluorescence

[0324] After centrifugation, the fluorescence intensity in the supernatant was measured. The higher the fluorescence intensity, the higher the activity of the phase separation reversal polypeptide or its variant in reversing phase separation.

[0325] 1.3 Experimental results

[0326] The results of the in vitro depolymerization experiment are as follows Figure 1 and Figure 2 shown, where the higher the value on the y-axis, the stronger the depolymerization ability of the polypeptide. Figure 1 It shows that at the same concentration, the depolymerization abilities of variant 2016# and variant 2017# are both stronger than that of the phase separation reversal polypeptide 0009#, and the depolymerization ability of variant 2017# is stronger than that of variant 2016#. Figure 2 It shows that at the same concentration, the depolymerization abilities of variant 2017#, variant 2019#, variant 2020# and variant 2021# are all significantly improved compared to the phase separation reversal polypeptide 0009#; as the concentration of the variant increases, the depolymerization ability of variant 2019# is the most prominent.

[0327] Example 2: Determination of the ability to reverse intracellular phase separation

[0328] 2.1 Expression of phase separation and phase separation reversal proteins in cells

[0329] Plasmids expressing any protein with phase separation ability (for example, FUS525 protein) and phase separation reversal polypeptides or their variants are transfected into cells, and any cell such as HEK293 or U2OS can be used.

[0330] Experimental group: Transfect plasmids expressing various variants of the phase separation reversal polypeptide 0009#.

[0331] Blank control group: Transfect plasmids expressing the phase separation reversal polypeptide 0009#.

[0332] Negative control group (NC): Do not transfect any plasmid expressing the phase separation reversal polypeptide.

[0333] 2.2 Evaluate the activity of the reversal polypeptide in cells by the proportion of aggregated cells in transfected positive cells. The lower the intracellular aggregation ratio, the higher the activity of the phase separation reversal polypeptide.

[0334] 2.3 Experimental results:

[0335] The results of the cell depolymerization experiment are as follows Figure 3 and Figure 4 shown, where the y-axis represents the percentage of cells with aggregation, and the lower the value, the stronger the depolymerization ability of the polypeptide). As Figure 3 shown, when variants 2016# and 2017# are added to cells overexpressing the aggregated protein FUS525, the depolymerization ability of variant 2017# is stronger than that of variant 2016#. As Figure 4As shown, the depolymerization activity of the variants obtained by transfecting with the insertion of one cysteine residue (#2017) and two cysteine residues (#2019) is superior to that of the original sequence (0009#). The depolymerization activity of the variants obtained by inserting three cysteine residues (#2020) and inserting one cysteine residue and one threonine residue (2021#) is poorer than that of the original sequence (0009#).

[0336] Although the present invention has been specifically shown and described with reference to specific embodiments (some of which are preferred embodiments), those skilled in the art should understand that various changes in form and detail can be made therein without departing from the spirit and scope of the present invention as disclosed herein.

Claims

1. A variant of a phase separation reversal polypeptide, wherein the amino acid sequence of the variant is a sequence selected from the group consisting of: MCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 55), MCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 56), MCCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 57), MTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 60), and MSCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 58).

2. A method for improving the phase separation reversal ability of a phase separation reversal polypeptide, wherein the method comprises changing the phase separation reversal polypeptide into a variant of the phase separation reversal polypeptide; wherein the amino acid sequence of the variant of the phase separation reversal polypeptide is a sequence selected from the group consisting of: MCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 55), MCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 56), MCCCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 57), MTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHHC (SEQ ID NO: 60), and MSCTEPQEESEEEVEEPEERQQTPEVVPDDSGTFYDQTVSNDLEGGRKKRRQRRRGSGHHHHHHHH (SEQ ID NO: 58).

3. A method of reversing, inhibiting, reducing, and / or preventing abnormal protein aggregation in cells in vitro, wherein the method comprises introducing into the cells the variant according to claim 1.

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