Compositions and methods for regulating molecules

Abstract

Described herein are compositions for modulating a molecule in a cell. Also described herein are methods for modulating a molecule in a cell.

Description

[0001] Cross-references

[0002] This patent application claims priority to international application No. PCT / CN2022 / 101584 filed on June 27, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporated by Reference

[0004] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] Modulating molecules in cells is essential for regulating cellular function or maintaining overall cellular homeostasis or health. For example, molecules can be regulated (e.g., enriched) at specific locations in the cell where they are close to each other, thereby initiating an enzymatic reaction or signaling cascade. Summary of the invention

[0006] Therefore, compositions and methods for regulating molecules are needed. In some aspects, an engineered polypeptide is described herein, comprising: a functional domain; and an condensate conversion domain, wherein the condensate conversion domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof. In some embodiments, the engineered polypeptide forms a first condensate in a cell by operatively contacting at least one additional condensate conversion domain. In some embodiments, after contacting an condensate regulator, the engineered polypeptide operatively contacts at least one additional condensate conversion domain by the engineered polypeptide to form a first condensate in the cell. In some embodiments, the first condensate mediates liquid-liquid phase separation (LLPS). In some embodiments, the first condensate comprises a liquid condensate. In some embodiments, the first condensate allows molecular exchange between the inside and outside of the first condensate. In some embodiments, the first condensate allows molecular movement inside the first condensate. In some embodiments, after removing the condensate regulator, the first condensate is reversed. In some embodiments, when the engineered polypeptide contacts a second condensate regulator, the first condensate is reversed. In some embodiments, the first condensate comprises a liquid condensate or a liquid-like condensate. In some embodiments, when the engineered polypeptide is contacted with a third coacervate regulator, the first coacervate is converted to a second coacervate. In some embodiments, after removing the third coacervate regulator, the second coacervate is reversed back to the first coacervate. In some embodiments, when the engineered polypeptide is contacted with a fourth coacervate regulator, the second coacervate is reversed back to the first coacervate. In some embodiments, the second coacervate comprises a solid-like coacervate or a semi-solid-like coacervate. In some embodiments, the second coacervate comprises a gel-like coacervate. In some embodiments, the second coacervate inhibits molecular exchange between the inside and outside of the second coacervate. In some embodiments, the second coacervate inhibits the movement of molecules within the second coacervate. In some embodiments, the nuclear receptor or its fragment comprises an androgen receptor (AR) or a fragment thereof. In some embodiments, the nuclear receptor or its fragment comprises an amino acid sequence at least 70% identical to SEQ ID NO: 1. In some embodiments, the nuclear receptor or its fragment comprises an amino acid sequence at least 70% identical to SEQ ID NO: 2. In some embodiments, the nuclear receptor or its fragment comprises an amino acid sequence at least 70% identical to SEQ ID NO: 4. In some embodiments, the nuclear receptor or fragment thereof does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO:3. In some embodiments, the nuclear receptor or fragment thereof comprises an estrogen receptor (ER) or fragment thereof. In some embodiments, the nuclear receptor or fragment thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:5. In some embodiments, the nuclear receptor or fragment thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:6.In some embodiments, the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO: 8. In some embodiments, the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO: 7. In some embodiments, the condensate switching domain comprises at least one modification. In some embodiments, the condensate switching domain comprises at least one modification compared to any one of SEQ ID NOs: 1-10. In some embodiments, the at least one modification reduces the binding between the engineered polypeptide and the nucleic acid by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, compared to the binding between the control polypeptide without the at least one modification and the nucleic acid. In some embodiments, at least one modification eliminates the binding between the engineered polypeptide and the nucleic acid. In some embodiments, at least one modification reduces the binding between the engineered polypeptide and the ligand by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, compared to the binding between the control polypeptide without the at least one modification and the ligand. In some embodiments, at least one modification eliminates the binding between the engineered polypeptide and the ligand. In some embodiments, the ligand is an endogenous ligand. In some embodiments, the ligand is an exogenous ligand. In some embodiments, at least one modification includes at least one truncation. In some embodiments, at least one truncation includes the deletion of a nucleic acid binding domain or a fragment thereof. In some embodiments, at least one modification includes at least one amino acid substitution. In some embodiments, at least one modification includes at least one truncation and at least one amino acid substitution. In some embodiments, compared to SEQ ID NO: 1, at least one modification includes an A574D mutation or a W742C mutation. In some embodiments, compared to SEQ ID NO: 1, at least one modification includes an A574D mutation and a W742C mutation. In some embodiments, compared to SEQ ID NO: 1, at least one modification includes an A574D mutation or a W742L mutation. In some embodiments, compared to SEQ ID NO: 1, at least one modification includes an A574D mutation and a W742L mutation. In some embodiments, compared to SEQ ID NO: 1, at least one modification includes a K632A mutation, a K633A mutation, a W742C mutation, or a combination thereof. In some embodiments, at least one modification comprises a K632A mutation, a K633A mutation, and a W742L mutation compared to SEQ ID NO: 1. In some embodiments, at least one modification comprises a truncation of amino acid residues 617-633 or a W742C mutation compared to SEQ ID NO: 1. In some embodiments, at least one modification comprises a truncation of amino acid residues 617-633 and a W742C mutation compared to SEQ ID NO: 1.In some embodiments, at least one modification comprises a truncation of amino acid residues 617-633 or a W742L mutation compared to SEQ ID NO: 1. In some embodiments, at least one modification comprises a truncation of amino acid residues 617-633 and a W742L mutation compared to SEQ ID NO: 1. In some embodiments, at least one modification comprises a truncation of amino acid residues 1-293 compared to SEQ ID NO: 1; a truncation of amino acid residues 556-666, a W742C mutation, or a combination thereof. In some embodiments, at least one modification comprises a truncation of amino acid residues 1-293 compared to SEQ ID NO: 1; a truncation of amino acid residues 556-666, and a W742C mutation. In some embodiments, at least one modification comprises a truncation of amino acid residues 1-293 compared to SEQ ID NO: 1; a truncation of amino acid residues 556-666, a W742L mutation, or a combination thereof. In some embodiments, at least one modification comprises a truncation of amino acid residues 1-293 compared to SEQ ID NO: 1; a truncation of amino acid residues 556-666, and a W742L mutation. In some embodiments, the at least one modification comprises a truncation of amino acid residues 44-163 compared to SEQ ID NO: 1; a truncation of amino acid residues 226-347; a truncation of amino acid residues 560-670, a W742L mutation, or a combination thereof. In some embodiments, the at least one modification comprises a truncation of amino acid residues 44-163 compared to SEQ ID NO: 1; a truncation of amino acid residues 226-347; a truncation of amino acid residues 560-670, and a W742L mutation. In some embodiments, compared to SEQ ID NO: 1, at least one modification comprises an A574D mutation; a W742C mutation; a W742L mutation; a K632A mutation; a K633A mutation; a truncation of amino acid residues 617-633; a truncation of amino acid residues 44-163; a truncation of amino acid residues 226-347; a truncation of amino acid residues 560-670; a truncation of amino acid residues 1-293; a truncation of amino acid residues 556-666; or a combination thereof. In some embodiments, compared to SEQ ID NO: 5, at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, a G521R mutation, or a combination thereof. In some embodiments, compared to SEQ ID NO: 5, at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, and a G521R mutation. In some embodiments, at least one modification compared to SEQ ID NO:5 includes K210A, R211E, L379R, C400V, M543A, and L544A mutations.In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 or a L379R mutation compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 and a L379R mutation compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 or a G521R mutation compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 and a G521R mutation compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 and a C400V, M543A, L544A mutation, or a combination thereof compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a truncation of amino acid residues 250-274 and C400V, M543A, and L544A mutations compared to SEQ ID NO: 5. In some embodiments, at least one modification comprises a K210A mutation; an R211E mutation; an L379R mutation; a G521R mutation; a C400V mutation; an M543A mutation; an L544A mutation; a truncation of amino acid residues 250-274; or a combination thereof. In some embodiments, at least one modification comprises a truncation of amino acid residues 44-163; a truncation of amino acid residues 226-347; a truncation of amino acid residues 560-670, or a combination thereof compared to SEQ ID NO: 9. In some embodiments, at least one modification comprises a truncation of amino acid residues 44-163; a truncation of amino acid residues 226-347; and a truncation of amino acid residues 560-670 compared to SEQ ID NO: 9. In some embodiments, compared to SEQ ID NO: 10, at least one modification includes truncation of amino acid residues 250-274. In some embodiments, the cohesion regulator includes a functional domain ligand. In some embodiments, the functional domain ligand includes a functional domain agonist. In some embodiments, the functional domain ligand includes a functional domain antagonist. In some embodiments, the functional domain ligand includes a functional domain inverse agonist. In some embodiments, the cohesion regulator includes a nuclear receptor ligand. In some embodiments, the cohesion regulator includes a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist. In some embodiments, the cohesion regulator includes an androgen receptor agonist, an androgen receptor antagonist, or an androgen receptor inverse agonist. In some embodiments, the cohesion regulator includes an androgen receptor ligand, which includes bicalutamide.In some embodiments, the coagulation regulator includes an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist. In some embodiments, the coagulation regulator includes an estrogen receptor ligand, which includes tamoxifen. In some embodiments, the second coagulation regulator, the third coagulation regulator, or the fourth coagulation regulator includes a nuclear receptor ligand. In some embodiments, the second coagulation regulator, the third coagulation regulator, or the fourth coagulation regulator includes a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist. In some embodiments, the second coagulation regulator, the third coagulation regulator, or the fourth coagulation regulator includes an androgen receptor agonist, an androgen receptor antagonist, or an androgen receptor inverse agonist. In some embodiments, the second coagulation regulator, the third coagulation regulator, or the fourth coagulation regulator includes enzalutamide. In some embodiments, the second coagulation regulator includes enzalutamide. In some embodiments, the second coagulation regulator, the third coagulation regulator, or the fourth coagulation regulator includes an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist. In some embodiments, the second coacervate regulator, the third coacervate regulator, or the fourth coacervate regulator includes fulvestrant. In some embodiments, the third coacervate regulator includes fulvestrant. In some embodiments, the engineered polypeptide forms a first coacervate or a second coacervate in the cytoplasm of the cell. In some embodiments, the engineered polypeptide forms a first coacervate or a second coacervate in the organelle of the cell. In some embodiments, the engineered polypeptide forms a first coacervate or a second coacervate in the nucleus. In some embodiments, the functional domain includes a kinase domain. In some embodiments, the functional domain includes a phosphatase domain. In some embodiments, the functional domain includes a transcriptional activation domain. In some embodiments, the functional domain includes a DNA binding domain. In some embodiments, the functional domain includes a metabolic enzyme domain. In some embodiments, the functional domain includes a receptor. In some embodiments, the receptor includes a CAR or a fragment thereof. In some embodiments, the receptor includes a TCR or a fragment thereof.

[0007] In some aspects, described herein are engineered polynucleotides encoding engineered polypeptides described herein. In some embodiments, the engineered polynucleotides include vectors. In some embodiments, the vectors include viral vectors. In some aspects, described herein are cells comprising engineered polypeptides or engineered polynucleotides described herein.

[0008] In some aspects, a system is described herein, comprising: an engineered polypeptide comprising a functional domain and an accretion conversion domain, wherein the accretion conversion domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof; and an accretion modulator, wherein the engineered polypeptide, upon contact with the accretion modulator, forms a first accretion comprising the engineered polypeptide in operative contact with at least one other accretion conversion domain. In some embodiments, the system further comprises a second accretion modulator, a third accretion modulator, or a fourth accretion modulator.

[0009] In some aspects, a pharmaceutical composition is described herein, comprising an engineered polypeptide, an engineered polynucleotide, a cell or a system as described in any of the preceding claims. In some embodiments, the pharmaceutical composition is formulated to be administered to a subject in need by one or a combination of the following: intrathecal, intraocular, intravitreal, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular system, intracerebral parenchyma, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, intrarectal, oral, sublingual, transdermal, inhalation, aerosol inhalation, intracavitary-gastrointestinal route. In some embodiments, the pharmaceutical composition comprises at least one excipient. In some embodiments, the pharmaceutical composition comprises at least one additional active ingredient.

[0010] In some aspects, a method for forming a first condensate in a cell is described herein, the method comprising contacting the cell with an engineered polypeptide, an engineered polynucleotide, or a system as described in any one of the preceding claims, wherein the first condensate is formed when the engineered polypeptide is in operative contact with at least one other condensate conversion domain. In some embodiments, the engineered polypeptide forms a first condensate in the cell by operative contact with at least one additional condensate conversion domain. In some embodiments, the engineered polypeptide forms a first condensate in the cell by operative contact with at least one additional condensate conversion domain after contact with the condensate regulator by the engineered polypeptide. In some embodiments, the first condensate mediates liquid-liquid phase separation (LLPS). In some embodiments, the first condensate includes a liquid condensate. In some embodiments, the first condensate allows molecular exchange between the inside and outside of the first condensate. In some embodiments, the first condensate allows molecular movement inside the first condensate. In some embodiments, after removing the condensate regulator, the first condensate is reversed. In some embodiments, when the engineered polypeptide is in contact with the second condensate regulator, the first condensate is reversed. In some embodiments, when the engineered polypeptide is contacted with a third coacervate regulator, the first coacervate is converted to a second coacervate. In some embodiments, after removing the third coacervate regulator, the second coacervate is reversed back to the first coacervate. In some embodiments, when the engineered polypeptide is contacted with a fourth coacervate regulator, the second coacervate is reversed back to the first coacervate. In some embodiments, the second coacervate includes a solid-like coacervate. In some embodiments, the second coacervate includes a semi-solid-like coacervate. In some embodiments, the second coacervate includes a gel-like coacervate. In some embodiments, the second coacervate inhibits the exchange of molecules between the inside and outside of the second coacervate. In some embodiments, the second coacervate inhibits the movement of molecules within the second coacervate.

[0011] In some aspects, a method for increasing enzyme activity in a cell is described herein, comprising contacting an engineered polypeptide or an engineered polynucleotide of any of the preceding claims with an aggregate regulator, so that when the engineered polypeptide is in operative contact with at least one other aggregate conversion domain, a first aggregate is formed, wherein the first aggregate enriches the local enzyme concentration. In some aspects, a method for increasing enzyme activity in a cell is also described herein, comprising contacting an engineered polypeptide or an engineered polynucleotide of any of the preceding claims with an aggregate regulator, so that when the engineered polypeptide is in operative contact with at least one other aggregate conversion domain, a first aggregate is formed, wherein the first aggregate brings two or more molecules with enzyme activity into close proximity, thereby increasing enzyme activity. In some embodiments, the two or more molecules comprise at least one enzyme. In some embodiments, the two or more molecules comprise at least one biomolecule. In some embodiments, the two or more molecules comprise at least one nucleic acid. In some embodiments, at least one nucleic acid comprises DNA, RNA, or a combination thereof. In some embodiments, the two or more molecules comprise at least one protein. In some embodiments, at least one protein comprises a transcription factor. In some embodiments, the two or more molecules comprise at least one polysaccharide. In some embodiments, the two or more molecules include at least one lipid. In some embodiments, the two or more molecules include at least one metabolite.

[0012] In some aspects, described herein is a method for regulating the concentration of at least one molecule in a cell, the method comprising contacting the cell with an engineered polypeptide or engineered polynucleotide of any of the preceding claims, wherein after the engineered polypeptide is contacted with an aggregate regulator, a first aggregate is formed when operatively contacted with at least one other aggregate conversion domain, wherein the concentration of at least one molecule inside the first aggregate is different from the concentration of at least one molecule outside the first aggregate. In some embodiments, the concentration of the at least one molecule inside the first aggregate is increased compared to the concentration of at least one molecule outside the first aggregate. In some embodiments, the concentration increases by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the concentration of at least one molecule inside the first aggregate is reduced compared to the concentration of at least one molecule outside the first aggregate. In some embodiments, the concentration decreases by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the viscosity of the first coacervate is at least increased by 10%, at least increased by 20%, at least increased by 30%, at least increased by 40%, at least increased by 50%, at least increased by 60%, at least increased by 70%, at least increased by 80%, at least increased by 90%, or at least increased by 99% compared to a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the viscosity of the first coacervate is at least reduced by 10%, at least reduced by 20%, at least reduced by 30%, at least reduced by 40%, at least reduced by 50%, at least reduced by 60%, at least reduced by 70%, at least reduced by 80%, at least reduced by 90%, or at least reduced by 99% compared to a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the surface tension of the first coacervate is at least increased by 10%, at least increased by 20%, at least increased by 30%, at least increased by 40%, at least increased by 50%, at least increased by 60%, at least increased by 70%, at least increased by 80%, at least increased by 90%, or at least increased by 99% compared to a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the surface tension of the first coacervate is at least reduced by 10%, at least reduced by 20%, at least reduced by 30%, at least reduced by 40%, at least reduced by 50%, at least reduced by 60%, at least reduced by 70%, at least reduced by 80%, at least reduced by 90%, or at least reduced by 99% compared to a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the turbidity of the first aggregate is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to a control first aggregate formed by a control polypeptide without at least one modification.In some embodiments, the turbidity of the first coacervate is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% lower than that of a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the first coacervate increases the rate of molecular exchange between its interior and exterior by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the rate of molecular exchange between the interior and exterior of a control first coacervate formed by a control polypeptide without at least one modification. In some embodiments, the first condensate reduces the molecular exchange rate between the interior and exterior of the first condensate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to the molecular exchange rate between the interior and exterior of the control first condensate formed by the control polypeptide without the at least one modification. In some embodiments, the first condensate is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% more likely to accumulate in the cytoplasm of the cell than the control first condensate formed by the control polypeptide without the at least one modification. In some aspects, described herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an engineered polypeptide, engineered polynucleotide, cell or system of any one of the preceding claims. In some embodiments, the engineered polypeptide, engineered polynucleotide, cell or system of any of the preceding claims forms a first aggregate, a second aggregate, a third aggregate or a combination thereof for use in treating a disease or disorder in a subject.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] Figure 1 An example of directing a signaling cascade within a cell by regulating (e.g., enriching) a molecule within the cell is shown. By concentrating the molecule within the cell to a certain location, the signaling cascade moves primarily in the direction of D, rather than being evenly distributed between C and D due to the molecule being unregulated (e.g., passively diffusing within the cell).

[0016] Figure 2Examples of switching between different condensates within cells mediated by the condensate switching domains and condensate regulators described herein are shown.

[0017] Figure 3A and Figure 3B Fluorescence images showing the transformation of condensates within cells. Figure 3C and Figure 3D Western blot images showing signaling cascades regulated by condensate formation or condensate turnover in cells. Figure 3A It is demonstrated that when an aggregate switching domain comprising an androgen receptor (AR) or a fragment thereof is contacted with an aggregate modulator comprising dihydrotestosterone (DHT), a first aggregate is formed (wherein the first aggregate is a phase that is not identical to its surrounding phase or medium). Figure 3B The formation of the first condensate is demonstrated when an condensate switching domain comprising an estrogen receptor (ER) or a fragment thereof is contacted with an condensate modulator comprising bicalutamide (Bica). Figure 3B It was further shown that the first aggregates can be dissolved or reversed by contacting the aggregate-switching domain comprising AR or a fragment thereof with another aggregate-modulating agent comprising enzalutamide (Enza). Figure 3C Western blot showing the phosphorylation of proteins modulated by the first aggregate formation when the aggregate switching domain comprising AR or ER or fragments thereof is contacted with an aggregate modulator comprising bicalutamide (Bica) or another aggregate modulator comprising fulvestrant, respectively. Figure 3D Western blot results showing regulation of protein phosphorylation by formation of a first aggregate or switching from a first aggregate to a second aggregate. An aggregate switching domain comprising an androgen receptor (AR) or a fragment thereof is contacted with an aggregate modulator comprising bicalutamide (Bica), or with an aggregate modulator comprising bicalutamide (Bica) and another aggregate modulator comprising enzalutamide (Enza) in sequence.

[0018] The novel features of the present disclosure are set forth with particularity in the appended claims.The features and advantages of the present disclosure will be better understood by reference to the following detailed description which sets forth illustrative embodiments. DETAILED DESCRIPTION

[0019] Overview

[0020] In some aspects, described herein are compositions comprising the engineered polypeptides described herein for forming a first aggregate in a cell. Also described herein are methods of forming a first aggregate using the engineered polypeptides described herein.

[0021] In some aspects, a first coacervate is formed when an engineered polypeptide is contacted with a coacervate regulator described herein. In some embodiments, a first coacervate can be formed spontaneously (e.g., without contacting an engineered polypeptide with a coacervate regulator described herein). In some embodiments, a first coacervate allows molecular exchange between the inside and outside of a first coacervate. In some embodiments, a first coacervate regulates the concentration of a molecule while allowing molecular exchange between the inside and outside of a first coacervate. In some embodiments, a first coacervate allows molecular movement inside a first coacervate. In some embodiments, a first coacervate reduces the exchange of at least one molecule described herein between the inside and outside of a first coacervate. In some embodiments, a first coacervate inhibits the exchange of at least one molecule described herein between the inside and outside of a first coacervate. In some embodiments, a first coacervate reduces the movement of at least one molecule described herein inside a first coacervate. In some embodiments, a first coacervate inhibits the movement of at least one molecule described herein inside a first coacervate. In some embodiments, when a coacervate regulator is removed, a first coacervate is reversed. In some embodiments, when an engineered polypeptide is contacted with a second coacervate regulator described herein, a first coacervate is reversed.

[0022] In some embodiments, the formation of the first coacervate in the cell regulates molecules in the cell. For example, the first coacervate can enrich the molecule in the first coacervate, and then increase the enzyme activity by bringing the molecules involved in the enzyme activity into close proximity, thereby initiating the enzyme activity. In some embodiments, the first coacervate can regulate the concentration of molecules in the cell by isolating the molecules in the first coacervate, thereby reducing the concentration of molecules in the cell that are not part of the first coacervate.

[0023] In some embodiments, when the engineered polypeptide is contacted with at least one additional coacervate regulator (e.g., a third coacervate regulator described herein), the first coacervate can be converted into a second coacervate. In some embodiments, the formation of the first coacervate or the second coacervate in the cell can isolate molecules in the first coacervate or the second coacervate, thereby reducing the concentration of molecules that are not part of the coacervate in the cell and reducing the enzyme activity associated with the molecules isolated by the first coacervate or the second coacervate. In some embodiments, the second coacervate includes a solid-like coacervate, a semi-solid-like coacervate, a gel-like coacervate, or a combination thereof. In some embodiments, the second coacervate allows molecular exchange between the inside and outside of the second coacervate. In some embodiments, the second coacervate regulates the concentration of molecules while allowing molecular exchange between the inside and outside of the second coacervate. In some embodiments, the second coacervate allows the movement of molecules inside the second coacervate. In some embodiments, the second coacervate reduces the exchange of at least one molecule described herein between the inside and outside of the second coacervate. In some embodiments, the second coacervate inhibits the exchange of at least one molecule described herein between the inside and outside of the second coacervate. In some embodiments, the second coacervate reduces the movement of at least one molecule described herein inside the second coacervate. In some embodiments, the second coacervate inhibits the movement of at least one molecule described herein within the second coacervate. In some embodiments, the second coacervate can be reversed by removing the third coacervate modulator. In some embodiments, the second coacervate can be reversed by contacting the engineered polypeptide with a fourth coacervate modulator.

[0024] In some aspects, an engineered polypeptide is described herein, comprising a functional domain and a coacervate conversion domain, wherein the coacervate conversion domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof. In some embodiments, after contacting a coacervate regulator, the engineered polypeptide is operatively contacted with at least one additional coacervate conversion domain by the engineered polypeptide (e.g., the engineered polypeptide is operatively contacted with at least one additional coacervate conversion domain from other engineered polypeptide molecules) to form a first coacervate in the cell. In some embodiments, the first coacervate is different from the surrounding phase. For example, the first coacervate can have a fluidity or viscosity different from the surrounding phase, wherein the surrounding phase is the cytoplasm. In some embodiments, the first coacervate mediates liquid-liquid coacervate separation (LLPS). In some embodiments, the first coacervate comprises a liquid coacervate. In some embodiments, the first coacervate comprises a liquid-like coacervate.

[0025] In some embodiments, the first coacervate regulates molecules in the cell by enriching the molecules in the first coacervate. In some embodiments, the first coacervate allows molecular exchange between the inside and outside of the first coacervate. In some embodiments, the first coacervate regulates the concentration of molecules while allowing molecular exchange between the inside and outside of the first coacervate. In some embodiments, the first coacervate allows molecules inside the first coacervate to move. In some embodiments, the first coacervate, by regulating the concentration of molecules, can subsequently regulate enzyme activity or signal cascades associated with regulatory molecules in the cell.

[0026] In some embodiments, the first coacervate can be converted into a second coacervate by contacting the engineered polypeptide with at least one additional coacervate regulator. In some embodiments, in contrast to the first coacervate, the second coacervate does not allow the movement of molecules in the second coacervate or the exchange of molecules between the inside and outside of the second coacervate. In some embodiments, compared with the first coacervate, the second coacervate reduces the movement of molecules in the second coacervate or the exchange of molecules between the inside and outside of the second coacervate. In some embodiments, the second coacervate is a solid coacervate, a semi-solid coacervate or a gel coacervate. In some aspects, the solid coacervate, the semi-solid coacervate or the gel coacervate includes an arbitrary shape and is able to maintain shape memory for a period of time. In some aspects, the solid coacervate, the semi-solid coacervate or the gel coacervate includes shear elasticity, which does not occur in the first coacervate. In some embodiments, the second coacervate has a different fluidity or viscosity from the first coacervate. In some embodiments, the second coacervate inhibits the enzyme activity or signal cascade in the cell by preventing molecules associated with the enzyme activity or signal cascade in the second coacervate. In some embodiments, when the engineered polypeptide is contacted with a fourth aggregate modulator, the second aggregate can be reversed into the first aggregate.

[0027] In some embodiments, the engineered polypeptide comprising the condensate conversion domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of a nuclear receptor or a fragment thereof. In some embodiments, the nuclear receptor or its fragment comprises an androgen receptor (AR) or a fragment thereof. In some embodiments, the nuclear receptor or its fragment comprises an estrogen receptor (ER) or a fragment thereof. In some embodiments, the condensate conversion domain comprising the amino acid sequence of a nuclear receptor or its fragment comprises at least one modification. In some embodiments, at least one modification comprises at least one amino acid substitution. In some embodiments, at least one modification comprises at least one truncation. In some embodiments, at least one truncation comprises deleting the entire domain, such as the DNA binding domain (DBD) of a nuclear receptor described herein. In some embodiments, at least one modification comprises at least one amino acid substitution and at least one truncation. In some embodiments, compared to the full-length amino acid sequence of a nuclear receptor described herein, at least one modification comprises at least one amino acid substitution and at least one truncation. In some embodiments, compared to the full-length amino acid sequence of an androgen receptor described herein, at least one modification comprises at least one amino acid substitution and at least one truncation. In some embodiments, compared to the full-length amino acid sequence of an estrogen receptor described herein, the at least one modification comprises at least one amino acid replacement and at least one truncation.

[0028] In some embodiments, the at least one modification reduces the binding between the engineered polypeptide and the nucleic acid as compared to the binding between a control polypeptide without the at least one modification and the nucleic acid. In some embodiments, the at least one modification eliminates the binding between the engineered polypeptide and the nucleic acid. In some embodiments, the at least one modification reduces the binding between the engineered polypeptide and the ligand of the engineered polypeptide as compared to the binding between a control polypeptide without the at least one modification and the ligand. In some embodiments, the at least one modification eliminates the binding between the engineered polypeptide and the ligand. The ligand of the engineered polypeptide can be an endogenous ligand or an exogenous ligand. In some embodiments, the cohesion modulator can be a functional domain ligand, such as a functional domain agonist, a functional domain antagonist, or a functional domain inverse agonist.

[0029] In some embodiments, described herein are engineered polynucleotides encoding engineered polypeptides described herein. In some embodiments, engineered polynucleotides include vectors, such as viral vectors or plasmids. In some embodiments, described herein are cells comprising engineered polypeptides or engineered polynucleotides described herein. For example, engineered polynucleotides can be used as part of a vector to transduce cells. In some embodiments, described herein is a system comprising: an engineered polypeptide comprising a functional domain and an condensate conversion domain, wherein the condensate conversion domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof; and an condensate regulator, wherein the engineered polypeptide, after contacting the condensate regulator, is operatively contacted with at least one other condensate conversion domain by the engineered polypeptide to form a first condensate. In some embodiments, the system further comprises at least one additional condensate regulator, such as a second condensate regulator, a third condensate regulator, a fourth condensate regulator, or a combination thereof. In some embodiments, described herein is a pharmaceutical composition comprising an engineered polypeptide, an engineered polynucleotide, a cell, or a system described herein.

[0030] In some embodiments, described herein is a method for forming a first coacervate in a cell, the method comprising contacting the cell with an engineered polypeptide, an engineered polynucleotide, or a system, wherein the first coacervate is formed when the engineered polypeptide is in operative contact with at least one other coacervate conversion domain after contact with a coacervate regulator. In some embodiments, the method comprises causing the first coacervate to be converted into a second coacervate when the engineered polypeptide is in contact with at least one additional coacervate regulator. In some embodiments, the method comprises reversing the second coacervate into the first coacervate by contacting the engineered polypeptide with a third coacervate regulator. In some aspects, also described herein is a method for increasing enzyme activity in a cell, the method comprising contacting the engineered polypeptide with a coacervate regulator to form a first coacervate when the engineered polypeptide is in operative contact with at least one other coacervate conversion domain, wherein the first coacervate brings two or more enzyme-active molecules into close proximity, thereby increasing enzyme activity. Figure 1An example of directing an intracellular signal cascade by regulating (e.g., enriching) molecules in the cell is shown. By enriching the molecules in the cell to a certain position, the signal cascade moves mainly in the D direction, rather than evenly distributing the signal cascade between C and D due to the molecules being unregulated (e.g., passive diffusion in the cell). In some aspects, the method described herein regulates the concentration of at least one molecule in the cell. In some embodiments, the concentration of at least one molecule inside the first coagulation body increases relative to the concentration of at least one molecule outside the first coagulation body. In some embodiments, the concentration of at least one molecule inside the first coagulation body decreases relative to the concentration of at least one molecule outside the first coagulation body. In some embodiments, the composition or method can treat a disease or disorder in a subject in need. In some embodiments, a method for treating a disease or disorder in a subject is described herein.

[0031] Engineered Peptides

[0032] In some embodiments, an engineered polypeptide is described herein, comprising a functional domain and a coacervate conversion domain, wherein the coacervate conversion domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator as described herein, the engineered polypeptide forms a first coacervate. The first coacervate comprises a phase different from the surrounding phase. For example, the first coacervate may include a liquid coacervate having different fluidity or viscosity compared to its surrounding phase. In some embodiments, the first coacervate may be reversed by removing the coacervate regulator. In some embodiments, the first coacervate may be reversed by contacting the engineered polypeptide with at least one additional coacervate regulator (e.g., a second coacervate regulator as described herein).

[0033] In some embodiments, the first coacervate is converted to a second coacervate by contacting the engineered polypeptide with at least one additional coacervate modifier (e.g., a third coacervate modifier). In some embodiments, the second coacervate can be reversed back to the first coacervate by removing the third coacervate modifier. In some embodiments, the second coacervate is reversed back to the first coacervate by contacting the engineered polypeptide with a fourth coacervate modifier.

[0034] In some embodiments, the engineered polypeptide can be directly introduced into the cell. In some embodiments, the engineered polypeptide can be delivered into the cell by the engineered polynucleotide described herein. In some embodiments, the formation of the first coacervate can regulate the enzyme activity or signal cascade reaction in the cell. In some embodiments, the formation of the first coacervate or the second coacervate can treat a disease or condition of the subject.

[0035] In some embodiments, the engineered polypeptide can be part of a system. For example, the engineered polypeptide is part of a system, wherein the system further comprises at least one coacervate regulator as described herein. In some embodiments, the engineered polypeptide, the engineered polynucleotide encoding the engineered polypeptide, or the system can be formulated into a pharmaceutical composition. In some embodiments, the engineered polypeptide, the engineered polynucleotide encoding the engineered polypeptide, or the system can be part of a kit.

[0036] Functional domain

[0037] In some embodiments, engineered polypeptides comprising functional domains are described herein. In some embodiments, functional domains are operatively contacted with cohesive domains described herein. In some embodiments, functional domains are covalently linked to cohesive domains. In some embodiments, functional domains include ligand binding domains. In some embodiments, functional domains can be contacted with functional domain ligands, wherein functional domain ligands are one of cohesive modulators described herein. In some embodiments, functional domain ligands include functional domain agonists, functional domain antagonists, or functional domain inverse agonists. In some embodiments, functional domains include receptors. In some embodiments, functional domains include extracellular domains. In some embodiments, functional domains include transmembrane domains. In some embodiments, functional domains include cytoplasmic domains. In some embodiments, functional domains include extracellular domains, transmembrane domains, cytoplasmic domains, or combinations thereof.

[0038] In some embodiments, the functional domain includes a kinase domain (e.g., a PKA domain or a PKC domain). In some embodiments, the functional domain includes a phosphatase domain (e.g., a diacylglycerol pyrophosphate phosphatase, DGPP, domain). In some embodiments, the functional domain includes a transactivation domain. Non-limiting examples of transactivation domains or DNA binding domains include the domain of a transcription factor, the domain of a transcription co-regulator, the domain of an epigenetic regulator, or a combination thereof. In some embodiments, the functional domain includes a transactivation domain or a DNA binding domain. In some embodiments, the functional domain includes a transactivation domain or a metabolic enzyme domain (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase domain, or a combination thereof). In some embodiments, the functional domain includes a receptor domain, such as a chimeric antigen receptor (CAR) domain or a T cell receptor (TCR) domain. In some embodiments, the functional domain includes a metabolic enzyme. In some embodiments, the CAR domain includes at least a portion of a Notch receptor, a G protein-coupled receptor (GPCR), an integrin receptor, a cadherin receptor, a receptor tyrosine kinase, a death receptor, an immune receptor, or a combination thereof. In some embodiments, the immune receptor includes a T cell receptor (TCR). In some embodiments, the receptor is endogenous to the cell. In some embodiments, the receptor is exogenous to the cell. Other examples of functional domains may include proteins involved in TCR clusters, nephrin clusters, actin plaques, adhesion plaques, synaptic density, stress granules, RNA transport granules, U bodies, P bodies, Balbiani bodies, P particles, cGAS condensates, lysates, Cajal bodies, GEMs, nuclear spots, OPT domains, PcG bodies, PML bodies, histone locus bodies, paraspots, or perinuclear compartments. In some embodiments, the functional domain includes a gene editing enzyme, such as 2 types of CRISPR / Cas, modified versions thereof, or mutant versions thereof. In some embodiments, the gene editing enzyme includes a mutant of Cas9 or Cas9. In some embodiments, the gene editing enzyme includes SaCas9, CjCas9, SuperFi-Cas9, Cas12a, Cas14, CasΦ, or CasIscB.

[0039] Condensate switching domain

[0040] In some aspects, an engineered polypeptide comprising a coacervate switching domain is described herein. In some embodiments, the coacervate switching domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the amino acid sequence of a nuclear receptor or a fragment thereof. For example, the coacervate switching domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the amino acid sequence of SEQ ID NO: 1-10 (Table 1). In some embodiments, the coacervate switching domain does not comprise a DNA binding domain (DBD). For example, the coacervate switching domain described herein does not include a DBD encoded by SEQ ID NO: 3 or SEQ ID NO: 7 in some aspects.

[0041] Table 1. Exemplary amino acid sequences of condensate switching domains

[0042]

[0043]

[0044]

[0045] In some embodiments, the condensate conversion domain comprises at least one modification compared to the wild-type amino acid sequence of a nuclear receptor or its fragment. In some embodiments, the nuclear receptor or its fragment includes an androgen receptor (AR) or its fragment. In some embodiments, the nuclear receptor or its fragment includes an estrogen receptor (ER) or its fragment. In some embodiments, compared with the binding between a similar polypeptide without the at least one modification and a nucleic acid, the at least one modification reduces the binding between the engineered polypeptide and the nucleic acid by at least 70%, at least 80%, at least 90%, at least 95% or at least 99%. In some embodiments, at least one modification eliminates the binding between the engineered polypeptide and the nucleic acid. In some embodiments, at least one modification includes deleting the DBD from the condensate conversion domain, wherein the condensate conversion domain is no longer able to bind to the nucleic acid. In some embodiments, compared with the binding between a similar polypeptide without at least one modification and a ligand, at least one modification reduces the binding between the engineered polypeptide and the ligand by at least 70%, at least 80%, at least 90%, at least 95% or at least 99%. In some embodiments, at least one modification eliminates the binding between the engineered polypeptide and the ligand. In some embodiments, the ligand is an endogenous ligand. In some embodiments, the ligand is an exogenous ligand. In some embodiments, at least one modification increases the formation of the first coacervate or the second coacervate by increasing the binding affinity between the coacervate switching domain and at least one other coacervate switching domain. Figure 2Examples of transitions between different phases within cells mediated by the condensate switching domains and condensate regulators described herein are shown.

[0046] Androgen receptor

[0047] In some embodiments, the condensate conversion domain described herein comprises an amino acid sequence or a fragment thereof of an androgen receptor (AR). AR is a member of the steroid and nuclear receptor superfamily and is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver, and central nervous system (CNS), with the highest expression levels observed in the prostate, adrenal glands, and epididymis. AR is a soluble protein that functions as an intracellular transcription factor. When AR binds to androgens (such as testosterone or dihydrotestosterone (DHT)) and is activated, AR undergoes conformational changes and post-translational modifications, dimerization, nuclear translocation, and ultimately binds to the regulatory region of the target gene DNA, which is called the androgen response element.

[0048] Wild-type AR contains three different domains, including an androgen-independent N-terminal domain (NTD), a DNA binding domain (DBD), and an androgen-dependent ligand binding domain (LBD). The full-length wild-type AR has an amino acid sequence of SEQ ID NO: 1, and the NTD in the full-length wild-type AR spans the first 559 amino acid residues. The NTD of AR has a high degree of intrinsic disorder and contains only a small amount of α-helix and β-folded structure. Disordered regions that lack fixed or ordered secondary and tertiary structures in proteins are also called intrinsic disordered regions (IDRs). IDRs can range from completely unstructured to partially structured. It was found that the NTD of AR contains a large number of IDRs. Without being bound by any theory, the phase separation or condensate formation of AR is believed to be driven by multivalent interactions of adhesion or association mediated by IDRs. In addition, it is known that the NTD of AR contains an activation function-1 (AF1) region, which is essential for AR transcriptional activation and exists in different forms of AR variants. The term "androgen receptor" or "AR" as used herein encompasses wild-type AR or AR variants (e.g., AR comprising at least one modification). The wild-type AR used herein refers to the full-length AR, an exemplary sequence of which is shown in SEQ ID NO: 1. The AR variants used herein encompass all different forms of AR that retain at least part of the NTD and can be phase separated or form a coacervate. Examples of AR variants include, but are not limited to, mutants, fragments, fusions, and splice variants. In some embodiments, the AR variant is resistant to at least one androgen deprivation therapy. The term "androgen deprivation therapy" or "ADT" as used herein refers to a therapy that suppresses androgens by reducing androgen levels or inhibiting the biological function of androgens (e.g., inhibiting AR signaling). ADT includes both surgical treatment (e.g., surgical castration) and drug therapy. Examples of ADT medications include, but are not limited to, LHRH agonists, such as leuprolide (Lupron, Eligard), goserelin (Zoladex), triptorelin (Trelstar), and histrelin (Vantas); LHRH antagonists, such as degarelix (Firmagon) and Relugolix (Orgovyx); medications that reduce adrenal androgen levels, such as abiraterone (Zytiga) and ketoconazole (Nizoral); androgen receptor antagonists, such as flutamide (Eulexin), bicalutamide (Casodex), and nilutamide (Nilandron); and other antiandrogens, such as enzalutamide (Xtandi), apalutamide (Erleada), and darolutamide (Nubeqa).

[0049] In some embodiments, the AR variant comprises one or more mutations. More than 800 different AR mutations have been found in patients with androgen insensitivity syndrome and prostate cancer. In the AR gene, four different types of mutations have been detected that can inactivate AR, including: single point mutations that cause amino acid substitutions or premature stop codons; nucleotide insertions or deletions that cause frameshifts and premature terminations; complete or partial deletions of genes; and intron mutations that cause alternative splicing. In some embodiments, the AR variant comprises one or more mutations in the ligand binding domain (LBD). In some embodiments, one or more mutations in the LBD result in a gain of function of AR. In some embodiments, the AR variant comprises one or more mutations at a residue selected from the following groups: A574, K632, K633, L702, V716, V731, W742, H875, F877, T878, D880, L882, S889, D891, E894, M896, E898, and T919, wherein the numbering refers to SEQ ID NO: 1. In some embodiments, the one or more mutations are selected from the group consisting of A574D, K632A, K633A, L702H, V716M, V731M, W742L / C, H875Y / Q, F877L, T878A / S, D880E, L882I, S889G, D891H, E894K, M896V / T, E898G, and T919S.

[0050] In some embodiments, the AR variant lacks all or part of the DBD. For example, the AR variant lacks the DBD encoded by SEQ ID NO: 3. In some embodiments, the AR variant lacks all or part of the LBD and has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the condensate conversion domain comprises at least one modification, and the at least one modification comprises an A574D mutation or a W742C mutation compared to SEQ ID NO: 1. In some embodiments, the condensate conversion domain comprises at least one modification, and the at least one modification comprises an A574D mutation and a W742C mutation compared to SEQ ID NO: 1. In some embodiments, the condensate conversion domain comprises at least one modification, and the at least one modification comprises an A574D mutation or a W742L mutation compared to SEQ ID NO: 1. In some embodiments, the condensate conversion domain comprises at least one modification, and the at least one modification comprises an A574D mutation and a W742L mutation compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a K632A mutation, a K633A mutation, a W742C mutation, or a combination thereof, compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a K632A mutation, a K633A mutation, and a W742C mutation, compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a K632A mutation, a K633A mutation, a W742L mutation, or a combination thereof, compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a K632A mutation, a K633A mutation, and a W742L mutation, compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a truncation of amino acid residues 617-633 or a W742C mutation, compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, comprising a truncation of amino acid residues 617-633 and a W742C mutation compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, comprising a truncation of amino acid residues 617-633 or a W742L mutation compared to SEQ ID NO: 1.In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a truncation of amino acid residues 617-633 and a W742L mutation compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, a W742C mutation, or a combination thereof compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, and the at least one modification comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, and a W742C mutation compared to SEQ ID NO: 1. In some embodiments, the coacervate switching domain comprises at least one modification, compared to SEQ ID NO: 1, comprising a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, a W742L mutation, or a combination thereof. In some embodiments, the coacervate switching domain comprises at least one modification, compared to SEQ ID NO: 1, comprising a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, and a W742L mutation. In some embodiments, the coacervate switching domain comprises at least one modification, compared to SEQ ID NO: 1, comprising a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, a W742L mutation, or a combination thereof. In some embodiments, the cohesin switch domain comprises at least one modification, comprising a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, and a W742L mutation compared to SEQ ID NO: 1.

[0051] In some embodiments, the coacervate switching domain comprises at least one modification compared to SEQ ID NO: 9, including a truncation of amino acid residues 44-163; a truncation of amino acid residues 226-347; a truncation of amino acid residues 560-670, or a combination thereof. In some embodiments, the coacervate switching domain comprises at least one modification, including a truncation of amino acid residues 44-163; a truncation of amino acid residues 226-347; and a truncation of amino acid residues 560-670, compared to SEQ ID NO: 9.

[0052] In some embodiments, when the condensate conversion domain is contacted with the condensate regulator described herein, the condensate conversion domain comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to AR or its fragment can form a first condensate. The first condensate includes a non-membrane encapsulated compartment formed by phase separation (including all stages of phase separation) of one or more condensate conversion domains described herein. In some embodiments, the first condensate includes a liquid-liquid condensate separation (LLPS) body. In some embodiments, the first condensate regulates molecules in the cell by enriching molecules in the first condensate. In some embodiments, the first condensate can be converted into a second condensate by contacting the engineered polypeptide with at least one additional condensate regulator. In some embodiments, the second condensate can be reversed back to the first condensate by contacting the engineered polypeptide with a third condensate regulator. In some embodiments, the condensate regulator includes an AR agonist, an AR antagonist, or an AR inverse agonist.

[0053] In some embodiments, when the engineered polypeptide is contacted with an aggregate modulator as described herein, a first aggregate is formed by contacting at least one other aggregate switching domain. For example, the engineered polypeptide may comprise an IDR-containing fragment of the NTD of the AR, wherein the IDR-containing fragment is tau-1 or tau-5. Tau-1 encompasses residues from amino acid positions 102 to 371 of the AR, numbered relative to SEQ ID NO: 1. Tau-5 encompasses residues from amino acid positions 330 to 448 of the AR, numbered relative to SEQ ID NO: 1. In some embodiments, the IDR-containing fragment is contained within the region of amino acid positions 102 to 371, positions 102 to 300, positions 102 to 250, positions 102 to 200, positions 102 to 150, positions 150 to 300, positions 200 to 300, positions 250 to 300, numbered relative to SEQ ID NO: 1. In some embodiments, the fragment containing the IDR is contained within the region of amino acid positions 330 to 448, positions 330 to 420, positions 330 to 400, positions 330 to 380, positions 330 to 360, positions 350 to 448, positions 380 to 448, positions 400 to 448, or positions 400 to 420, which are numbered relative to SEQ ID NO: 1. In some embodiments, the length of the fragment containing the IDR is about or at least 20 residues, 25 residues, 30 residues, 40 residues, or 50 residues. In some embodiments, the cohesion switching domain binds to residues within the IDR-containing fragment provided herein. In some embodiments, the cohesion switching domain binds to the IDR of the AR, or binds to a region outside the IDR but can allosterically affect the IDR. Binding to the IDR can be determined using suitable methods known in the art. In some embodiments, the region outside the IDR is located within the NTD of the AR.

[0054] In some embodiments, when the engineered polypeptide is contacted with the coacervate regulator described herein, it is operatively contacted with at least one other coacervate conversion domain to form a first coacervate. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising a nuclear receptor ligand (including a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist), a first coacervate is formed. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising an androgen receptor agonist, an androgen receptor antagonist, or an androgen receptor inverse agonist, a first coacervate is formed. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising an androgen receptor antagonist (e.g., bicalutamide), a first coacervate is formed.

[0055] In some embodiments, the formation of the first coagulation can be inhibited or reversed by contacting the engineered polypeptide with a coagulation regulator as described herein. In some embodiments, the first coagulation can be inhibited or reversed by contacting the engineered polypeptide with a coagulation regulator comprising a nuclear receptor ligand (e.g., a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist). In some embodiments, the first coagulation can be inhibited or reversed by contacting the engineered polypeptide with a coagulation regulator comprising an androgen receptor agonist, an androgen receptor antagonist, or an androgen receptor inverse agonist. In some embodiments, the first coagulation can be inhibited or reversed by contacting the engineered polypeptide with an androgen receptor antagonist (e.g., enzalutamide). In some embodiments, the first coagulation can be inhibited or reversed by contacting the engineered polypeptide with an androgen receptor agonist (e.g., DHT).

[0056] Estrogen receptor

[0057] In some embodiments, the condensate conversion domain described herein comprises an amino acid sequence or a fragment thereof of an estrogen receptor (ER). ER is a member of the steroid and nuclear receptor superfamily and is primarily expressed in estrogen target tissues, such as the endometrium, breast cancer cells, ovarian stromal cells, and hypothalamus. ER is a soluble protein that functions as an intracellular transcription factor. When bound to and activated by estrogen, ER undergoes conformational changes and post-translational modifications, dimerization, nuclear translocation, and ultimately binds to regulatory regions of target gene DNA, which are referred to as estrogen response elements (EREs).

[0058] There are two types of wild-type ER: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). ER is similar to AR, having a DNA binding domain (DBD) and an estrogen-dependent ligand binding domain (LBD). The amino acid sequence of the full-length wild-type estrogen receptor (ER) is shown in SEQ ID NO:5. The terms "estrogen receptor" or "ER" used herein encompass wild-type ER or ER variants (e.g., ER comprising at least one modification). The wild-type ER used herein refers to the full-length ER, an exemplary sequence of which is SEQ ID NO:5. The "ER variants" used herein encompass all different forms of the estrogen receptor (ER), which retain at least part of the N-terminal domain and are capable of phase separation or formation of condensates. Examples of ER variants include, but are not limited to, mutants, fragments, fusions, and splice variants. In some embodiments, the ER variant is resistant to at least one estrogen deprivation therapy.

[0059] In some embodiments, the ER variant comprises one or more mutations. In some embodiments, the ER variant comprises one or more mutations in the ligand binding domain (LBD). In some embodiments, one or more mutations in the LBD result in ER gain of function. In some embodiments, the ER variant comprises one or more mutations at a residue selected from K210A, R211E, L379R, G521R, C400V, M543A, and L544A, wherein the numbering is relative to SEQ ID NO: 5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274, or has an L379R mutation, compared to SEQ ID NO: 5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274 and has an L379R mutation, compared to SEQ ID NO: 5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274 and has an L379R mutation, compared to SEQ ID NO: 5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274, or has a G521R mutation, compared to SEQ ID NO: 5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274 and has a G521R mutation compared to SEQ ID NO:5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274, a C400V mutation, an M543A mutation, an L544A mutation, or a combination thereof compared to SEQ ID NO:5. In some embodiments, the ER variant comprises a truncation of amino acid residues 250-274, a C400V mutation, an M543A mutation, and an L544A mutation compared to SEQ ID NO:5. In some embodiments, the ER variant comprises a K210A mutation, an R211E mutation, an L379R mutation, a G521R mutation, a C400V mutation, an M543A mutation, an L544A mutation, a truncation of amino acid residues 250-274, or a combination thereof compared to SEQ ID NO:5.

[0060] In some embodiments, the ER variant lacks all or part of the DBD. For example, the ER variant lacks the DBD encoded by SEQ ID NO:7. In some embodiments, the ER variant lacking all or part of the DBD comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:8. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:5, comprising K210A, R211E, and G521R mutations. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:5, comprising K210A, R211E, L379R, C400V, M543A, and L544A mutations. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:5, comprising truncation of amino acid residues 250-274 and L379R mutation. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:5, including truncation of amino acid residues 250-274 and G521R mutation. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:5, including truncation of amino acid residues 250-274 and C400V, M543A and L544A mutations. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:9, including truncation of amino acid residues 44-163, truncation of amino acid residues 226-347 and truncation of amino acid residues 560-670. In some embodiments, the engineered polypeptide comprises at least one modification compared to SEQ ID NO:10, including truncation of amino acid residues 250-274.

[0061] In some embodiments, when the condensate conversion domain is contacted with the condensate regulator described herein, the condensate conversion domain comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the ER or its fragment can form a first condensate. The first condensate includes a non-membrane encapsulated compartment formed by phase separation (including all stages of phase separation) of one or more condensate conversion domains described herein. In some embodiments, the first condensate includes liquid-liquid condensate separation (LLPS). In some embodiments, the first condensate regulates molecules in the cell by enriching molecules in the first condensate. In some embodiments, the first condensate can be converted into a second condensate by contacting the engineered polypeptide with at least one additional condensate regulator. In some embodiments, the second condensate can be reversed back to the first condensate by contacting the engineered polypeptide with a third condensate regulator. In some embodiments, the condensate regulator includes a nuclear receptor ligand, which includes an ER agonist, an ER antagonist or an ER inverse agonist.

[0062] In some embodiments, when the engineered polypeptide is contacted with the coacervate regulator described herein, it is operatively contacted with at least one other coacervate conversion domain to form a first coacervate. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising a nuclear receptor ligand (including a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist), a first coacervate is formed. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist, a first coacervate is formed. In some embodiments, when the engineered polypeptide is contacted with a coacervate regulator comprising an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist, a first coacervate is formed.

[0063] In some embodiments, the first coacervate can be converted into a second coacervate by contacting the engineered polypeptide with an additional coacervate regulator described herein. In some embodiments, the first coacervate can be converted into a second coacervate by contacting the engineered polypeptide with a coacervate regulator comprising a nuclear receptor ligand (e.g., a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist). In some embodiments, the first coacervate can be converted into a second coacervate by contacting the engineered polypeptide with a coacervate regulator comprising an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist. In some embodiments, the first coacervate can be converted into a second coacervate by contacting the engineered polypeptide with an estrogen receptor antagonist (e.g., Fulvestrant).

[0064] Aggregate regulator

[0065] In some aspects, coacervate regulators are described herein. In some embodiments, coacervate regulators induce the formation of coacervates as described herein. For example, when a coacervate regulator is in contact with an engineered polypeptide as described herein, the first coacervate or the second coacervate can be induced to form by making the engineered polypeptide operatively contacted with at least one other coacervate conversion domain. In some embodiments, coacervate regulators can reverse the coacervates described herein. For example, the first coacervate or the second coacervate can be reversed by contacting a coacervate regulator with an engineered polypeptide, wherein the engineered polypeptide is part of the first coacervate or the second coacervate. In some embodiments, one or more coacervate regulators can be contacted with the same engineered polypeptide to induce the formation or conversion of coacervates. For example, a first coacervate regulator can be contacted with an engineered polypeptide, thereby operatively contacting the engineered polypeptide with at least one other coacervate conversion domain to form a first coacervate. In addition, additional coacervate regulators can be contacted with the engineered polypeptide to reverse the first coacervate or convert the first coacervate into a second coacervate. Figure 2 Examples of conversion between different aggregates within a cell mediated by the aggregate switching domains and aggregate modulators described herein are illustrated. In some embodiments, the aggregate modulator can be the second aggregate modulator, the third aggregate modulator, or the fourth aggregate modulator described herein.

[0066] In some embodiments, the coagulation regulator includes a molecule, such as a compound, a small molecule, a peptide, a nucleic acid, a sugar, a lipid, or a combination thereof. In some embodiments, the coagulation regulator is a ligand endogenous to the cell. In some embodiments, the coagulation regulator is a ligand exogenous to the cell. In some embodiments, the coagulation regulator includes a functional domain ligand. In some embodiments, the functional domain ligand may be a functional domain agonist. In some embodiments, the functional domain ligand may be a functional domain antagonist. In some embodiments, the functional domain ligand may be a functional domain inverse agonist. In some embodiments, the functional domain ligand may be an antigen bound to a functional domain comprising a CAR or TCR. Other exemplary functional domain ligands may include receptor tyrosine kinase ligands, such as insulin growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fibroblast growth factor (FGF).

[0067] In some embodiments, the condensate modulator includes a nuclear receptor ligand. In some embodiments, the nuclear receptor ligand is an endogenous ligand of the cell. In some embodiments, the nuclear receptor ligand is an exogenous ligand of the cell. In some embodiments, the nuclear receptor ligand includes a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist. For example, the nuclear receptor ligand can bind to and activate the nuclear receptor, including lipophilic substances such as endogenous hormones, vitamins A and D, and exogenous compound hormones. In some cases, the nuclear receptor ligand can be synthetic. For example, the nuclear receptor ligand can be a synthetic compound for binding to a condensate conversion domain comprising a nuclear receptor or a fragment thereof. In some embodiments, the synthetic compound can be bound to a condensate conversion domain comprising an orphan or adopted orphan nuclear receptor or a fragment thereof. Other nuclear receptor ligands for binding to condensate conversion domains comprising orphan receptors, such as FXR (farnesoid X receptor), LXR (liver X receptor), and PPAR (peroxisome proliferator-activated receptor), can include many metabolic intermediates with relatively low affinity, such as fatty acids, bile acids, or sterols.

[0068] In some embodiments, the cohesion regulator includes an androgen receptor agonist. In some embodiments, the cohesion regulator includes an androgen receptor antagonist. In some embodiments, the cohesion regulator includes an androgen receptor inverse agonist. In some embodiments, the cohesion regulator includes an endogenous androgen (e.g., testosterone, dihydrotestosterone, androstenedione, androstenediol, or dehydroepiandrosterone); synthetic androgens (e.g., methyltestosterone, methandrostenolone, nandrolone, trenbolone, oxandrolone, or stanozolol); selective androgen receptor modulators (e.g., andara or enroxolone); steroidal antiandrogens (e.g., cyproterone acetate, chlormadinone acetate, spironolactone, or oxendolone); nonsteroidal antiandrogens (e.g., flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, or RU-58841); or N-terminal domain antiandrogens (e.g., bisphenol A, EPI-001, ralaniten, or JN compounds). In some embodiments, the coacervate modulator is bicalutamide. In some embodiments, bicalutamide, when in contact with an engineered polypeptide described herein (e.g., an engineered polypeptide comprising a coacervate conversion domain containing an AR or a fragment thereof), causes the engineered polypeptide to be in operative contact with at least one other coacervate conversion domain to form a first coacervate. In some embodiments, the coacervate modulator is enzalutamide. In some embodiments, when enzalutamide is in contact with an engineered polypeptide, the formation of a first coacervate formed by the operative contact of the engineered polypeptide with at least one other coacervate conversion domain can be reversed.

[0069] In some embodiments, the coagulation regulator includes an estrogen receptor agonist. In some embodiments, the coagulation regulator includes an estrogen receptor antagonist. In some embodiments, the coagulation regulator includes an estrogen receptor inverse agonist. In some embodiments, the coagulation regulator includes endogenous estrogens (e.g., estradiol, estrone, estriol or estraterol); natural estrogens (e.g., conjugated estrogens); synthetic estrogens (e.g., ethinyl estradiol or diethylstilbestrol); mixed estrogens (agonist and antagonist modes of action); phytoestrogens (e.g., coumestrol, soy flavonoids, genistein or kurostol); selective estrogen receptor modulators (e.g., tamoxifen, clomiphene or raloxifene); antiestrogens (e.g., fulvestrant, ICI-164384 or ethoxytamoxifen). In some embodiments, the coagulation regulator is tamoxifen. In some embodiments, when tamoxifen is contacted with an engineered polypeptide described herein (e.g., an engineered polypeptide comprising an accretion conversion domain, the accretion conversion domain containing an estrogen receptor (ER) or a fragment thereof), the engineered polypeptide is brought into operative contact with at least one other accretion conversion domain, thereby forming a first accretion. In some embodiments, the accretion modulator is fulvestrant. In some embodiments, when fulvestrant is contacted with an engineered polypeptide, the first accretion formed by the operative contact of the engineered polypeptide with at least one other accretion conversion domain is converted into a second accretion, wherein the first accretion and the second accretion are different accretion. In some embodiments, the first accretion allows molecular exchange between the inside and outside of the first accretion. In some embodiments, the second accretion reduces molecular exchange between the inside and outside of the second accretion. Therefore, the transition from the first accretion to the second accretion can reduce or inhibit the enzyme activity associated with the molecule regulated by the first accretion or the second accretion.

[0070] Methods for regulating molecules

[0071] In some aspects, the method for delivering engineered polypeptides, systems comprising engineered polypeptides or engineered polynucleotides encoding engineered polypeptides described herein to cells is described herein. In some aspects, the method includes delivering the engineered polypeptides, systems or engineered polynucleotides to cells directly or indirectly. In some aspects, the method includes delivering engineered polynucleotides to cells, and then cells can express engineered polypeptides described herein. In some aspects, engineered polynucleotides can be delivered to cells by any transfection method described herein. In some aspects, engineered polynucleotides can be delivered to cells by using expression vectors (e.g., viral vectors). With regard to expression vectors, vectors can be easily introduced into cells described herein by any method in the art. For example, expression vectors can be converted into cells by physical, chemical or biological methods.

[0072] Physical methods for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells can include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, gene guns, electroporation, etc. Methods for preparing cells containing vectors and / or exogenous nucleic acids are suitable for the methods herein. One method for introducing engineered polynucleotides or vectors encoding the engineered polynucleotides into host cells is calcium phosphate transfection.

[0073] Chemical methods for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells can include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNA), liposomes or lipid nanoparticles. A typical colloidal system that can be used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). There are also other advanced nucleic acid targeted delivery methods available, such as using targeted nanoparticles or other suitable submicron-sized delivery systems to deliver engineered polynucleotides or vectors encoding engineered polynucleotides.

[0074] In the case of using non-viral delivery system, typical delivery vector is liposome.Consider using lipid formulations to introduce oligonucleotide or the vector encoding this oligonucleotide into cell (in vitro, in vitro or in vivo).On the other hand, engineered polynucleotide or the vector encoding this engineered polynucleotide can be combined with lipid.In some respects, the engineered polynucleotide relevant to lipid or the vector encoding this engineered polynucleotide is encapsulated in the aqueous phase inside of liposome, is dispersed in the lipid bilayer of liposome, is attached to liposome by the connecting molecule that is all associated with liposome and oligonucleotide, is embedded in liposome, is compounded with liposome, is dispersed in the solution containing lipid, is mixed with lipid, is combined with lipid, is included in lipid as suspension, comprises or is compounded with micelle or is otherwise relevant with lipid.Lipid, lipid / DNA or lipid / expression vector related composition are not limited to any specific structure in solution.For example, in some respects, they exist with double-layer structure, micelle or " folding " structure.Or, they are just dispersed in solution, may form the aggregate of size or shape inhomogeneity.Lipid is fatty substance, is naturally occurring or synthetic lipid in some respects. For example, lipids include naturally occurring fat droplets in the cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are usually stored at a temperature of about -20°C. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that covers a variety of monolayer and multilayer lipid carriers formed by closed lipid bilayers or aggregates. Liposomes generally have a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and encapsulate water and dissolved solutes between the lipid bilayers. However, compositions whose structure in solution is different from the normal vesicle structure are also included. For example, in some aspects, the lipids present a micellar structure or exist only as a heterogeneous aggregate of lipid molecules. Liposomal transfection reagent-nucleic acid complexes are also contemplated.

[0075] In some cases, non-viral delivery methods include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, exosomes, polycations or lipid-cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virosomes, and agent-enhanced polypeptide or DNA uptake. In some aspects, the delivery method includes conjugating or encapsulating the compositions or oligonucleotides described herein with at least one polymer (e.g., a natural polymer or a synthetic material). The polymer can be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, amine-containing polyoxaesters, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, grafted) of a variety of different monomers, such as two or more of lactic acid, lactide, glycolic acid, glycolide, ε-caprolactone, trimethylene carbonate, p-dioxanone, etc. For example, the scaffold can be composed of a polymer comprising glycolic acid and lactic acid, such as a polymer having a glycolic acid to lactic acid ratio of 90 / 10 or 5 / 95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragments derived from these components, elastin, laminin, decorin, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0076] In some aspects, the engineered polynucleotides described herein can be delivered to cells as a vector (e.g., a viral vector). Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. In some embodiments, other viral vectors are derived from slow viruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV), poxvirus vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some cases, retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or mouse stem cell virus (MSCV) genomes. In some cases, retroviral vectors also include slow viral vectors, such as vectors derived from human immunodeficiency virus (HIV) genomes. In some cases, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotypes. In some cases, the viral vector is a chimeric viral vector, comprising viral parts from two or more viruses. In other cases, the viral vector is a recombinant viral vector.

[0077] In some cases, the engineered polynucleotides encoding the engineered polypeptides described herein can be administered to a subject in need thereof by using a transgenic cell, wherein the transgenic cell is produced by first introducing the engineered polynucleotides into an allogeneic or autologous cell. In some cases, the cell can be isolated. In some aspects, the cell can be isolated from a subject.

[0078] In some embodiments, after the engineered polypeptide is contacted with the coacervate modulator, the engineered polypeptide can form a first coacervate or a second coacervate by operatively contacting at least one other coacervate conversion domain. The formation of the first coacervate or the second coacervate can be determined based on a suitable method, such as transfer diffusion nuclear magnetic resonance (STD NMR), affinity analysis, mass spectrometry, pull-down analysis, or live cell imaging by deconvolution microscopy, structured illumination microscopy, turbidity detection, optical tweezers system, microfluidics system or interference microscopy.

[0079] In some embodiments, the formation of the first coagulation or the second coagulation can be determined by live cell imaging. For example, the engineered polypeptide can be coupled to a detectable marker such as a fluorescent molecule so that the engineered polypeptide can be visualized under a microscope, and the fluorescence intensity can indicate the formation of the first coagulation or the second coagulation. For example, when the first coagulation or the second coagulation is not formed, the fluorescence intensity of the labeled engineered polypeptide is usually evenly distributed in the nucleus or cytoplasm. However, when the engineered polypeptide is contacted with a coagulation regulator, the formation of the first coagulation or the second coagulation can be observed under background contrast, forming bright spots or spots, wherein the bright spots or spots indicate the presence of the first coagulation or the second coagulation.

[0080] In some embodiments, the first condensate or the second condensate is formed mainly in the nucleus. In some embodiments, the first condensate or the second condensate is formed in the nucleus. In some embodiments, the first condensate or the second condensate is formed mainly in the organelle of the cell. In some embodiments, the first condensate or the second condensate is formed in the organelle of the cell. In some embodiments, the first condensate or the second condensate is formed mainly in the cytoplasm of the cell. In some embodiments, the first condensate or the second condensate is formed in the cytoplasm of the cell. In some embodiments, the first condensate or the second condensate is formed mainly in the cytoplasmic membrane of the cell. In some embodiments, the first condensate or the second condensate is formed in the cytoplasmic membrane of the cell. In some embodiments, since the engineered polypeptide has at least one modification, the first condensate or the second condensate is formed in the cytoplasm. For example, an engineered polypeptide comprising a condensate conversion domain containing an AR variant or an ER variant forms a first condensate or a second condensate in the cytoplasm, which is opposite to the condensate formed by the wild-type AR or ER, which occurs in the nucleus of the cell.

[0081] In some embodiments, the first coacervate can be used to sense a rapid, adaptive, or reversible response. In some embodiments, the first coacervate can be used to buffer the concentration of molecules, such as molecules regulated by the first coacervate. When the concentration of the regulated molecule increases to exceed the saturation concentration, it will cause the regulated molecule to coagulate. In some embodiments, the first coacervate can be used to locally concentrate molecules in various compartments or locations within the cell. Increasing the local concentration of key enzymes or protein complexes can accelerate biochemical reactions. In some embodiments, the first coacervate can be used to prevent reactions or for inactivation. If a key component is recruited into the first coacervate, but all other components of the enzymatic reaction or signaling event remain outside the first coacervate, the reaction or signaling event will be inhibited or slowed down. In some embodiments, the first coacervate can be used to form a physicochemical and mechanical filter, in which the pore size is determined by the number and dynamics of cross-links between the pores of the first coacervate.

[0082] In some embodiments, the first coacervate comprises a liquid coacervate. In some embodiments, the liquid coacervate comprises a liquid-liquid coacervate separation phase (LLPS). In some embodiments, the liquid coacervate comprises a coacervate. In some embodiments, the liquid coacervate comprises a membraneless compartment. In some embodiments, the first coacervate comprising a liquid coacervate can be fused with other liquid coacervates or droplets in a cell. In some embodiments, the first coacervate comprising a liquid coacervate does not have a clear shape. In some embodiments, the first coacervate comprising a liquid coacervate has a spherical shape. In some embodiments, the first coacervate comprising a liquid coacervate has an amorphous shape. In some embodiments, the first coacervate comprises a fluidity determined by turbidity, viscosity, surface tension, or a combination thereof.

[0083] In some embodiments, the first coacervate can regulate the molecules in the cell by accumulating the molecules in the first coacervate, thereby increasing the concentration of molecules in the cell. In some embodiments, the increase in the concentration of the regulating molecules in the first coacervate can increase the enzyme activity associated with the regulated molecules. In some embodiments, the first coacervate allows the molecules inside the first coacervate to move. In some embodiments, the first coacervate allows the exchange of molecules between the inside and outside of the first coacervate. The molecules regulated by the first coacervate (or the second coacervate) can be exogenous molecules, endogenous molecules, or a combination thereof. In some embodiments, the regulated molecules include at least one enzyme, at least one compound, at least one biomolecule, at least one nucleic acid, at least one lipid, at least one protein, at least one sugar, at least one metabolite, or a combination thereof. In some embodiments, the regulated molecules include at least one transcription factor.

[0084] In some embodiments, the first coacervate increases enzyme activity by regulating and bringing two or more molecules into close proximity in the first coacervate, thereby increasing the enzyme activity associated with the regulated molecule. In some embodiments, the first coacervate regulates the concentration of the molecule in the cell by enriching or isolating at least one molecule inside it. In some embodiments, the concentration of at least one molecule inside the first coacervate increases relative to the concentration of at least one molecule outside the first coacervate. In some embodiments, the concentration increases by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the concentration of at least one molecule inside the first coacervate decreases relative to the concentration of at least one molecule outside the first coacervate. In some embodiments, the concentration decreases by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the viscosity of the first coacervate is at least increased by 10%, at least increased by 20%, at least increased by 30%, at least increased by 40%, at least increased by 50%, at least increased by 60%, at least increased by 70%, at least increased by 80%, at least increased by 90%, or at least increased by 99% compared to a comparable first coacervate formed by a comparable polypeptide without at least one modification. In some embodiments, the viscosity of the first coacervate is at least decreased by 10%, at least decreased by 20%, at least decreased by 30%, at least decreased by 40%, at least decreased by 50%, at least decreased by 60%, at least decreased by 70%, at least decreased by 80%, at least decreased by 90%, or at least decreased by 99% compared to a comparable first coacervate formed by a comparable polypeptide without at least one modification. In some embodiments, the first coacervate comprises at least 10% increased surface tension, at least 20% increased surface tension, at least 30% increased surface tension, at least 40% increased surface tension, at least 50% increased surface tension, at least 60% increased surface tension, at least 70% increased surface tension, at least 80% increased surface tension, at least 90% increased surface tension, or at least 99% increased surface tension compared to a similar first coacervate formed by a similar polypeptide without at least one modification. In some embodiments, the first coacervate has at least 10% lower surface tension, at least 20% lower surface tension, at least 30% lower surface tension, at least 40% lower surface tension, at least 50% lower surface tension, at least 60% lower surface tension, at least 70% lower surface tension, at least 80% lower surface tension, at least 90% lower surface tension, or at least 99% lower surface tension compared to a similar first coacervate formed by a similar polypeptide without at least one modification.In some embodiments, the first coacervate comprises at least 10% increased turbidity, at least 20% increased turbidity, at least 30% increased turbidity, at least 40% increased turbidity, at least 50% increased turbidity, at least 60% increased turbidity, at least 70% increased turbidity, at least 80% increased turbidity, at least 90% increased turbidity, or at least 99% increased turbidity compared to a similar first coacervate formed by a similar polypeptide without at least one modification. In some embodiments, the first coacervate comprises at least 10% lower turbidity, at least 20% lower turbidity, at least 30% lower turbidity, at least 40% lower turbidity, at least 50% lower turbidity, at least 60% lower turbidity, at least 70% lower turbidity, at least 80% lower turbidity, at least 90% lower turbidity, or at least 99% lower turbidity compared to a similar first coacervate formed by a similar polypeptide without at least one modification. In some embodiments, the first coacervate increases the rate of molecular exchange between its interior and exterior by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the rate of molecular exchange between the interior and exterior of a similar first coacervate formed by a similar polypeptide without at least one modification. In some embodiments, the first coacervate decreases the rate of molecular exchange between the interior and exterior of the first coacervate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the rate of molecular exchange between the interior and exterior of a similar first coacervate formed by a similar polypeptide without at least one modification. In some embodiments, the likelihood that the first aggregate accumulates in the cytoplasm of a cell is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to a similar first aggregate formed by a similar polypeptide without at least one modification.

[0085] In some embodiments, the first coacervate can be converted into a second coacervate as described herein. For example, the first coacervate can be converted into a second coacervate by contacting an engineered polypeptide comprising an ER or fragment thereof coacervate switching domain with a coacervate modulator comprising fulvestrant. In some embodiments, the second coacervate includes a coacervate that reduces molecular movement within the second coacervate. In some embodiments, the second coacervate includes a coacervate that reduces molecular exchange between the inside and outside of the second coacervate. In some embodiments, the second coacervate includes a solid coacervate. In some embodiments, the second coacervate includes a semi-solid coacervate. In some embodiments, the second coacervate includes a gel coacervate. In embodiments, the second coacervate can be reversed back to the first coacervate by contacting the engineered polypeptide with at least one additional coacervate modulator. In some embodiments, the second coacervate can be dissolved by contacting the engineered polypeptide with at least one additional coacervate modulator.

[0086] Treatment

[0087] Provided herein are methods for treating a disease or condition in a subject by administering an engineered polypeptide, an engineered polynucleotide, a cell transduced with an engineered polynucleotide, a system, a pharmaceutical composition, or a combination thereof to the subject. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, inhaled, etc.). In some embodiments, the subject is human. In some embodiments, the disease or condition includes: CAR-related diseases such as hematological malignancies, malignant tumors, leukemia, multiple myeloma, malignant lymphoma, etc.; kinase-related diseases such as autoimmune diseases, heteroimmune diseases, cancer or thromboembolic diseases; TCR-related diseases such as hematological malignancies, malignant tumors, leukemia, pancreatic malignancies, malignant lymphoma, etc.; metabolic diseases such as protein metabolism disorders, sugar metabolism disorders, diabetes, ester metabolism disorders, water and electrolyte metabolism disorders, inorganic element metabolism disorders, purine metabolism disorders, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy deficiency, malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency, osteoporosis; transcription factor-related diseases such as breast cancer, prostate cancer, uterine fibroids, lung cancer, head and neck cancer, leukemia, pancreatic malignancies, hematological malignancies, malignant tumors, lymphoma, etc.

[0088] In some embodiments, engineered polypeptide, engineered polynucleotide, cells transduced with the engineered polynucleotide, systems, pharmaceutical compositions or combinations thereof can be applied to the subject with suitable dosage, mode of administration and frequency according to the expected effect. In some embodiments, this application is carried out at least once in a period of time (for example, every 2 days, twice a week, once a week, weekly, three times a month, twice a month, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is applied twice or more times (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times) in a period of time. In some embodiments, this application includes being carried out by various forms and approaches with a therapeutically effective amount, such as oral or topical application. In some embodiments, engineered polypeptides, engineered polynucleotides, cells transduced with engineered polynucleotides, systems, pharmaceutical compositions, or combinations thereof can be administered parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, subarachnoidally, intraocularly, intrasternally, ophthalmologically, endothelially, topically, intranasally, intrapulmonaryly, rectally, intraarterially, intrathecally, inhaled, intralesionally, intradermally, epidurally, intracapsularly, subcapsularly, intracardially, transtracheally, subcutaneously, subarachnoidally, or intraspinally (e.g., injected or infused). In some embodiments, compositions can be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa administration). In some embodiments, engineered polypeptides, engineered polynucleotides, cells transduced with engineered polynucleotides, systems, pharmaceutical compositions, or combinations thereof are delivered by a variety of administration routes.

[0089] Engineered polypeptides, engineered polynucleotides, cells transduced with engineered polynucleotides, systems, pharmaceutical compositions, or combinations thereof can be administered in a localized manner, for example, by direct injection of the agent into an organ, optionally in the form of a reservoir or sustained release formulation or implant. Engineered polypeptides, engineered polynucleotides, cells transduced with engineered polynucleotides, systems, pharmaceutical compositions, or combinations thereof can be provided in the form of a rapid release formulation, an extended release formulation, or an intermediate release formulation.

[0090] The engineered polypeptides, engineered polynucleotides, cells transduced with engineered polynucleotides, systems, pharmaceutical compositions, or combinations thereof provided herein can be administered in combination with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. Actual dosage levels may vary to obtain a dosage that achieves the desired therapeutic response for a specific subject, composition, and mode of administration without causing toxicity to the subject. The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic and / or preventive response). For example, a single large dose may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased according to the urgency of the treatment situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and dosage uniformity. The dosage unit form used herein refers to a physically independent unit suitable for providing to a subject (e.g., a subject for immunization or a subject for treatment) as a unit dose; each unit contains a calculated predetermined amount of active agent to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. In some embodiments, the engineered polypeptide, engineered polynucleotide, cell transduced with an engineered polynucleotide, system, pharmaceutical composition, or combination thereof can be repeatedly administered to a subject in need thereof.

[0091] In some cases, administration of an engineered polypeptide, an engineered polynucleotide, a cell transduced with an engineered polynucleotide, a system, a pharmaceutical composition, or a combination thereof is sufficient to alleviate at least one symptom of a disease or condition, treat a disease or condition, and / or eliminate a disease or condition. In some cases, improvement of a disease or condition can be determined by any of the diagnostic tests provided.

[0092] Pharmaceutical composition

[0093] Pharmaceutical compositions comprising engineered polypeptides, engineered polynucleotides, cells transduced with engineered polypeptides, systems, or combinations thereof are described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent, or nebulizer. In some embodiments, the pharmaceutical composition comprises at least one coacervate modulator. In some embodiments, the pharmaceutical composition comprises at least one other active agent. In some embodiments, the active agent may be a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an anti-hormone agent, an anti-angiogenic agent, a cardioprotective agent, or a checkpoint inhibitor.

[0094] In implementing the treatment or use methods provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is applied to a subject suffering from a disease, disorder or condition to be treated, such as cancer. In some embodiments, the subject is a human being. The therapeutically effective amount may vary greatly due to the severity of the disease, the age and relative health of the subject, the effectiveness of the pharmaceutical composition used, and other factors. In some aspects, the pharmaceutical composition described herein can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0095] The pharmaceutical compositions described herein can be administered to a subject by an appropriate route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal administration. The compositions described herein can include but are not limited to aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multi-particulate formulations, and mixed immediate release and controlled release formulations.

[0096] The pharmaceutical composition can be manufactured in a conventional manner, for example, by conventional mixing, dissolving, granulating, making dragees, grinding, emulsifying, encapsulating, embedding or compression processes. The pharmaceutical composition may include at least one exogenous therapeutic agent as an active ingredient, which is in the form of a free acid or free base, or in the form of a pharmaceutically acceptable salt. In addition, the methods and compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds with the same type of activity. In some embodiments, the therapeutic agent exists in a non-solvated form, or in a solvated form with a pharmaceutically acceptable solvent (e.g., water, ethanol, etc.). The solvated form of the therapeutic agent is also considered to be disclosed herein.

[0097] In certain embodiments, the pharmaceutical compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as mercurophene and thimerosal; stabilized chlorine dioxide; quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride. In some embodiments, the pharmaceutical compositions described herein benefit from antioxidants, metal chelators, thiol-containing compounds and other general stabilizers.

[0098] Pharmaceutical composition described herein can be formulated into any suitable dosage form, including but not limited to aqueous oral dispersion, liquid, gel, syrup, elixir, slurry, suspension, solid oral dosage form, aerosol, controlled release preparation, fast dissolving preparation, effervescent preparation, lyophilized preparation, tablet, powder, pill, dragee, capsule, delayed release preparation, extended release preparation, pulse release preparation, multi-granule preparation and mixed quick release and controlled release preparation.On the one hand, therapeutic agent as discussed herein, for example therapeutic agent is formulated into the pharmaceutical composition suitable for intramuscular, subcutaneous or intravenous injection.On the one hand, preparation suitable for intramuscular, subcutaneous or intravenous injection includes physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for rehydration into sterile injectable solution or dispersion. The example of suitable aqueous and non-aqueous carrier, diluent, solvent or excipient comprises water, ethanol, polyol (propylene glycol, polyethylene glycol, glycerine, Cremophor etc.), their suitable mixture, vegetable oil (for example olive oil) and injectable organic ester such as ethyl oleate.Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using a surfactant.In some embodiments, the preparation suitable for subcutaneous injection also contains additives such as preservative, wetting agent, emulsifier and dispersant.Can ensure to prevent the growth of microorganisms by various antibacterial and antifungal agents (for example parahydroxybenzoate, chlorobutanol, phenol, sorbic acid etc.).In some cases, it is necessary to add isotonic agent, for example sugar, sodium chloride etc..By using the medicament with delayed absorption effect such as aluminum monostearate and gelatin etc., the long-acting absorption of injection medicine can be realized.

[0099] For intravenous injection or infusion or infusion, the pharmaceutical compositions described herein are formulated as aqueous solutions, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants suitable for the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably containing physiologically compatible buffers or excipients. Such excipients are known.

[0100] Parenteral injection may involve a push injection or a continuous infusion. Pharmaceutical compositions for injection may be present in unit dosage form, such as in ampoules or multidose containers, and preservatives may be added. The compositions described herein may be in a form suitable for parenteral injection, such as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and may contain a formulation agent, such as a suspending agent, a stabilizer and / or a dispersant. On the one hand, the active ingredient is in powder form so that it can be mixed with a suitable carrier (e.g., sterile pyrogen-free water) before use.

[0101] For administration by inhalation, the therapeutic agent will be formulated into an aerosol, mist or powder for use. The pharmaceutical compositions described herein can be conveniently delivered in the form of an aerosol spray from a pressurized bag or nebulizer, and use a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For pressurized aerosols, the dosage unit can be determined by providing a valve that can deliver a quantitative amount. Capsules and cartridges for inhalers or insufflators can be formulated, such as (for example only) gelatin, which contains a powder mixture of the therapeutic agent described herein and a suitable powder matrix (such as lactose or starch). The preparation containing a certain ingredient is prepared as a saline solution, using benzyl alcohol or other suitable preservatives, fluorocarbons and / or other solubilizers or dispersants known in the art. Preferably, these compositions and preparations are prepared using suitable non-toxic pharmaceutically acceptable ingredients. The selection of a suitable carrier depends on the exact nature of the desired nasal dosage form, such as a solution, suspension, ointment or gel. In addition to the active ingredient, the nasal dosage form generally contains a large amount of water. Minor amounts of other ingredients may optionally be included, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents or buffers, and other stabilizers and solubilizers.Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0102] By mixing one or more solid excipients with one or more compositions described herein, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores, a pharmaceutical preparation for oral administration is obtained. Suitable excipients include, for example, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; or others, such as polyvinyl pyrrolidone (PVP or povidone) or calcium phosphate. If necessary, disintegrants such as cross-linked sodium carboxymethylcellulose, polyvinyl pyrrolidone, agar, alginic acid or its salts, such as sodium alginate, can be added. In some embodiments, dyes or pigments are added to tablets or dragee coatings for identification or characterization of different combinations of active therapeutic agent doses.

[0103] In some embodiments, the pharmaceutical composition of the exogenous therapeutic agent is in the form of a capsule, including a push-in capsule made of gelatin, and a soft sealed capsule made of gelatin and a plasticizer (e.g., glycerol or sorbitol). The push-in capsule contains the active ingredient and a filler (e.g., lactose), a binder (e.g., starch), and / or a lubricant (e.g., talc or magnesium stearate) and an optional stabilizer. In a soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. For example, a capsule can be prepared by placing a large amount of a mixture of a therapeutic agent formulation inside a capsule. In some embodiments, the preparation (non-aqueous suspension and solution) is placed in a soft gelatin capsule. In other embodiments, the preparation is placed in a standard gelatin capsule or a non-gelatin capsule, such as a capsule containing HPMC. In other embodiments, the preparation is placed in a dusting capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents are sprinkled on food before consumption.

[0104] The pharmaceutical composition for oral administration has a dosage suitable for such administration. On the one hand, the solid oral dosage form is prepared by mixing the composition with one or more of the following: antioxidants, flavoring agents and carrier materials, such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents and diluents. In some embodiments, the solid dosage form disclosed herein is in the form of tablets (including suspension tablets, fast-dissolving tablets, bite-disintegrating tablets, rapidly disintegrating tablets, effervescent tablets or caplets), pills, powders, capsules, solid dispersions, solid solutions, bioerodible dosage forms, controlled release formulations, pulse release dosage forms, multi-particulate dosage forms, microspheres, micropellets, and granules. In other embodiments, the composition is in the form of a powder. Tableting is a solid dosage form formed by compressing the mixture of the above formula. In various embodiments, the tablet will contain one or more flavoring agents. In other embodiments, the tablet will be wrapped with a film after the final tableting. In some embodiments, the film coating can provide delayed release of the therapeutic agent from the preparation. In other embodiments, the film coating contributes to patient compliance. The weight of the film coating is usually about 1% to 3% of the tablet weight. In some embodiments, solid dosage forms (e.g., tablets, effervescent tablets, and capsules) are prepared by mixing particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk mixed composition. The mixture is easily divided into unit dosage forms of equal efficacy, such as tablets, pills, and capsules. In some embodiments, a single unit dose includes a film coating. These preparations are manufactured by conventional formulation techniques.

[0105] In another aspect, the dosage form includes a microcapsule formulation. In some embodiments, one or more other compatible materials are present in the microcapsule material. Non-limiting examples of materials include pH regulators, corrosion inhibitors, defoamers, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0106] Liquid dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels and syrups. In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as: (a) a disintegrant; (b) a dispersant; (c) a wetting agent; (d) at least one preservative, (e) a viscosity increasing agent, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further comprises a crystal formation inhibitor.

[0107] In some embodiments, the pharmaceutical composition described herein is a self-emulsifying drug delivery system (SEDDS). An emulsion is an immiscible phase dispersed in another phase, usually in the form of droplets. In general, an emulsion is produced by violent mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously forms an emulsion when added to excess water without any external mechanical dispersion or stirring. An advantage of SEDDS is that the droplets can be distributed throughout the solution by gentle mixing. In addition, water or aqueous coacervates are optionally added before administration, which ensures the stability of unstable or hydrophobic active ingredients. Therefore, SEDDS provides an effective drug delivery system for oral and parenteral administration of hydrophobic active ingredients. In some embodiments, SEDDS improves the bioavailability of hydrophobic active ingredients.

[0108] Buccal formulations can be administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein may also include a bioerodible (hydrolyzable) polymer carrier, which is also used to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the composition can be in the form of tablets, lozenges, or gels formulated in a conventional manner.

[0109] For intravenous administration, the pharmaceutical composition is optionally formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants are formulated with ingredients appropriate to the barrier to be permeated. For other parenteral injections, appropriate formulations include aqueous or non-aqueous solutions, preferably containing physiologically compatible buffers or excipients.

[0110] Parenteral injections may optionally include push injections or continuous infusions. Injectable dosage forms may optionally be presented in unit dosage form, such as ampoules or multidose containers, and preservatives may be added. In some embodiments, the compositions described herein are in a form suitable for parenteral injection, as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulations such as suspending agents, stabilizers and / or dispersants. Compositions for parenteral administration include aqueous solutions of agents that regulate carotid body activity in water-soluble form. In addition, suspensions of agents that regulate carotid body activity, such as oily injection suspensions, may be optionally prepared as needed.

[0111] Conventional formulation techniques include a combination of one or more of the following methods: (1) dry blending, (2) direct compression, (3) grinding, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) melt granulation. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Wurster coating), tangential coating, top spraying, tableting, extrusion, etc.

[0112] In some embodiments, a composition comprising particles of a therapeutic agent and at least one dispersing agent or suspending agent is provided for oral administration to a subject. The formulation can be a powder and / or granules for suspension, and after mixing with water, a substantially uniform suspension can be obtained.

[0113] In addition, the pharmaceutical composition optionally contains one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethylaminomethane); and buffers such as citrate / glucose, sodium bicarbonate, and ammonium chloride. The content of such acids, bases, and buffers should maintain the pH value of the composition within an acceptable range.

[0114] In addition, the pharmaceutical composition optionally comprises one or more salts in an amount such that the osmotic pressure of the composition is within an acceptable range. Such salts include salts having sodium, potassium or ammonium cations and chloride, citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfuric acid, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0115] Other pharmaceutical compositions optionally contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as mercurophene and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0116] In one embodiment, the aqueous suspensions and dispersions described herein remain in a uniform state for at least 4 hours. In one embodiment, the aqueous suspension is resuspended into a uniform suspension by physical agitation that lasts less than 1 minute. In another embodiment, no agitation is required to maintain a uniform aqueous dispersion.

[0117] Aerosols for nasal administration are typically an aqueous solution that is injected into the nasal cavity in the form of drops or sprays. Nasal solutions are similar to nasal secretions in that they are generally isotonic and lightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range may also be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0118] Reagent test kit

[0119] In some embodiments, disclosed herein are kits for using the engineered polypeptides, engineered polynucleotides, systems, pharmaceutical compositions described herein. In some embodiments, the kits disclosed herein can be used to treat a disease or condition in a subject. In some embodiments, the kits comprise a combination of a set of materials or components in addition to the engineered polypeptides, engineered polynucleotides, systems, pharmaceutical compositions.

[0120] In some embodiments, the kit described herein includes instructions for administering engineered polypeptides, engineered polynucleotides, systems, and pharmaceutical compositions to a subject to treat a disease or condition of the subject. In some embodiments, the kit also includes additional therapeutic agents. In some embodiments, the kit described herein includes components for forming a first coacervate or a second coacervate or for determining the formation of a first coacervate or a second coacervate. In some embodiments, the kit described herein includes components for determining a molecule regulated by a first coacervate or a second coacervate. In some embodiments, the kit includes components for determining the concentration of an enzyme activity or a regulatory molecule associated with a regulatory molecule. In some embodiments, the kit includes components for performing protein blotting, enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, and qPCR detection. The exact nature of the components configured in the kit depends on its intended use. For example, some embodiments are configured to treat a disease or condition disclosed herein in a subject.

[0121] The kit may include instructions for use. In some embodiments, the kit includes instructions for administering an engineered polypeptide, engineered polynucleotide, system, or pharmaceutical composition to a subject in need thereof. Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipettes, or measuring tools, bandage materials, or other useful appliances. The materials or components assembled in the kit can be stored and provided to the practitioner in any convenient and appropriate manner to maintain their operability and practicality. For example, the ingredients can be dissolved, dehydrated, or lyophilized; they can be provided at room temperature, refrigerated, or frozen temperatures. These components are typically contained in suitable packaging materials.

[0122] The use of absolute or ordinal terms, such as "will," "will not," "should," "should not," "must not," "must not," "first," "initially," "next," "subsequently," "before," "after," "last," and "ultimately" are not intended to limit the scope of the embodiments disclosed herein, but are merely exemplary.

[0123] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "includes," "comprising," "having," "having," "with," or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0124] As used herein, the phrases "at least one", "one or more", and "and / or" are open-ended expressions that are both connective and selective in semantics. For example, each of the expressions "at least one of A, B, and C", "at least one of A, B, or C", "one or more of A, B, and C", "one or more of A, B, or C", and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0125] As used herein, "or" may mean "and", "or" or "and / or", and may be used exclusively or inclusively. For example, the term "A or B" may mean "A or B", "is A not B", "is B not A", and "A and B". In some cases, the context may dictate the specific meaning.

[0126] Any systems, methods, software, and platforms described herein are modular. Therefore, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of action.

[0127] When referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximate value within the experimental variability range (or within the statistical experimental error range), and the number or numerical range can vary from the number or numerical range, for example, from 1% to 15% of the number or numerical range. In the example, the term "about" refers to ±10% of the specified number or value.

[0128] The terms "increase", "increased", or "improvement" used herein generally represent an increase in a statistically significant amount. In some aspects, the term "increase" or "increased" means an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100% or any increase between 10-100%. Other examples of "increase" include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times or more compared to a reference level.

[0129] The term "reduce", "reduce" or "reduce" used herein generally refers to the reduction of statistically significant amount. In some respects, "reduce" or "reduce" means to reduce at least 10% compared to a reference level, for example, reduce at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% reduction (for example, there is no level or undetectable level compared to a reference level), or any reduction between 10-100% compared to a reference level. In the context of a marker or symptom, these terms refer to the statistically significant decline of this level. The reduction amount can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to the level in the acceptable normal range for an individual not suffering from a particular disease.

[0130] The terms "individual" or "subject" are used interchangeably and encompass mammals. Non-limiting examples of mammals include any member of the class of mammals: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents such as rats, mice and guinea pigs, etc. The mammal can be a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, such as a rodent, such as a rat or a mouse. "Patient" as used herein refers to a subject suffering from or diagnosed with a disease or condition described herein.

[0131] The term "percent (%) identity" as used herein generally refers to the percentage of amino acid (or nucleic acid) residues that are identical between a candidate sequence and a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent identity (e.g., gaps may be introduced in one or both of the candidate and reference sequences to achieve optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment, for determining percent identity can be achieved in a variety of well-known ways. The percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acid or nucleotide at the same position in the comparison sequence, and then dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0132] As used herein, the term "in vivo" may be used to describe events that occur in an organism, such as the body of a subject.

[0133] As used herein, the term "ex vivo" may be used to describe an event that occurs outside of an organism (e.g., a subject's body). An "ex vivo" experiment cannot be performed on a subject. Instead, it can be performed on a sample that has been separated from the subject. Ex vivo may be used to describe an event that occurs in intact cells outside of the subject.

[0134] As used herein, the term "in vitro" may be used to describe an event that occurs in a container used to hold laboratory reagents, such that the container is separated from the living biological source organism from which the material was obtained. In vitro assays may include cell-based assays, in which living or dead cells are employed. In vitro assays may also include cell-free assays, in which intact cells are not used.

[0135] As used herein, "treat," "therapeutic," or "therapy" refers to the alleviation or elimination of a disorder, disease, or condition; or one or more symptoms associated with a disorder, disease, or condition; or the alleviation or elimination of the cause of the disorder, disease, or condition itself. The desired effects of treatment may include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis.

[0136] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and generally refer to an amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), comprising a system of the present disclosure, sufficient to produce the desired activity upon administration to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay manifestation, prevent progression, alleviate or mitigate at least one symptom of a disease treated by the methods of the present disclosure.

[0137] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refer to pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. An ingredient can be "pharmaceutically acceptable" if it is compatible with the other ingredients in the pharmaceutical composition. It can also be suitable for contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, and with a reasonable benefit / risk ratio.

[0138] As used herein, the terms "administer", "give" and variants thereof refer to the introduction of a composition or medicament into a subject, and include simultaneous and sequential introduction of a composition or medicament. The introduction of a composition or medicament into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic or topical. Management includes self-management and management by others. Suitable routes of administration can enable the composition or medicament to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or medicament into the vein of the subject. Administration can be performed by any suitable route. In some embodiments, administration is intravenous administration. In some embodiments, administration is pulmonary administration. In some embodiments, administration is inhalation.

[0139] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The present invention is not limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein should not be construed as restrictive. Those skilled in the art will now appreciate many variations, changes and substitutions without departing from the present invention. In addition, it should be understood that the various aspects of the present invention are not limited to the specific descriptions, configurations or relative proportions described herein, which depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted when practicing the present invention. Therefore, the present invention should also encompass any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and are intended to encompass methods and structures within the scope of these claims and their equivalents.

[0140] Example

[0141] The following illustrative examples represent embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.

[0142] Example 1. Engineered polypeptide-mediated aggregate conversion

[0143] The engineered polypeptides comprising the AR or ER condensate switching domain described herein are further fused to a reporter gene (GFP) and transduced into cells to monitor the formation of the first condensate or the conversion of the first condensate to the second condensate. Briefly, 293T cells (ATCC) were transfected with AR (SEQ ID NO: 1)-(△617-633)-W742L plasmid by Lipofectamine 3000 transfection reagent for 48 hours, and cell images were taken using a Leica THUNDER imaging system ( Figure 3B Left), and then the cells were photographed 6 hours after the addition of bicalutamide ( Figure 3B Middle), and then enzalutamide was added to the cells for 6 hours and the pictures were taken ( Figure 3BRight). The engineered polypeptides containing the AR or ER condensate conversion domain described in this article were further fused with a reporter gene (GFP) and a functional domain, and then transduced into cells to test changes in the signal cascade reaction after the first condensate was formed. Briefly, 293T cells (ATCC) were transfected with AR (SEQ ID NO: 1)-(△44-163, 226-347, 560-670)-W742L-GFP-functional domain plasmid or ER (SEQ ID NO: 5)-(△250-274)-L379R-GFP-functional domain plasmid using Lipofectamine 3000 reagent for 24 hours. After adding bicalutamide or fulvestrant for 1 hour, the cells were harvested and the phosphorylation levels of downstream signaling proteins were detected by Western blot ( Figure 3C ). The engineered polypeptide containing the AR condensate conversion domain described in this article was further fused with a reporter gene (GFP) and a functional domain, and then transduced into cells to test changes in the signal cascade reaction when the first condensate is formed or converted from the first condensate to the second condensate. Briefly, the AR (SEQ ID NO: 1)-(△44-163, 226-347, 560-670)-W742L-GFP-functional domain plasmid was transfected into 293T cells (ATCC) using Lipofectamine 3000 reagent for 24 hours, and after adding bicalutamide for 1 hour, or adding bicalutamide for 1 hour and enzalutamide for 3 hours, the cells were harvested and the phosphorylation levels of downstream signal proteins were detected by Western blot ( Figure 3D ).

[0144] Although the aforementioned disclosure has been described in more detail for the purpose of clarity and understanding, it will be clear to those skilled in the art by reading the disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all techniques and equipment described above can be used in various combinations. All publications, patents, patent applications and / or other documents cited in this application are incorporated by reference in their entirety for all purposes, and the degree of citation is as if each individual publication, patent, patent application and / or other document is individually and separately indicated as being used for all purposes and incorporated herein by reference.

Claims

1. An engineered polypeptide comprising: Functional domains; and The condensate switching domain comprises the amino acid sequence of a nuclear receptor or a fragment thereof.

2. The engineered polypeptide of claim 1, wherein the engineered polypeptide forms a first condensate within a cell by being in operative contact with at least one additional condensate switching domain.

3. The engineered polypeptide of claim 1, wherein after contacting with an aggregate regulator, the engineered polypeptide forms a first aggregate in the cell through operative contact between the engineered polypeptide and at least one additional aggregate conversion domain.

4. The engineered polypeptide of claim 2 or 3, wherein the first condensate mediates liquid-liquid phase separation (LLPS).

5. The engineered polypeptide of claim 2 or 3, wherein the first condensate comprises a liquid condensate.

6. The engineered polypeptide of claim 2 or 3, wherein the first aggregate allows for exchange of molecules between the interior and exterior of the first aggregate.

7. The engineered polypeptide of claim 2 or 3, wherein the first condensate allows molecules to move within the first condensate.

8. The engineered polypeptide of claim 3, wherein upon removal of the aggregate modulator, the first aggregate is reversed.

9. The engineered polypeptide of claim 2 or 3, wherein the first aggregate is reversed when the engineered polypeptide is contacted with a second aggregate modulator.

10. The engineered polypeptide of claim 2 or 3, wherein the first coacervate comprises a liquid coacervate or a liquid-like coacervate.

11. The engineered polypeptide of any of the preceding claims, wherein when the engineered polypeptide is contacted with a third aggregate modulator, the first aggregate is converted to a second aggregate.

12. The engineered polypeptide of claim 11, wherein upon removal of the third aggregate modulator, the second aggregate reverts back to the first aggregate.

13. The engineered polypeptide of claim 11, wherein when the engineered polypeptide is contacted with a fourth aggregate modulator, the second aggregate is reversed back to the first aggregate.

14. The engineered polypeptide of claim 11, wherein the second aggregate comprises a solid-like aggregate or a semi-solid-like aggregate.

15. The engineered polypeptide of claim 11, wherein the second aggregate comprises a gel-like aggregate.

16. The engineered polypeptide of claim 11, wherein the second aggregate inhibits the exchange of molecules between the interior and exterior of the second aggregate.

17. The engineered polypeptide of claim 11, wherein the second aggregate inhibits the movement of molecules within the second aggregate.

18. The engineered polypeptide of any of the preceding claims, wherein the nuclear receptor or fragment thereof comprises an androgen receptor (AR) or a fragment thereof.

19. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:

1.

20. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

2.

21. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

4.

22. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO:

3.

23. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO:

9.

24. The engineered polypeptide of any one of claims 1-17, wherein the nuclear receptor or fragment thereof comprises an estrogen receptor (ER) or a fragment thereof.

25. The engineered polypeptide of any one of claims 1-17, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

5.

26. The engineered polypeptide of any one of claims 1-17, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

6.

27. The engineered polypeptide of any one of claims 1-17, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

8.

28. The engineered polypeptide of any one of claims 1-17, wherein the nuclear receptor or fragment thereof comprises an amino acid sequence at least 70% identical to SEQ ID NO:

7.

29. The engineered polypeptide of claim 18, wherein the nuclear receptor or fragment thereof does not comprise an amino acid sequence that is at least 70% identical to SEQ ID NO:

10.

30. The engineered polypeptide of any of the preceding claims, wherein the condensate switching domain comprises at least one modification.

31. The engineered polypeptide of any of the preceding claims, wherein the coacervate switch domain comprises at least one modification compared to any one of SEQ ID NOs: 1-10.

32. An engineered polypeptide as described in claim 30 or 31, wherein the at least one modification reduces the binding between the engineered polypeptide and the nucleic acid by at least 70%, at least 80%, at least 90%, at least 95% or at least 99% compared to the binding between a comparable polypeptide without the at least one modification and the nucleic acid.

33. The engineered polypeptide of claim 30 or 31, wherein at least one modification eliminates binding between the engineered polypeptide and a nucleic acid.

34. An engineered polypeptide as described in claim 30 or 31, wherein the at least one modification reduces the binding between the engineered polypeptide and the ligand by at least 70%, at least 80%, at least 90%, at least 95% or at least 99% compared to the binding between a comparable polypeptide not having the at least one modification and the ligand.

35. The engineered polypeptide of claim 30 or 31, wherein at least one modification eliminates binding between the engineered polypeptide and a ligand.

36. The engineered polypeptide of claim 34 or 35, wherein the ligand is an endogenous ligand.

37. The engineered polypeptide of claim 34 or 35, wherein the ligand is an exogenous ligand.

38. The engineered polypeptide of claim 30 or 31, wherein the at least one modification comprises at least one truncation.

39. The engineered polypeptide of claim 38, wherein at least one truncation comprises a deletion of a nucleic acid binding domain or a fragment thereof.

40. The engineered polypeptide of claim 30 or 31, wherein the at least one modification comprises at least one amino acid substitution.

41. The engineered polypeptide of claim 30 or 31, wherein the at least one modification comprises at least one truncation and at least one amino acid substitution.

42. The engineered polypeptide of claim 31, wherein the at least one modification comprises an A574D mutation or a W742C mutation compared to SEQ ID NO:

1.

43. The engineered polypeptide of claim 31, wherein the at least one modification comprises an A574D mutation and a W742C mutation compared to SEQ ID NO:

1.

44. The engineered polypeptide of claim 31, wherein the at least one modification comprises an A574D mutation or a W742L mutation compared to SEQ ID NO:

1.

45. The engineered polypeptide of claim 31, wherein the at least one modification comprises an A574D mutation and a W742L mutation compared to SEQ ID NO:

1.

46. ​​The engineered polypeptide of claim 31, wherein the at least one modification comprises a K632A mutation, a K633A mutation, a W742C mutation, or a combination thereof compared to SEQ ID NO:

1.

47. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K632A mutation, a K633A mutation, and a W742C mutation compared to SEQ ID NO:

1.

48. The engineered polypeptide of claim 31, wherein the at least one modification compared to SEQ ID NO: 1 comprises a K632A mutation, a K633A mutation, a W742L mutation; or a combination thereof.

49. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K632A mutation, a K633A mutation, and a W742L mutation compared to SEQ ID NO:

1.

50. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 617-633 or a W742C mutation compared to SEQ ID NO:

1.

51. The engineered polypeptide of claim 31, wherein the at least one modification comprises truncation of amino acid residues 617-633 and a W742C mutation compared to SEQ ID NO:

1.

52. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 617-633 or a W742L mutation compared to SEQ ID NO:

1.

53. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 617-633 and a W742L mutation compared to SEQ ID NO:

1.

54. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, a W742L mutation, or a combination thereof compared to SEQ ID NO:

1.

55. The engineered polypeptide of claim 31, wherein at least one modification comprises a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, and a W742L mutation compared to SEQ ID NO:

1.

56. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, a W742C mutation, or a combination thereof compared to SEQ ID NO:

1.

57. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, and a W742C mutation compared to SEQ ID NO:

1.

58. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, a W742L mutation, or a combination thereof compared to SEQ ID NO:

1.

59. The engineered polypeptide of claim 31, wherein the at least one modification compared to SEQ ID NO: 1 comprises a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, and a W742L mutation.

60. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, a W742L mutation, or a combination thereof compared to SEQ ID NO:

1.

61. The engineered polypeptide of claim 31, wherein the at least one modification compared to SEQ ID NO: 1 comprises a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, and a W742L mutation.

62. The engineered polypeptide of claim 31, wherein the at least one modification comprises an A574D mutation, a W742C mutation, a W742L mutation; a K632A mutation, a K633A mutation, a truncation of amino acid residues 617-633, a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, a truncation of amino acid residues 560-670, a truncation of amino acid residues 1-293, a truncation of amino acid residues 556-666, or a combination thereof, compared to SEQ ID NO:

1.

63. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, a G521R mutation, or a combination thereof compared to SEQ ID NO:

5.

64. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, and a G521R mutation compared to SEQ ID NO:

5.

65. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, a C400V mutation, a M543A mutation, a L544A mutation, or a combination thereof compared to SEQ ID NO:

5.

66. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, a C400V mutation, a M543A mutation, and a L544A mutation compared to SEQ ID NO:

5.

67. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274 or a L379R mutation compared to SEQ ID NO:

5.

68. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274 and a L379R mutation compared to SEQ ID NO:

5.

69. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274 or a G521R mutation compared to SEQ ID NO:

5.

70. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274 and a G521R mutation compared to SEQ ID NO:

5.

71. The engineered polypeptide of claim 31, wherein the at least one modification comprises truncation of amino acid residues 250-274, a C400V mutation, an M543A mutation, an L544A mutation, or a combination thereof compared to SEQ ID NO:

5.

72. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274, a C400V mutation, a M543A mutation, and a L544A mutation compared to SEQ ID NO:

5.

73. The engineered polypeptide of claim 31, wherein the at least one modification comprises a K210A mutation, a R211E mutation, a L379R mutation, a G521R mutation, a C400V mutation, a M543A mutation, a L544A mutation, a truncation of amino acid residues 250-274, or a combination thereof, compared to SEQ ID NO:

5.

74. The engineered polypeptide of claim 31, wherein the at least one modification comprises truncation of amino acid residues 44-163, truncation of amino acid residues 226-347, truncation of amino acid residues 560-670, or a combination thereof, compared to SEQ ID NO:

9.

75. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 44-163, a truncation of amino acid residues 226-347, and a truncation of amino acid residues 560-670 compared to SEQ ID NO:

9.

76. The engineered polypeptide of claim 31, wherein the at least one modification comprises a truncation of amino acid residues 250-274 compared to SEQ ID NO:

10.

77. An engineered polypeptide as described in any of the preceding claims, wherein the aggregate modulator comprises a functional domain ligand.

78. The engineered polypeptide of claim 77, wherein the functional domain ligand comprises a functional domain agonist.

79. The engineered polypeptide of claim 77, wherein the functional domain ligand comprises a functional domain antagonist.

80. The engineered polypeptide of claim 79, wherein the functional domain ligand comprises a functional domain inverse agonist.

81. The engineered polypeptide of any of the preceding claims, wherein the aggregate modulator comprises a nuclear receptor ligand.

82. The engineered polypeptide of claim 81, wherein the aggregate modulator comprises a nuclear receptor agonist, a nuclear receptor antagonist, or a nuclear receptor inverse agonist.

83. The engineered polypeptide of any one of claims 1-76, wherein the aggregate modulator comprises an androgen receptor agonist, an androgen receptor antagonist, or an androgen receptor inverse agonist.

84. The engineered polypeptide of any one of claims 1-76, wherein the aggregate modulator comprises an androgen receptor ligand comprising bicalutamide.

85. The engineered polypeptide of any one of claims 1-76, wherein the aggregate modulator comprises an estrogen receptor agonist, an estrogen receptor antagonist, or an estrogen receptor inverse agonist.

86. The engineered polypeptide of any one of claims 1-76, wherein the aggregate modulator comprises an estrogen receptor ligand comprising tamoxifen.

87. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregation modulator, the third aggregation modulator or the fourth aggregation modulator comprises a nuclear receptor ligand.

88. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregation modulator, the third aggregation modulator or the fourth aggregation modulator comprises a nuclear receptor agonist, a nuclear receptor antagonist or a nuclear receptor inverse agonist.

89. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregate modulator, the third aggregate modulator or the fourth aggregate modulator comprises an androgen receptor agonist, an androgen receptor antagonist or an androgen receptor inverse agonist.

90. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregation modulator, the third aggregation modulator or the fourth aggregation modulator comprises enzalutamide.

91. The engineered polypeptide of claim 90, wherein the second aggregate modulator comprises enzalutamide.

92. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregate modulator, the third aggregate modulator or the fourth aggregate modulator comprises an estrogen receptor agonist, an estrogen receptor antagonist or an estrogen receptor inverse agonist.

93. The engineered polypeptide of claim 9, 10 or 12, wherein the second aggregation modulator, the third aggregation modulator or the fourth aggregation modulator comprises fulvestrant.

94. The engineered polypeptide of claim 93, wherein the third aggregate modulator comprises fulvestrant.

95. The engineered polypeptide of any of the preceding claims, wherein the engineered polypeptide forms the first aggregate or the second aggregate in the cytoplasm of a cell.

96. The engineered polypeptide of any of the preceding claims, wherein the engineered polypeptide forms the first aggregate or the second aggregate in the cytoplasmic membrane of a cell.

97. The engineered polypeptide of any one of claims 1-94, wherein the engineered polypeptide forms the first condensate or the second condensate in an organelle of a cell.

98. The engineered polypeptide of any one of claims 1-94, wherein the engineered polypeptide forms the first condensate or the second condensate in a cell nucleus.

99. An engineered polypeptide as described in any of the preceding claims, wherein the functional domain comprises a kinase domain.

100. The engineered polypeptide of any one of claims 1-98, wherein the functional domain comprises a phosphatase domain.

101. The engineered polypeptide of any one of claims 1-98, wherein the functional domain comprises a transactivation domain.

102. The engineered polypeptide of any one of claims 1-98, wherein the functional domain comprises a DNA binding domain.

103. The engineered polypeptide of any one of claims 1-98, wherein the functional domain comprises a metabolic enzyme domain.

104. The engineered polypeptide of any one of claims 1-98, wherein the functional domain comprises a receptor.

105. The engineered polypeptide of claim 104, wherein the receptor comprises a CAR or a fragment thereof.

106. The engineered polypeptide of claim 104, wherein the receptor comprises a TCR or a fragment thereof.

107. An engineered polynucleotide encoding the engineered polypeptide of any one of the preceding claims.

108. The engineered polynucleotide of claim 107, comprising a vector.

109. The engineered polynucleotide of claim 108, wherein the vector comprises a viral vector.

110. A cell comprising the engineered polypeptide or engineered polynucleotide of any one of the preceding claims.

111. A system comprising: An engineered polypeptide comprising a functional domain and an accreta switching domain, wherein the accreta switching domain comprises an amino acid sequence of a nuclear receptor or a fragment thereof; as well as Aggregate regulator, Wherein, the engineered polypeptide forms the first aggregate after contacting with the aggregate regulator, and the first aggregate comprises the engineered polypeptide in operative contact with at least one other aggregate switching domain.

112. The system of claim 111, further comprising a second coagulation regulator, a third coagulation regulator, or a fourth coagulation regulator.

113. A pharmaceutical composition comprising the engineered polypeptide, engineered polynucleotide, cell or system of any one of the preceding claims.

114. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition is formulated for administration to a subject in need thereof by an intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraventricular, subcutaneous, subretinal, suprachoroidal, intratumoral, pulmonary, intratracheal, intraperitoneal, intravesical, intravaginal, intrarectal, oral, sublingual, transdermal, inhalation, nebulized inhalation, intraluminal-GI route, or a combination thereof.

115. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition comprises at least one excipient.

116. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition comprises at least one additional active ingredient.

117. A method for forming a first aggregate in a cell, the method comprising contacting the cell with an engineered polypeptide, engineered polynucleotide or system as described in any of the preceding claims, wherein the first aggregate is formed when the engineered polypeptide is in operative contact with at least one other aggregate conversion domain.

118. The method of claim 117, wherein the engineered polypeptide forms the first accretasome in a cell by being in operative contact with at least one additional accretasome switching domain.

119. The method of claim 117, wherein the engineered polypeptide, after contacting the aggregate modulator, forms the first aggregate in the cell by operatively contacting the engineered polypeptide with at least one additional aggregate switching domain.

120. The method of claim 118 or 119, wherein the first condensate mediates liquid-liquid phase separation (LLPS).

121. The method of claim 118 or 119, wherein the first condensate comprises a liquid condensate.

122. The method of claim 118 or 119, wherein: The first coacervate allows for molecular exchange between the interior and exterior of the first coacervate.

123. A method as claimed in claim 118 or 119, wherein the first condensate allows the movement of molecules within the first condensate.

124. The method of claim 119, wherein the first aggregate reverses after removal of the aggregate modulator.

125. The method of claim 119, wherein the first aggregate is reversed when the engineered polypeptide is contacted with a second aggregate modulator.

126. The method of any of the preceding claims, wherein the first aggregate is converted to a second aggregate when the engineered polypeptide is contacted with a third aggregate modulator.

127. A method as claimed in claim 126, wherein after removing the third aggregate regulator, the second aggregate is reversed back to the first aggregate.

128. The method of claim 127, wherein when the engineered polypeptide is contacted with a fourth aggregate modulator, the second aggregate is reversed back to the first aggregate.

129. The method of claim 127, wherein the second agglomerate comprises a solid-like agglomerate.

130. The method of claim 127, wherein the second coacervate comprises a semisolid-like coacervate.

131. The method of claim 127, wherein the second coacervate comprises a gel-like coacervate.

132. A method as claimed in claim 127, wherein the second coagulation inhibits molecular exchange between the interior and the exterior of the second coagulation.

133. A method as claimed in claim 127, wherein the second condensate inhibits the movement of molecules within the second condensate.

134. A method for increasing enzyme activity in a cell, the method comprising contacting an engineered polypeptide or engineered polynucleotide as described in any of the preceding claims with an aggregate regulator to form a first aggregate when the engineered polypeptide is in operative contact with at least one other aggregate conversion domain, wherein the first aggregate is enriched in local enzyme concentration.

135. A method for increasing enzyme activity in a cell, the method comprising contacting the engineered polypeptide or engineered polynucleotide of any of the preceding claims with an aggregate regulator to form a first aggregate when the engineered polypeptide is in operative contact with at least one other aggregate conversion domain, wherein the first aggregate brings two or more molecules with enzyme activity into close proximity, thereby increasing the enzyme activity.

136. The method of claim 135, wherein the two or more molecules include at least one enzyme.

137. The method of claim 135, wherein the two or more molecules include at least one biomolecule.

138. The method of claim 135, wherein the two or more molecules comprise at least one nucleic acid.

139. The method of claim 135, wherein the at least one nucleic acid comprises DNA, RNA, or a combination thereof.

140. The method of claim 135, wherein the two or more molecules include at least one protein.

141. The method of claim 140, wherein the at least one protein comprises a transcription factor.

142. The method of claim 135, wherein the two or more molecules comprise at least one polysaccharide.

143. The method of claim 135, wherein the two or more molecules comprise at least one lipid.

144. The method of claim 135, wherein the two or more molecules comprise at least one metabolite.

145. A method for regulating the concentration of at least one molecule in a cell, the method comprising contacting the cell with an engineered polypeptide or an engineered polynucleotide as described in any of the preceding claims, wherein when the engineered polypeptide is contacted with an aggregate regulator, a first aggregate is formed when it is operatively contacted with at least one other aggregate conversion domain, wherein the concentration of the at least one molecule inside the first aggregate is different from the concentration of the at least one molecule outside the first aggregate.

146. A method as claimed in claim 145, wherein the concentration of the at least one molecule inside the first agglomerate is increased relative to the concentration of the at least one molecule outside the first agglomerate.

147. The method of claim 146, wherein the concentration is increased by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

148. A method as claimed in claim 147, wherein the concentration of the at least one molecule inside the first agglomerate is reduced relative to the concentration of the at least one molecule outside the first agglomerate.

149. The method of claim 148, wherein the concentration is reduced by at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

150. The method of any one of claims 117-149, wherein the viscosity of the first coacervate is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to a comparable first coacervate formed from a comparable polypeptide not having at least one modification.

151. The method of any one of claims 117-149, wherein the viscosity of the first coacervate is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to a comparable first coacervate formed from a comparable polypeptide not having at least one modification.

152. A method as described in any of claims 117-151, wherein the first aggregate comprises at least a 10% increase in surface tension, at least a 20% increase in surface tension, at least a 30% increase in surface tension, at least a 40% increase in surface tension, at least a 50% increase in surface tension, at least a 60% increase in surface tension, at least a 70% increase in surface tension, at least an 80% increase in surface tension, at least a 90% increase in surface tension, or at least a 99% increase in surface tension compared to a comparable first aggregate formed by a comparable polypeptide not having at least one modification.

153. The method of any one of claims 117-151, wherein the first aggregate comprises at least 10% lower surface tension, at least 20% lower surface tension, at least 30% lower surface tension, at least 40% lower surface tension, at least 50% lower surface tension, at least 60% lower surface tension, at least 70% lower surface tension, at least 80% lower surface tension, at least 90% lower surface tension, or at least 99% lower surface tension as compared to a comparable first aggregate formed from a comparable polypeptide not having at least one modification.

154. The method of any one of claims 117-153, wherein the first aggregate comprises at least a 10% increase in turbidity, at least a 20% increase in turbidity, at least a 30% increase in turbidity, at least a 40% increase in turbidity, at least a 50% increase in turbidity, at least a 60% increase in turbidity, at least a 70% increase in turbidity, at least an 80% increase in turbidity, at least a 90% increase in turbidity, or at least a 99% increase in turbidity, compared to a comparable first aggregate formed from a comparable polypeptide not having at least one modification.

155. The method of any one of claims 117-153, wherein the first aggregate comprises at least 10% lower turbidity, at least 20% lower turbidity, at least 30% lower turbidity, at least 40% lower turbidity, at least 50% lower turbidity, at least 60% lower turbidity, at least 70% lower turbidity, at least 80% lower turbidity, at least 90% lower turbidity, or at least 99% lower turbidity compared to a comparable first aggregate formed from a comparable polypeptide not having at least one modification.

156. A method as described in any of claims 117-144, wherein the first aggregate increases the rate of molecular exchange between the interior and the exterior of the first aggregate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to the rate of molecular exchange between the interior and the exterior of a comparable first aggregate formed by a comparable polypeptide not having the at least one modification.

157. A method as described in any of claims 117-155, wherein the first aggregate reduces the rate of molecular exchange between the interior and the exterior of the first aggregate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to the rate of molecular exchange between the interior and the exterior of a comparable first aggregate formed by a comparable polypeptide not having the at least one modification.

158. The method of any one of claims 117-157, wherein the likelihood of the first aggregate accumulating in the cytoplasm of a cell is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% compared to a comparable first aggregate formed by a comparable polypeptide not having at least one modification.

159. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the engineered polypeptide, engineered polynucleotide, cell or system of any one of the preceding claims.

160. The method of claim 159, wherein the engineered polypeptide, engineered polynucleotide, cell or system of any of the preceding claims forms a first aggregate, a second aggregate, a third aggregate or a combination thereof for treating a disease or condition in a subject.

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