Polymer assemblies for cargo delivery

Abstract

The present disclosure relates, at least in part, to self-assembling peptides comprising a plurality of self-assembly repeat units that promote the formation of larger scale structures, such as a hydrogel. In some embodiments, a polymer described herein comprises a self-assembling and a cargo molecule, such as a peptide, polypeptide, or protein. In some embodiments, a cargo is a therapeutic cargo. Polymer assemblies described herein, such as those comprised in hydrogels, are useful for delivering therapeutic cargos to cells and may be used to treat a disease, disorder, or condition in a subject. The present disclosure further relates to methods of producing polymer assemblies and administration of polymer assemblies to a subject in need thereof.

Description

[0001] POLYMER ASSEMBLIES FOR CARGO DELIVERY

[0002] RELATED APPLICATIONS

[0003] The application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application number 63 / 598,523, filed November 13, 2023, which is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant Number R35 GM133697, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (U119770241WO00-SEQ-PRW.xml; Size: 21,124 bytes; and Date of Creation: November 8, 2024) are herein incorporated by reference in its entirety.

[0008] BACKGROUND OF INVENTION

[0009] Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance.

[0010] SUMMARY OF INVENTION

[0011] Biologically active molecules, such as ones that are capable of target protein binding, affecting intracellular signaling, and / or catalytically modifying a substrate, may be sensitive to certain conditions that reduce or abolish their activities. For example, biologically active molecules may be sensitive to certain pH ranges, temperature ranges, oxidizing / reducing agents, etc. which are used in biopharmaceutical manufacturing methods. Delivery of active molecules to cells can be useful for inducing biological changes that result in physiologically-relevant outcomes. Methods of producing delivery systems that can deliver active molecules to cells may be useful for generating therapies for treating a subject.

[0012] Aspects of the disclosure relates to polymers comprising structural features (e.g., selfassembling peptides comprising self-assembly repeat units) that promote self-assembly under physiologically relevant conditions, such as non-denaturing conditions comprising neutral pH and / or non-extreme temperatures. As such, polymers described herein can be useful for producing delivery systems comprising cargos that retain their function following polymer selfassembly. In addition, polymers described herein may be expressed in cellular systems which make production of said polymers efficient and scalable.

[0013] In some aspects, the disclosure relates to a polymer comprising a self-assembling peptide fused to a cargo, wherein the self-assembling peptide comprises a plurality of self-assembly repeat units, wherein each self-assembly repeat unit of the plurality comprises an amide-bearing amino acid and an aromatic amino acid. In some embodiments, the cargo comprises a peptide, a polypeptide, or a protein. In some embodiments, a linker connects the cargo to the selfassembling peptide.

[0014] In some embodiments, the amide-bearing amino acid and the aromatic amino acid are contiguous in at least one self-assembly repeat unit of the plurality. In some embodiments, the amide-bearing amino acid is N-terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality. In other embodiments, the amide-bearing amino acid is C-terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality. In some embodiments, the amide-bearing amino acid in at least one selfassembly repeat unit of the plurality is asparagine (N). In some embodiments, the amide-bearing amino acid in at least one self-assembly repeat unit of the plurality is glutamine (Q). In some embodiments, the aromatic amino acid in at least one self-assembly repeat unit of the plurality is tryptophan (W). In some embodiments, the plurality of self-assembly repeat units comprises 2- 10 self-assembly repeat units.

[0015] In some embodiments, the polymer comprises a first sequence comprising one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality, a second sequence comprising one or more amino acids that are C-terminal to at least one selfassembly repeat unit of the plurality, and / or a third sequence comprising one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality and one or more amino acids that are C-terminal to the at least one self-assembly repeat unit. In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence and / or the third sequence comprises 2-10 amino acids. In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence and / or the third sequence comprises at least one glycine (G) amino acid. In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence and / or the third sequence comprises at least one hydroxyl-bearing amino acid. In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence and / or the third sequence comprises an amino acid sequence of GGGSGGGSGG (SEQ ID NO: 15). In some embodiments, the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit in the second sequence and / or the third sequence comprises 2 amino acids. In some embodiments, the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit in the second sequence and / or the third sequence comprises at least one hydroxylbearing amino acid. In some embodiments, each of the at least one hydroxyl-bearing amino acid that are N-terminal to the at least one self-assembly repeat unit in the first sequence and the third sequence is serine (S) and / or each of the at least one hydroxyl-bearing amino acid that are C- terminal to the at least one self-assembly repeat unit in the second sequence and the third sequence is threonine (T). In some embodiments, the third sequence of (iii) comprises GGGSGGGSGGNWTT (SEQ ID NO: 11). In some embodiments, the first sequence, the second sequence, and / or the third sequence is repeated one or more times. In some embodiments, the first sequence, the second sequence, and / or the third sequence is repeated nine times. In some embodiments, a cysteine (C) is N-terminal and / or C-terminal to a sequence comprising the plurality of self-assembly repeat units.

[0016] In some aspects, the disclosure relates to a nucleic acid comprising a sequence encoding a polymer described herein. In some embodiments, the sequence encoding the polymer is operably linked to at least one regulatory sequence. In some embodiments, the at least one regulatory sequence comprises an inducible regulatory sequence. In some embodiments, the nucleic acid is a vector. In further aspects, the disclosure relates to a cell or cell population thereof comprising a nucleic acid described herein.

[0017] In some aspects, the disclosure relates to a polymer assembly comprising a plurality of a polymer described herein. In some embodiments, the polymer is present at a concentration of at least O.lmM in the polymer assembly. In some embodiments, the polymer is present at a concentration greater than or equal to l.OmM in the polymer assembly. In some embodiments, the polymer assembly is comprised in a semi-solid material. In some embodiments, the semisolid material comprises a hydrogel.

[0018] In other aspects, the disclosure relates to methods. In some embodiments, a method comprises producing a polymer assembly described herein. In some embodiments, a method comprises generating a mixture comprising the polymer and a buffer, thereby assembling the polymers. In some embodiments, producing the polymer assembly comprises incubating the mixture at a temperature of about 4 °C to about 37 °C. In some embodiments, the mixture comprises the polymer at a concentration of about 1.0% to 2.0% (weight / volume). In some embodiments, the buffer comprises a pH of about 7.0 to about 8.0. In some embodiments, the method further comprises contacting a nucleic acid comprising a sequence encoding the polymer with one or more cells and obtaining the plurality of polymers from the one or more cells before generating the mixture. In some embodiments, the method further comprises contacting the one or more cells with an inducing agent which is capable of inducing expression of the polymer from the nucleic acid. In some embodiments, a method is an ex vivo method which comprises contacting a polymer assembly with at least one cell. In some embodiments, the method further comprises obtaining a biological sample, wherein the biological sample comprises the at least one cell or a descendant cell thereof. In some embodiments, a method comprises administering a polymer assembly to a subject. In some embodiments, administering the polymer assembly comprises injecting the subject with the polymer assembly. In some embodiments, the method further comprises obtaining a biological sample from the subject after administration of the hydrogel. In some embodiments, the biological sample comprises one or more cells from the subject. In some embodiments, the biological sample is a blood sample or a urine sample. In some embodiments, the method further comprises contacting the biological sample with one or more detection agents capable of binding to an analyte, wherein the biological sample comprises or is suspecting of comprising the analyte. In some embodiments, the analyte comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the one or more detection agents comprises the polymer or a fragment thereof. In some embodiments, the one or more detection agents comprises an antibody or an antigen-binding fragment capable of binding to the analyte. In some embodiments, the method further comprises detecting the analyte in the biological sample. In some embodiments, the method further comprises isolating and / or purifying the analyte from the biological sample. In further aspects, the disclosure relates to a composition comprising a polymer, a nucleic acid, a cell or cell population, and / or a polymer assembly described herein.

[0019] In other aspects, the disclosure relates to a kit comprising a polymer, a nucleic acid, a cell or cell population, a polymer assembly, and / or a composition described herein. In some embodiments, the polymer assembly is comprised in a syringe. In some embodiments, the syringe further comprises a pharmaceutically acceptable buffer.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] FIGs. 1A-1P show non-limiting embodiments of self-assembling peptides and methods for producing hydrogels. FIG. 1A shows a non-limiting embodiment of a polymer comprising a self-assembling peptide which is fused to a cargo molecule at its C-terminus. FIG. IB shows a non-limiting embodiment of a polymer comprising a self-assembling peptide which is fused to a cargo at its N-terminus. FIG. 1C shows a non-limiting embodiment of a polymer comprising a self-assembling peptide which is fused to a cargo via a linker that connects the N-terminus of the self-assembling peptide to the C-terminus of the cargo. FIG. ID shows a non-limiting embodiment of a polymer comprising a self-assembling peptide which is fused to a cargo via a linker that connects the C-terminus of the self-assembling peptide to the N-terminus of the cargo. FIG. IE shows a non-limiting embodiment of a polymer comprising a self-assembling peptide fused to a cargo, wherein the self-assembling peptide comprises a plurality of selfassembly repeat units. FIG. IF shows a non-limiting embodiment of a polymer comprising a self-assembling peptide fused to an enzymatic cargo, wherein the polymer comprises a selfassembling peptide comprising a plurality of self-assembly repeat units. FIG. 1G shows a nonlimiting embodiment of a polymer comprising a self-assembling peptide fused to an enzymatic cargo via a linker, wherein the polymer comprises a self-assembling peptide comprising a plurality of self-assembly repeat units. FIG. 1H shows a non-limiting embodiment of a selfassembling peptide comprising a plurality of self-assembly repeat units, wherein each selfassembly repeat unit comprises a hydrophilic amino acid and a hydrophobic amino acid. FIG. II shows a non-limiting embodiment of a self-assembling peptide comprising a first plurality of self-assembly repeat units comprising a first self-assembly repeat unit configuration and a second plurality of self-assembly repeat units comprising a second self-assembly repeat unit configuration. FIG. 1J shows a non-limiting embodiment of a self-assembling peptide comprising a plurality of self-assembly repeat units, wherein each self-assembly repeat unit comprises an amide -bearing amino acid and an aromatic amino acid, wherein three of the selfassembly repeat units are contiguous and wherein each self-assembly repeat unit is separated by a different number of amino acids. FIG. IK shows a non-limiting embodiment of a selfassembling peptide comprising a first, a second, and a third plurality of self-assembly repeat units. FIG. IL shows a non-limiting embodiment of a self-assembling peptide comprising a plurality of self-assembly repeat units, wherein each self-assembly repeat unit comprises an amide-bearing amino acid which is either asparagine or glutamine and an aromatic amino acid. FIG. IM shows a non-limiting embodiment of a self-assembling peptide comprising a plurality of self-assembly repeat units, wherein each self-assembly repeat unit comprises an amide- bearing amino acid which is either asparagine or glutamine and an aromatic amino acid which is either phenylalanine or tryptophan. FIG. IN shows non-limiting embodiments of a method comprising introducing a nucleic acid encoding a polymer into a bacterial cell population and purifying polymers expressed by the cell population. FIG. IO shows non-limiting embodiments of a method of expressing an inducible sequence encoding a polymer in a population of bacterial cells and purifying polymers expressed by the cell population in response to the inducing agent. FIG. IP shows a non-limiting embodiment of a method for producing a hydrogel, wherein purified polymers are dialyzed, subjected to storage and incubation, and then packed to form hydrogels.

[0022] FIGs. 2A-2B shows non-limiting embodiments of N-GlycoTags (NGT) structures which were modeled using AlphaFold. FIG. 2A shows non-limiting embodiments of of NGT structures as shown from the long axis. FIG. 2B shows non-limiting embodiment of NGT structures as shown from the short axis.

[0023] FIG. 3 shows the molecular mass of three different NGT fusion proteins, NGT-nanoLuc (nL), NGT-Superfolder Green Fluorescent Protein (sfGFP), and Indoleamine-2,3-Dioxygenase (IDO)-NGT, measured by MALDI-TOF.

[0024] FIG. 4 shows representative results from analyses of storage modulus (G’, closed circles) and loss modulus (G”, open circles) of NGT-sfGFP and NGT-nL at 37°C over a range of frequencies.

[0025] FIGs. 5A-5B show representative results from characterizing physical properties of hydrogels. FIG. 5A shows the representative results from analyses of viscosity of NGT-sfGFP and NGT-nL. The shear rate was measured at 100 s'1between dotted lines and the shear rate was measured at 0.5 s'1shear rate before and after dotted lines. FIG. 5B shows time-lapse images of a representative NGT-fusion protein being extruded from a 27G needle.

[0026] FIG. 6 shows representative results from analyses of storage modulus (G’, closed circles) and loss modulus (G”, open circles) of NGT-sfGFP and NGT-nL measured at 1000% strain in between dotted lines and at 0.3% strain before and after dotted lines.

[0027] FIGs. 7A-7B show representative results demonstrating that NGT-fusion protein gels are enzymatically active. FIG. 7A shows representative results from analyses of peak relative light units (RLU) of the NGT-Fusion Protein in a gel state, NGT-Fusion Protein in a non-gel state, and negative control (PBS). FIG. 7B shows fluorescence of a representative NGT-Fusion protein in a gel state before (left) and after (right) introduction of substrate.

[0028] FIG. 8 shows representative results from analyses of the molecular mass of N-glycotag- sfGFP and glycosylated N-glycotag-sfGFP after glycosylation by A. pleuroneumoniae N- glycosyltransferase (ApNGT).

[0029] FIG. 9 shows a transmittance chart representation of representative results from analyses of N-glycotag-sfGFP during thermal cycling with sodium thiocynate.

[0030] FIGs. 10A-10B show representative results from analyses of abrogation of N-glycotag- sfGFP gelation upon glycosylation. FIG. 10A shows liquid Glu-N-glycotag-sfGFP (left) and gelated N-glycotag-sfGFP (right) at 4°C. FIG. 10B shows the absorbance of N-glycotag-sfGFP, Glu-N-glycotag-sfGFP, and negative control (buffer) measured at 4°C and after 5, 10, and 15 minutes of incubation at 55 °C.

[0031] FIGs. 11A-11B show NGT-based fusion proteins were recovered as pure, full-length proteins.

[0032] FIGs. 12A-12B show (NGT)iosfGFP formed assemblies, whereas (NGT)2sfGFP, (NGT)ssfGFP, and (QGT)iosfGFP did not.

[0033] FIG. 13 shows 30 pM of (NGT)iosfGFP (left), sfGFP (middle), and (QGT)iosfGFP (right) after freeze-thaw.

[0034] FIGs.l4A-14B show polymers subjected to freeze-thaw conditions. FIG. 14A shows 500 pM (NGT)iosfGFP processed under different temperature conditions. FIG. 14B shows 500 pM (QGT)iosfGFP after being frozen at -80 °C and thawed at 4 °C.

[0035] FIG. 15 shows (NGT)iosfGFP tryptophan fluorescence emission peak red-shifted with increasing temperature.

[0036] FIGs. 16A-16F show no Trp Fl. shift was observed in any other group w.r.t temperature. FIG. 17 shows Congo Red (CR) staining absorbance comparison.

[0037] FIG. 18 shows (NGT)2sfGFP, (NGT)ssfGFP do not show 540 nm peak with Congo Red.

[0038] FIGs. 19A-19B show Congo Red Staining of (NGT)iosfGFP.

[0039] FIGs. 20A-20B show Congo Red Staining of (NFT)iosfGFP.

[0040] FIG. 21 shows (NGT)lOnL binds Thioflavin T.

[0041] FIGs. 22A-22B show Ac-GGNWTT-Am (SEQ ID NO: 1) is stained by CR.

[0042] FIG. 23 shows Ac-GGNWTT-Am (SEQ ID NO: 1) binds ThT.

[0043] FIG. 24 shows NGT-Ovalbumin creates sub-micron size particles, when suspended in PBS.

[0044] FIG. 25 shows data obtained from analyzing control samples used in analyses of cross presentation on a dendritic cell line (DC2.4s).

[0045] FIG. 26 shows NGT-Ovalbumin induces a concentration dependent increase in SIINFEKL-MHCI expression on DC2.4s

[0046] FIGs. 27A-27C show PEG interacts with NGT-Ovalbumin to form sub-micron peaks.

[0047] FIG. 28 shows NGT-OVA + PEG formulations do not yield SIINFEKL-MHCI presentation on DC2.4s.

[0048] DETAILED DESCRIPTION OF INVENTION

[0049] The present disclosure relates, at least in part, to polymers which are useful for delivering biological active molecules to cells. As used herein, a “polymer” may be used to refer to a molecule comprising a plurality of monomeric molecules (e.g., amino acids) that are linked together by one or more covalent bonds. Non-limiting embodiments of polymers are shown in FIGs. 1A-1M.

[0050] In some embodiments, a polymer comprises a “self-assembling peptide” comprising a plurality of “self-assembly repeat units” (e.g., repeated sequences of at least two amino acids in length). In some embodiments, self-assembling peptides adopt ordered, larger scale structures as a result of self-assembly repeat units forming intramolecular and / or intermolecular interactions, such as electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, pi-pi stacking, etc. that promote the formation of a polymer assembly. In some embodiments, self-assembly of polymers comprises phase separation events which occur when self-assembly repeat units comprising an amide-bearing amino acid and an aromatic amino acid form intermolecular and / or intramolecular interactions. A non-limiting example of a larger scale structure formed by self-assembling peptides is a “polymer assembly” which may be used to refer to a structure that is stably held together via one or more interactions between selfassembling peptides comprised therein. In some embodiments, polymer assemblies are comprised within and / or form semi-solid materials described herein, such as hydrogels.

[0051] In some embodiments, when self-assembling peptides are contacted with each other under certain conditions described herein, the resulting polymer assembly which forms will also comprise other molecules that are linked and / or interacting with the self-assembling peptides, such as a cargo molecule. A “cargo” refers to a molecule which is capable of binding to a target, such as a nucleic acid or a protein. In some embodiments, a cargo is a “therapeutic cargo” which refers to a molecule that leads to a physiological change (e.g., in a cell, such as a cell in a subject) that is associated with or expected to at least partially, if not fully, remedy at least one symptom associated with a disease, disorder, or condition. In some embodiments, a polymer comprises one or more cargos via fusion through a covalent bond to a self-assembling peptide (see, e.g., FIGs. 1A-1B and IF). In some embodiments, a polymer comprises a self-assembling peptide which is fused to a cargo via a linker (see, e.g., FIGs. 1C-1D and 1G). In some embodiments, a polymer comprises a self-assembling peptide which is fused to a cargo at the C- terminus of the self-assembling peptide (see, e.g., FIGs. 1A and 1C) and / or at the N-terminus of the self-assembling peptide (see, e.g., FIGs. IB and ID). In some embodiments, a polymer may comprise a first self-assembling peptide fused to the N-terminus of a cargo and a second selfassembling peptide fused to the C-terminus of the cargo. The first and second self-assembling peptides may have the same sequence in some embodiments, or a different sequence in other embodiments. In some embodiments, a polymer may comprise a first cargo fused to the N- terminus of a self-assembling peptide and a second cargo fused to the C-terminus of the selfassembling peptide. The first and second cargoes may be the same polypeptide in some embodiments or different polypeptides in other embodiments.

[0052] Non-limiting embodiments of methods of the present disclosure are shown in FIGs. 1N- 1P. In some embodiments, a method comprises mixing a polymer at a concentration in a buffer which is capable of promoting self-assembly and mixing polymers under physiologically- relevant conditions, such as under non-denaturing conditions (e.g., at about 4 °C to about 37 °C and / or in a buffer at a pH of about 7.0 to about 8.0). In some embodiments, said conditions are useful for producing a polymer assembly comprising an active cargo, such as an enzymatic cargo. Nucleic acids described herein may also be useful in a method of producing a polymer. For example, in some embodiments, methods described herein comprise introducing a nucleic acid comprising a sequence encoding a polymer into a cell or cell population thereof (e.g., a population comprising E. coli cells) and then obtaining the polymers (see, e.g., FIG. IN). In some embodiments, a nucleic acid comprises an inducible-regulatory sequence which is operably linked to a sequence encoding a polymer (see, e.g., FIG. 10). In some embodiments, polymers are isolated and / or purified prior to being used to produce a polymer assembly or a semi-solid material thereof, such as a hydrogel (see, e.g., FIG. IP).

[0053] In some embodiments, methods of the present disclosure comprise contacting polymers with a cell or cell population (e.g., a cell in culture or a cell in a subject). In some embodiments, a method comprises administering polymers to a subject (e.g., via injection). In some embodiments, a biological sample is obtained after administering a polymer (e.g., a biological sample comprising a cell that was contacted with polymers ex vivo or a cell obtained from a subject that was administered the polymers). In some embodiments, a method comprises assaying one or more analytes in a biological sample.

[0054] Accordingly, in some embodiments, a polymer (e.g., polypeptide) comprises a selfassembling peptide fused to a cargo (e.g., a peptide, a polypeptide, or a protein), wherein the self-assembling peptide comprises a plurality of self-assembly repeat units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeat units, such as 10-15, 15-20, 20-25, 25-30, or more than 30 repeat units), wherein each self-assembly repeat unit of the plurality comprises an amide-bearing amino acid (e.g., asparagine (N) or glutamine (Q)) and an aromatic amino acid (e.g., tryptophan (W) or phenylalanine (F)). In some embodiments, the amide-bearing amino acid is N-terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality. In some embodiments, the amide-bearing amino acid is C-terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality. In some embodiments, the self-assembling peptide is fused directly to the cargo. In some embodiments, the self-assembling peptide and the cargo are connected via a linker (e.g., a linker described herein). In some embodiments, the polymer comprises a sequence comprising one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, or more than 20 amino acids, such as a sequence comprising at least one glycine (G) amino acid and / or at least one hydroxyl-bearing amino acid (e.g., serine (S) or threonine (T)) which can include, but is not limited to, a sequence comprising GGGSGGGSGG (SEQ ID NO: 15)) that are N-terminal to at least one self-assembly repeat unit of the plurality (e.g., wherein the one or more amino acids, which are N-terminal to the at least one self-assembly repeat unit in the sequence, is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times), a sequence comprising one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10- 15, 15-20, or more than 20 amino acids, such as a sequence comprising at least one glycine (G) amino acid and / or at least one hydroxyl-bearing amino acid (e.g., serine (S) or threonine (T)) which can include, but is not limited to, a sequence comprising GGGSGGGSGG (SEQ ID NO: 15)) that are C-terminal to at least one self-assembly repeat unit of the plurality (e.g., wherein the one or more amino acid, which are C-terminal to the at least one self-assembly repeat unit in the sequence, is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times), and / or a sequence comprising one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, or more than 20 amino acids, such as a sequence comprising at least one glycine (G) amino acid and / or at least one hydroxyl-bearing amino acid (e.g., serine (S) or threonine (T)) which can include, but is not limited to, a sequence comprising GGGSGGGSGG (SEQ ID NO: 15)) that are N-terminal to at least one self-assembly repeat unit of the plurality and one or more amino acids that are C- terminal to the at least one self-assembly repeat unit (e.g., wherein the one or more amino acids, which are N-terminal to the at least one self-assembly repeat unit in the sequence, and the one or more amino acids, which are C-terminal to the at least one self-assembly repeat unit in the sequence, are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times).

[0055] Polymers

[0056] In some embodiments, a polymer (e.g., a polypeptide) comprises a plurality of monomers, wherein each monomer of the plurality is linked by covalent bonds. In some embodiments, a polymer comprises at least 3 monomers. In some embodiments, a polymer comprises 3-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-170, 170-180, 180-200, 200-250, 250-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500- 1600, 1600-1700, 1700-1800, 1800-1900, 2000-2200, 2200-2400, 2400-2600, 2600-2800, 2800- 3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-6000, 6000-7000, 7000-8000, 8000- 9000, or 9000-10000 monomers. In some embodiments, a polymer comprises more than 10,000 monomers.

[0057] In some embodiments, a polymer (e.g., polypeptide) comprises a molecular weight of at least 300 g / mol. In some embodiments, a polymer comprises a molecular weight of 300-1,000, 1000,-2,000, 2,000-3,000, 3,000-4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, 9,000-10,000, 10,000-12,000, 12,000-14,000, 14,000-16,000, 16,000-17,000, 17,000-18,000, 18,000-20,000, 20,000-25,000, 25,000-30,000, 30,000-40,000, 40,000-50,000, 50,000-60,000, 60,000-70,000, 70,000-80,000, 80,000-90,000, 90,000-100,000, 100,000- 110,000, 110,000-120,000, 120,000-130,000, 130,000-140,000, 140,000-150,000, 150,000- 160,000, 160,000-170,000, 170,000-180,000, 180,000-190,000, 200,000-220,000, 220,000- 240,000, 240,000-260,000, 260,000-280,000, 280,000-300,000, 300,000-350,000, 350,000- 400,000, 400,000-450,000, 450,000-500,000, 500,000-600,000, 600,000-700,000, 700,000- 800,000, 800,000-900,000, 900,000-1,000,000, 1,000,000-2,500,000, 2,500,000-5,000,000, 5,000,000-7,500,000, or 7,500,000-10,000,000 g / mol. In some embodiments, a polymer comprises a molecular weight of about 1,000, 2,000, 5,000, 7,500, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 125,000, 150,000, 200,000, 250,000, 250,000, or 300,000 g / mol. In some embodiments, a polymer comprises a molecular weight that is greater than 10,000,000 g / mol.

[0058] In some embodiments, a polymer (e.g., a polypeptide) comprises naturally occurring (e.g., amino acids), recombinant, and / or non-naturally occurring monomers (e.g., synthetic monomers). In some embodiments, a polymer comprises a peptide (e.g., a self-assembling peptide), polypeptide, or protein, wherein the constituent monomers comprise amino acids. In some embodiments, amino acids may be naturally-occurring amino acids. Non-limiting examples of naturally occurring amino acids include hydrophilic and hydrophobic amino acids. In some embodiments, a hydrophilic amino acid is a polar amino acid, such as an amide-bearing amino acid (e.g., asparagine (N) and glutamine (Q)), a hydroxyl-bearing amino acid (e.g., serine (S) and threonine (T)), or a thiol-bearing amino acid (e.g., cysteine (C)). In some embodiments, a hydrophilic amino acid is a charged amino acid, such as an acidic amino acid or anionic amino acid (e.g., aspartate (D) and glutamine (E)) or a basic amino acid or cationic amino acid (e.g., arginine (R), lysine (K), and histidine (H)). In some embodiments, an “amide-bearing amino acid” is asparagine (N) or glutamine (G). In some embodiments, a hydrophobic amino acid comprises a side group which is nonpolar, aliphatic, and / or aromatic, such as glycine (G), alanine (A), valine (V), leucine (L), methionine (M), isoleucine (I), phenylalanine (F), tyrosine (Y), tryptophan (Y), and proline (P). In some embodiments, an “aromatic amino acid” is tryptophan (W), phenylalanine (F), or tyrosine (Y). In some embodiments, amino acids may be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. In some embodiments, a polymer comprises one or more modified amino acids, such as those that have been modified post-translationally and / or modified by a non-natural or synthetic process. In some embodiments, a polymer comprises one or more glycosylated amino acid residues (e.g., O- linked glycosylated amino acid residues, such as at a GalNAcT2 site comprising an amino acid sequence of APTYAP) (SEQ ID NO: 16). In some embodiments, a polymer comprises one or more modified cysteines, such as cysteines involved in a disulfide bond or a cysteine that has been modified at its thiol group to comprise a synthetic molecule or moiety. In some embodiments, a polymer comprises a combination of monomeric molecules, such as a polymer comprising an amino acid sequence linked to a polynucleotide (e.g., a single- and / or doublestranded nucleic acid) and / or a synthetic molecule (e.g., such as a chemically modified nucleic acid, a hapten, drug molecule, or a synthetic polymer, such as polyethylene glycol (PEG)).

[0059] In some embodiments, a polymer (e.g., a polypeptide) a plurality of components (e.g., one or more self-assembling peptides and one or more cargos), wherein the plurality of components (indicated by brackets below) are configured in the polymer according to any of the following:

[0060] (i) [self-assembling peptide(s)]-[cargo(s)];

[0061] (ii) [cargo(s)]-[self-assembling peptide(s)];

[0062] (iii) [cargo(s)]-[self-assembling peptide(s)]-[cargo(s)];

[0063] (iv) [self-assembling peptide(s)]-[cargo(s)]-[self-assembling peptide(s)];

[0064] (v) [cargo(s)]-[self-assembling peptide(s)]-[cargo(s)]- [self-assembling peptide(s)];

[0065] (vi) [self-assembling peptide(s)]-[cargo(s)]-[ self-assembling peptide(s)]-[cargo(s)];

[0066] (vii) [cargo(s)]-[self-assembling peptide(s)]-[cargo(s)]- [self-assembling peptide(s)]- [cargo(s)];

[0067] (viii) [self-assembling peptide(s)]-[cargo(s)]-[self-assembling peptide(s)]-[cargo(s)]-

[0068] [self-assembling peptide(s)]; or

[0069] (ix) any combination of (i)-(vi).

[0070] In some embodiments, a polymer (e.g., a polypeptide) comprises a configuration of components as set forth in (i)-(ix) above, wherein one or more of the dashes separating each component indicated by brackets corresponds to a covalent bond. In some embodiments, a polymer comprises a configuration of components as set forth in (i)-(ix) above, wherein one or more of the dashes separating each component indicated by brackets corresponds to a linker described herein.

[0071] In some embodiments, when more than one cargo is comprised in a polymer, the polymer may comprise multiple copies of the same cargo or cargos that are different. In some embodiments, when more than one cargo is comprised in a polymer, at least two of the cargos may be linked directly via a covalent bond or via a linker described herein. In some embodiments, when more than one self-assembling peptide is comprised in a polymer, each of the self-assembling peptides may comprise the same sequence or a different sequence. In some embodiments, when more than one self-assembling cargo is comprised in a polymer, at least two self-assembling cargos may be linked directly via a covalent bond or via a linker described herein.

[0072] Self-Assembling Peptides, Self-Assembly Repeats, and Self-Assembly Repeat Units

[0073] In some embodiments, a self-assembling peptide comprises at least 4 amino acids. In some embodiments, a self-assembling peptide comprises 4-200 amino acids. In some embodiments, a self-assembling peptide comprises 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175-200, 200-300, 300-400, 400-500, 500- 600, 600-700, 700-800, 800-900, or 900-1000 amino acids. In some embodiments, selfassembling peptide comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids. In some embodiments, a self-assembling peptide comprises less than 150 amino acids, less than 100 amino acids, or less than 50 amino acids. In some embodiments, a self-assembling peptide comprises more than 1000 amino acids.

[0074] In some embodiments, self-assembling peptides described herein comprise a plurality of self-assembly repeat units. In some embodiments, a self-assembly repeat unit comprises at least two amino acids in length.

[0075] In some embodiments, self-assembling peptides comprise a plurality of self-assembly repeat units that are contiguous such that each self-assembly repeat unit is directly linked via a peptide bond in a “repeat” sequence or configuration (see, e.g., contiguous self-assembly repeat units in FIG. 1 J). In some embodiments, a self-assembling peptide comprises a plurality of selfassembly repeat units that are non-contiguous such that one or more amino acids separate some or all of the self-assembly repeat units in the plurality (see, e.g., non-contiguous self-assembly repeat units in FIG. 1J). In some embodiments, a self-assembling peptide comprises an amino acid sequence, wherein less than 100% of the amino acid sequence comprises amino acids which are comprised in self-assembly repeat units. As non-limiting examples, a self-assembling peptide comprising NWNWNWRNWNW (SEQ ID NO: 17), NWNWNWRNWKNWNW (SEQ ID NO: 18), or NWGLNRNWWKPDNWEFMRFS (SEQ ID NO: 19) would have 90.9% (10 out of 11 residues are comprised in self-assembly repeat units), 85.7% (12 out of 14 residues are comprised in selfassembly repeat units), or 30% (6 out of 20 residues are comprised in self-assembly repeat units) of its sequence are comprised in NW self-assembly repeat units, respectively. In some embodiments, a self-assembling peptide comprises an amino acid sequence, wherein about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,

[0076] 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

[0077] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,

[0078] 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,

[0079] 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence comprises amino acids in self-assembly repeat units. In some embodiments, a self-assembling peptide comprises an amino acid sequence, wherein between 10% and 50% (e.g., 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, etc.) of the amino acid sequence comprises amino acids in self-assembly repeat units. In some embodiments, a self-assembling peptide comprises an amino acid sequence, wherein 50-99% of the amino acid sequence comprises amino acids in self-assembly repeat units (e.g., 50-55%, 55- 60%, 60-70%, 70-80%, etc.).

[0080] In some embodiments, a self-assembling peptide comprises a plurality of self-assembly repeat units, wherein the plurality comprises at least 2 self-assembly repeat units. In some embodiments, a plurality of self-assembly repeat units comprises 2-1,000 self-assembly repeat units. In some embodiments, a plurality of self-assembly repeat units comprises 2-5, 5-10, 10- 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175- 200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 selfassembly repeat units. In some embodiments, a plurality of self-assembly repeat units comprises 3-5 self-assembly repeat units, 5-7 self-assembly repeat units, 7-9 self-assembly repeat units, 9- 12 self-assembly repeat units, or 12-15 self-assembly repeat units, 15-20 self-assembly repeat units, 20-25 self-assembly repeat units, or 25-30 self-assembly repeat units. In some embodiments, a plurality of self-assembly repeat units comprises 2-10 self-assembly repeat units, such as 3 self-assembly repeat units, 4 self-assembly repeat units, 5 self-assembly repeat units, 6 self-assembly repeat units, 7 self-assembly repeat units, 8 self-assembly repeat units, 9 self-assembly repeat units, or 10 self-assembly repeat units. In some embodiments, the number of self-assembly repeat units in a self-assembling peptide may be adjusted based on the size of the cargo molecule to which it is fused. As a non-limiting example, in some embodiments, a self-assembling peptide fused to a cargo molecule greater than 500 amino acids, 1,000 amino acids, 2,000 amino acids or more may comprise more than 10, 20, 30, or 40 self-assembly repeat units.

[0081] In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprises one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality, wherein the one or more amino acids do not comprise the sequence or the configuration of the self-assembly repeat units (see, e.g., self-assembly repeat unit “1” in FIG. IE). In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality comprises 2-100 amino acids (e.g., 2-4, 4-8, 8-12, 12-16, 16-20, 20-25, 25-30, 30-35, 35-40, 40- 45, 45-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids). In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality comprises 2-25 amino acids (e.g., 2-10 amino acids, such as 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids). In some embodiments, the sequence comprising the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality is repeated in the selfassembling peptide one or more times. In some embodiments, the sequence comprising the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality is repeated in the self-assembling peptide 1-200 times (e.g., 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, or 175-200 times). In some embodiments, the sequence comprising the one or more amino acids that are N- terminal to the at least one self-assembly repeat unit of the plurality is repeated in the selfassembling peptide 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times. In some embodiments, the sequence comprising the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality is comprised in a linker described herein. In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprises one or more amino acids that are C-terminal to at least one self-assembly repeat unit of the plurality, wherein the one or more amino acids do not comprise the sequence or the configuration of the self-assembly repeat units (see, e.g., self-assembly repeat unit “6” in FIG. IE). In some embodiments, the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit of the plurality comprises 2-100 amino acids (e.g., 2-4, 4-8, 8-12, 12-16, 16-20, 20-25, 25-30, 30-35, 35-40, 40- 45, 45-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids). In some embodiments, the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit of the plurality comprises 2-25 amino acids (e.g., 2-10 amino acids, such as 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids). In some embodiments, the sequence comprising the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit of the plurality is repeated in the selfassembling peptide one or more times. In some embodiments, the sequence comprising the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit of the plurality is repeated in the self-assembling peptide 1-200 times (e.g., 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, or 175-200 times). In some embodiments, the sequence comprising the one or more amino acids that are C- terminal to the at least one self-assembly repeat unit of the plurality is repeated in the selfassembling peptide 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times. In some embodiments, the sequence comprising the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit is comprised in a linker described herein.

[0082] In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprises one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality and one or more amino acids that are C-terminal to the at least one self-assembly repeat unit of the plurality, wherein the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit and the one or more amino acids that C-terminal to the at least one self-assembly repeat unit do not comprise the sequence or the configuration of the self-assembly repeat units (see, e.g., self-assembly repeat unit “6” in FIG. IE). In some embodiments, multiple self-assembly repeat units of the plurality comprise amino acids that are N-terminal to the multiple self-assembly repeat units and C-terminal to the multiple self-assembly repeat units (see, e.g., self-assembly repeat units “1-6” in FIG. IE). In some embodiments, the one or more amino acids found between each self-assembly repeat unit of the plurality are equal in length (see, e.g., “1stsequence” in FIG. IK). In some embodiments, the one or more amino acids found between each self-assembly repeat unit of the plurality comprises sequences of different lengths (see, e.g., FIG. 1J). In some embodiments, a self-assembling peptide may comprise at least one linker described herein, wherein the at least one linker is located between self-assembly repeat units.

[0083] In some embodiments, a self-assembly repeat unit comprises at least two amino acids. In some embodiments, a self-assembly repeat unit comprises two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids. In some embodiments, self-assembly repeat unit comprises more than six amino acids. In some embodiments, self-assembly repeat units comprise 7-10 amino acids, 10-15 amino acids, 15-20 amino acids, or more.

[0084] In some embodiments, amino acids in a self-assembly repeat units assemble into structures comprising beta strands, such as parallel and / or anti-parallel strands (see, e.g., FIGs. 2A-2B). In some embodiments, amino acids comprised in self-assembly repeat units form beta strands that interact to form parallel and / or anti-parallel beta sheets. In some embodiments, selfassembly repeat units form beta meanders, beta-alpha beta motifs, beta hairpins, Greek key motifs, beta solenoids (e.g., structures comprising beta rolls and / or beta helices, such as parallel beta helices), and / or psi-loop motifs (see, as a non-limiting example, FIGs. 2A-2B). Nonlimiting examples of tools which are useful for prediction of protein structure and may be used to model peptides, polypeptides, and / or proteins comprising amino acid sequences described herein include AlphaFold, Rossetta, I-TASSER, Robetta, Phyre2, RaptorX, and SWISS- MODEL.

[0085] In some embodiments, self-assembly repeat units comprise an arrangement of amino acids that adopt ordered structures which are stabilized by interactions involving the side chains of amino acids in the self-assembling peptide. In some embodiments, self-assembly repeat units drive folding events (e.g., via phase separation), wherein hydrophobic moieties in the selfassembling peptide (e.g., the side chains of aromatic amino acids, such as a phenylalanine and tryptophan) are positioned in three-dimensional space so that they form hydrophobic interactions, van der Waals interactions, pi-pi stacking interactions, or any combination thereof. In some embodiments, self-assembly repeat units drive folding events (e.g., via phase separation), wherein hydrophilic moieties in the self-assembling peptide (e.g., the side chains of hydrophilic amino acids, carbonyl groups in the peptide backbone, and amine groups in the peptide backbone) are positioned in three-dimensional space so that they form electrostatic interactions, hydrogen bonds, van der Waals interactions, or any combination thereof. In some embodiments, self-assembly repeat units comprise flexible and / or hydrophilic segments (e.g., segments that are serine- and / or glycine-rich) of sequences which promote folding of selfassembling peptides into a conformation that pushes hydrophobic side chains into close physical proximity. In some embodiments, self-assembly repeat units drive folding events such that the self-assembling peptide adopts a three-dimensional conformation comprising an interior and / or exterior aspect (or face) of the molecule which is substantially hydrophobic and a separate interior and / or exterior aspect (or face) of the molecule which is substantially hydrophilic.

[0086] In some embodiments, a self-assembly repeat unit (e.g., an amphiphilic self-assembly repeat unit) comprises at least one hydrophilic amino acid and at least one hydrophobic amino acid (see, e.g., FIGs. 1H-1I). In some embodiments, a hydrophilic amino acid and a hydrophobic amino are contiguous in a self-assembly repeat unit (e.g., are directly connected by a peptide bond, see, e.g., “3rdself-assembly repeat unit configuration” in FIG. IK). In some embodiments, a hydrophilic amino acid and a hydrophobic amino are non-contiguous in a self-assembly repeat unit (e.g., are separated by one or more amino acids, see, e.g., “1stself-assembly repeat unit configuration” and “2ndself-assembly repeat unit configuration” in FIG. IK). In some embodiments, a hydrophobic amino acid in a self-assembly repeat unit is N-terminal to a hydrophilic amino acid in a self-assembly repeat unit (see, e.g., “1stself-assembly repeat unit configuration” in FIG. II). In some embodiments, a hydrophobic amino acid in a self-assembly repeat unit is C-terminal to a hydrophilic amino acid in a self-assembly repeat unit (see, e.g., “2ndself-assembly repeat unit configuration” in FIG. II).

[0087] In some embodiments, a self-assembly repeat unit comprises an arrangement of amino acids that have a repeating or alternating chemistry. In some embodiments, a self-assembly repeat unit comprises an arrangement of amino acids which alternate in amino acid side chain chemistry to comprise one or more stretches of sequence comprising a configuration of: hydrophobic amino acid- hydrophilic amino acid-hydrophobic amino acid etc.; hydrophilic amino acid-hydrophobic amino acid-hydrophilic amino acid etc.) In some embodiments, a selfassembly repeat unit comprises two hydrophobic amino acids that are contiguous and immediately preceded (e.g., in the N-terminal direction) and / or immediately proceeded (e.g., in the C-terminal direction) by a hydrophilic amino acid. In some embodiments, a self-assembly repeat unit comprises two hydrophilic amino acids that are contiguous and immediately preceded (e.g., in the N-terminal direction)) and / or immediately proceeded (e.g., in the C- terminal direction) by a hydrophobic amino acid amino acid. In some embodiments, a selfassembly repeat unit comprises two contiguous hydrophobic amino acids that are immediately preceded (e.g., in the N-terminal direction) and / or proceeded (e.g., in the C-terminal direction) by two contiguous hydrophilic amino acids. In some embodiments, a self-assembly repeat unit comprises one or more stretches of sequence comprising 1-3 hydrophobic amino acids that are immediately preceded (e.g., in the N-terminal direction) and / or immediately proceeded (e.g., in the C-terminal direction) by 1-3 hydrophilic amino acids.

[0088] In some embodiments, a hydrophilic amino acid in a self-assembly repeat unit comprises an amide-bearing amino acid and a hydrophobic amino acid in the self-assembly repeat comprises an aromatic amino acid (see, e.g., FIGs. 1J-1M). In some embodiments, a selfassembling peptide comprises a plurality of self-assembly repeat units, wherein each selfassembly repeat unit is configured such that the amide-bearing amino acid and the aromatic amino acid are contiguous (see, e.g., the self-assembling peptide in FIG. IL). In some embodiments, a self-assembling peptide comprises one or more configurations of self-assembly repeat units wherein an amide-bearing amino acid and an aromatic amino acid are noncontiguous (see, e.g., the self-assembling peptide in FIG. IK). In some embodiments, a selfassembling peptide comprises a plurality of self-assembly repeat units, wherein two or more of the self-assembly repeat units are contiguous, and thus form a repeat (see, e.g., the contiguous self-assembly repeat units in FIG. 1 J). In some embodiments, a self-assembling peptide comprises a plurality of self-assembly repeat units, wherein two or more of the self-assembly repeat units are non-contiguous, and thus are interrupted by amino acids that do not comprise the sequence or configuration of the self-assembly repeat unit (see, e.g., the three left-most selfassembly repeat units in the self-assembling peptide in FIG. 1 J and the self-assembling peptide in FIG. IL). In some embodiments, a self-assembling peptide comprises a mix of contiguous and non-contiguous self-assembly repeat units, wherein each self-assembly repeat unit comprises an amide-bearing amino acid and an aromatic amino acid (see, e.g., the self-assembling peptide in FIG. 1J). In some embodiments, the amide-bearing amino acid in each self-assembly repeat unit is independently selected from N and Q and the aromatic amino acid in each self-assembly repeat unit is independently selected from W, F, and Q (see, e.g., the self-assembling peptide in FIG. IM). In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X2X3, wherein Xi, X2, and X3 each correspond to respective amino acids. In some embodiments, Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), and X3 is an aromatic amino acid (e.g., F or W). In some embodiments, Xi is an aromatic amino acid (e.g., F or W), X2is an amide-bearing amino acid (e.g., N or Q), and X3 is a hydrophobic amino acid (e.g., G). In some embodiments, Xi is an amide-bearing amino acid (e.g., N or Q), X2 is an aromatic amino acid (e.g., F or W), and X3 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, Xi is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T), X2 is an aromatic amino acid (e.g., F or W), and X3 is an amide-bearing amino acid (e.g., N or Q).

[0089] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X2X3, wherein Xi, X2, and X3 each correspond to respective amino acids. In some embodiments, each Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), and X3 is an aromatic amino acid (e.g., F or W). In some embodiments, each Xi is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T), X2 is an aromatic amino acid (e.g., F or W), and X3 is an amide-bearing amino acid (e.g., N or Q).

[0090] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X2X3X3, wherein Xi, X2, and X3 each correspond to respective amino acids. In some embodiments, Xi is an amide-bearing amino acid (e.g., N or Q), X2 is an aromatic amino acid (e.g., F or W), and each X3 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, Xi is an aromatic amino acid (e.g., F or W), X2 is an amide- bearing amino acid (e.g., N or Q), and each X3 is a hydrophobic amino acid (e.g., G).

[0091] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids. In some embodiments, Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), X3 is an aromatic amino acid (e.g., F or W), and X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, Xi is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T), X2 is an aromatic amino acid (e.g., F or W), X3 is an amide-bearing amino acid (e.g., N or Q), and X4 is a hydrophobic amino acid (e.g., G).

[0092] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids. In some embodiments, each Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), X3 is an aromatic amino acid (e.g., F or W), and each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, each Xi is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T), X2 is an aromatic amino acid, X3 is an amide-bearing amino acid, and each X4 is a hydrophobic amino acid (e.g., G).

[0093] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X2X3X4X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids. In some embodiments, each Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), X3 is an aromatic amino acid (e.g., F or W), and each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, each Xi is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T), X2 is an aromatic amino acid, X3 is an amide-bearing amino acid, and each X4 is a hydrophobic amino acid (e.g., G).

[0094] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X2X3X4X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids. In some embodiments, each Xi is a hydrophobic amino acid (e.g., G), X2 is an amide-bearing amino acid (e.g., N or Q), X3 is an aromatic amino acid (e.g., F or W), and each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T). In some embodiments, each Xi is a hydroxyl-bearing amino acid (e.g., T), X2 is an aromatic amino acid, X3 is an amide-bearing amino acid, and each X4 is a hydrophobic amino acid (e.g., G).

[0095] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids. In some embodiments, each Xi is a hydroxyl-bearing amino acid and each X2 is a hydrophobic amino acid. In some embodiments, each Xi is a serine. In some embodiments, each X2 is glycine. In some embodiments, each Xi is a hydrophobic amino acid and each X2 is a hydroxyl-bearing amino acid. In some embodiments, each Xi is glycine and each X2 is serine. In some embodiments, X3 is an amide-bearing amino acid and X4 is an aromatic amino acid. In some embodiments, X3 is asparagine and X4 is tryptophan. In some embodiments, X3 is an aromatic amino acid and X4 is an amide-bearing amino acid. In some embodiments, X3 is tryptophan and X4 is asparagine. In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprising the plurality of self-assembly repeat units comprises the sequence X1X1X1X2X1X1X1X2X2X3X4, wherein X1X1X1X2X1X1X1X2X2X3X4 is repeated in the self-assembling peptide 1, 2, 3, 4, 5, 6, 7, 8, or 9 times. In some embodiments, X1X1X1X2X1X1X1X2X2X3X4 is repeated in the selfassembling peptide 10 times or more.

[0096] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X2X2X2X3X2X2X2X3X3X4X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids. In some embodiments, each Xi and X2 is a hydroxyl-bearing amino acid and each X3 is hydrophobic amino acid. In some embodiments, each Xi is threonine. In some embodiments, each X2 is serine. In some embodiments, each X3 is glycine. In some embodiments, X4 is an amide-bearing amino acid and X5 is an aromatic amino acid. In some embodiments, X4 is an aromatic amino acid and X5 is an amide-bearing amino acid. In some embodiments, X4 is tryptophan and X5 is asparagine. In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprising the plurality of self-assembly repeat units comprises the sequence X1X1X2X2X2X3X2X2X2X3X3X4X5, wherein X1X1X2X2X2X3X2X2X2X3X3X4X5 is repeated in the self-assembling peptide 1, 2, 3, 4, 5, 6, 7, 8, or 9 times. In some embodiments, X1X1X2X2X2X3X2X2X2X3X3X4X5 is repeated in the self-assembling peptide 10 times or more.

[0097] In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X2X3X4X5X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids. In some embodiments, each Xi is a hydroxyl-bearing amino acid, each X2 is a hydrophobic amino acid, and each X5 is a hydroxyl-bearing amino acid. In some embodiments, each Xi is serine. In some embodiments, each X2 is glycine. In some embodiments, each Xi is a hydrophobic amino acid, each X2 is a hydroxyl-bearing amino acid, and each X5 is a hydroxylbearing amino acid. In some embodiments, each Xi is glycine and each X2 is serine. In some embodiments, X3 is an amide-bearing amino acid and X4 is an aromatic amino acid. In some embodiments, X3 is asparagine and X4 is tryptophan. In some embodiments, X3 is an aromatic amino acid and X4 is an amide-bearing amino acid. In some embodiments, X3 is tryptophan and X4 is asparagine. In some embodiments, each X5 is threonine. In some embodiments, a selfassembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprising the plurality of self-assembly repeat units comprises the sequence X1X1X1X2X1X1X1X2X2X3X4X5X5, wherein X1X1X1X2X1X1X1X2X2X3X4X5X5 is repeated in the self-assembling peptide 1, 2, 3, 4, 5, 6, 7, 8, or 9 times. In some embodiments, X1X1X1X2X1X1X1X2X2X3X4X5X5 is repeated in the self-assembling peptide 10 times or more. In some embodiments, a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X1X1X3X4X5X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids. In some embodiments, each Xi is a hydroxyl-bearing amino acid, each X2 is a hydrophobic amino acid, and each X5 is a hydroxyl-bearing amino acid. In some embodiments, each Xi is serine and each X2 is glycine. In some embodiments, each Xi is a hydrophobic amino acid, each X2 is a hydroxyl-bearing amino acid, and each X5 is a hydroxylbearing amino acid. In some embodiments, each Xi is glycine and each X2 is serine. In some embodiments, X3 is an amide-bearing amino acid and X4 is an aromatic amino acid. In some embodiments, X3 is asparagine. In some embodiments, X3 is glutamine. In some embodiments, X4 is phenylalanine. In some embodiments, X4 is tryptophan. In some embodiments, X3 is asparagine and X4 is tryptophan. In some embodiments, X3 is an aromatic amino acid and X4 is an amide-bearing amino acid. In some embodiments, X3 is tryptophan and X4 is asparagine. In some embodiments, each X5 is threonine. In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence comprising the plurality of self-assembly repeat units comprises the sequence X1X1X1X2X1X1X1X2X1X1X3X4X5X5, wherein X1X1X1X2X1X1X1X2X1X1X3X4X5X5 is repeated in the self-assembling peptide 1, 2, 3, 4, 5, 6, 7, 8, or 9 times. In some embodiments, X1X1X1X2X1X1X1X2X1X1X3X4X5X5 is repeated in the self-assembling peptide 10 times or more.

[0098] In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence is immediately preceded by at least one amino acid (e.g., wherein the at least one amino acid is N-terminal to the sequence comprising the plurality of self-assembly repeat units). In some embodiments, the at least one amino acid is a cysteine (C). In some embodiments, the at least one amino acid is a cysteine which comprises a disulfide bond (e.g., a disulfide bond between another cysteine in the selfassembling peptide or to a cysteine in the cargo molecule) or another thiol group modification. In some embodiments, the at least one amino acid is comprised in a linker described herein.

[0099] In some embodiments, a self-assembling peptide comprises a sequence comprising a plurality of self-assembly repeat units, wherein the sequence is immediately proceeded by at least one acid (e.g., wherein the at least one amino acid is C-terminal to the sequence comprising the plurality of self-assembly repeat units). In some embodiments, the at least one amino acid is a cysteine (C). In some embodiments, the at least one amino acid is a cysteine which comprises a disulfide bond (e.g., a disulfide bond between another cysteine in the self-assembling peptide or to a cysteine in the cargo molecule) or another thiol group modification. In some embodiments, the at least one amino acid is comprised in a linker described herein.

[0100] In some embodiments, a sequence comprising a plurality of self-assembly repeat units comprises an amino acid sequence set forth in SEQ ID NO: 3.

[0101] In some embodiments, a polymer comprises one or more amino acid sequences described in Table 1 below.

[0102] Table 1. Non-limiting Examples of Amino Acid Sequences

[0103] Cargo Molecules

[0104] In some embodiments, a self-assembling peptide is capable of interacting with one or more cargo molecules. In some embodiments, a self-assembling peptide is capable of interacting with one or more cargos via a non-covalent bond, such as electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, pi-pi stacking, or any combination thereof.

[0105] In some embodiments, a comprises a cargo comprises molecular weight of at least 1 g / mol. In some embodiments, a cargo comprises a molecular weight of 500-1,000, 1000,-2,000, 2,000-3,000, 3,000-4,000, 4,000-5,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, 9,000-10,000, 10,000-12,000, 12,000-14,000, 14,000-16,000, 16,000-17,000, 17,000-18,000, 18,000-20,000, 20,000-25,000, 25,000-30,000, 30,000-40,000, 40,000-50,000, 50,000-60,000, 60,000-70,000, 70,000-80,000, 80,000-90,000, 90,000-100,000, 100,000-110,000, 110,000- 120,000, 120,000-130,000, 130,000-140,000, 140,000-150,000, 150,000-160,000, 160,000- 170,000, 170,000-180,000, 180,000-190,000, 200,000-220,000, 220,000-240,000, 240,000- 260,000, 260,000-280,000, 280,000-300,000, 300,000-350,000, 350,000-400,000, 400,000- 450,000, 450,000-500,000, 500,000-600,000, 600,000-700,000, 700,000-800,000, 800,000- 900,000, 900,000-1,000,000, 1,000,000-2,500,000, or 2,500,000-5,000,000 g / mol. In some embodiments, a cargo comprises a molecular weight of approximately 1,000, 2,000, 5,000, 7,500, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 125,000, or 150,000, g / mol. In some embodiments, a cargo may comprise a molecular weight of that is greater than 5,000,000 g / mol.

[0106] In some embodiments, a cargo (e.g., a therapeutic cargo) comprises a peptide, polypeptide, or a protein. In some embodiments, a cargo comprises a peptide, wherein the peptide comprises approximately 50 amino acids or less in length (e.g., 1-10 amino acids, 10-20 amino acids, etc.). In some embodiments, a cargo comprises a polypeptide or a protein, wherein the polypeptide or protein comprises approximately 1,500 amino acids or less in length (e.g., 100-250 amino acids, 250-500 amino acids, 500-750 amino acids, 750 amino acids, etc.). In other embodiments, a polypeptide or protein comprises more than 1,500 amino acids in length (e.g., 2,000 amino acids, 3,000 amino acids, 4,000 amino acids, etc.). Non-limiting examples of peptide, polypeptide, or a protein that may be comprised in a cargo include a cell- or tissuetargeting peptide or protein, a cell-penetrating peptide or protein, an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a nanobody, a single-chain antibody, such as an scFv, etc.), an antigen-binding fragment, an antigenic peptide or protein (e.g., one comprising an amino acid sequence comprised in an analyte described herein), an enzyme (e.g., indoleamine 2,3-dioxygenase, urease, a protease, signaling protein, transcriptional regulator, RNA-guided nuclease or a variant thereof, etc.) or an enzymatic domain, an enzyme substrate, an extracellular matrix protein or a fragment thereof, a transmembrane receptor or a fragment thereof, a toxin or a fragment thereof, hormones, receptors (e.g., chimeric antigen receptors), a peptibody, a growth factor, a clotting factor, a cytokine, a chemokine, an activating or inhibitory peptide capable of targeting a cell surface receptor or ion channel, a thrombolytic, a bone morphogenetic protein, an Fc-fusion protein, an anticoagulant, and a detectable marker.

[0107] In some embodiments, a cargo is selected for the purposes of vaccine production against a pathogen. In some embodiments, a cargo is selected for the purposes of vaccine production is an immunogenic protein or an immunogenic fragment thereof (e.g., an immunogenic peptide) comprising an antigen of a pathogen. In some embodiments, the pathogen is pathogenic (e.g., infects and / or causes one or more symptoms of a disease, disorder, or condition) to humans. In some embodiments, an immunogenic protein or an immunogenic fragment thereof comprises an amino acid sequence of a peptide, polypeptide, or protein associated with a virus, a bacteria, a fungus, or a parasite. In some embodiments, an immunogenic protein or an immunogenic fragment thereof comprises an amino acid sequence of a peptide, polypeptide, or protein associated with Adenoviridae, Picomaviridae, Herpesviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Poxviridae, Rhabdoviridae, Togaviridae, Mycobacterium tuberculosis, Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella, Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocytis, Stachybotrus, Bacillus anthracis, Clostridium botulinum, Mycobacterium leprae, Yersinia pestis, Rickettsia prowazekii, Bartonella spp., malaria, amoebiasis, babesiosis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Chagas disease, leishmaniasis, African trypanosomiasis (sleeping sickness), Acanthamoeba keratitis, or primary amoebic meningoencephalitis (naegleriasis). In some embodiments, a cargo comprises one or more moieties and / or molecules which are not a peptide, polypeptide, or protein. As a non-limiting example, in some embodiments, cargos may comprise one or more synthetic moieties and / or molecules (e.g., a small molecule or a polynucleotide). In some embodiments, a cargo is a synthetic and / or engineered molecule (e.g., a modified nucleic acid, a small molecule, such as a drug molecule, a hapten, etc.). In some embodiments, a cargo comprises a nucleic acid. In some embodiments, a nucleic acid comprised in a cargo comprises approximately 2-10,000 nucleotides. In some embodiments, a cargo comprises a nucleic acid that is single- stranded, double- stranded, or a nucleic acid that comprises one or more stretches of sequence that are single- stranded and one or more stretches of sequence that are double-stranded. In some embodiments, a cargo comprises an inhibitory nucleic acid (e.g., an antisense oligonucleotide or an interfering RNA, such as a siRNA, a shRNA, a miRNA, etc.). In some embodiments, a cargo comprises two different types of molecules that are associated with each other via one or more non-covalent interactions, one or more covalent interactions, or a combination of such interactions. As a non-limiting example, in some embodiments, a cargo comprises an antibody-drug conjugate or an antigen-binding fragment-drug conjugate. As a further non-limiting example, in some embodiments, a cargo comprises a conjugate, such as a peptide-oligonucleotide (e.g., a single- stranded oligonucleotide) conjugate.

[0108] In some embodiments, a cargo comprises a compound (e.g., small molecules, such as a drug molecules) that is therapeutic for a disease, disorder, or condition. In some embodiments, a cargo comprises an anti-proliferative compound, an anti-cancer compound, an anti-angiogenesis compound, a steroidal or non-steroidal anti-inflammatory compound, an immunosuppressant compound, an anti-bacterial compound, an anti-viral compound, a cardiovascular compound, a cholesterol-lowering compound, an anti-diabetic compound, an anti-allergic compound, a contraceptive compound, an pain-relieving compound, an anesthetic compound, an anticoagulant compound, an enzyme-inhibiting compound, a steroidal compound, or an analgesic compound. In some embodiments, the compound is conjugated to a peptide, polypeptide, or protein which is linked to the self-assembling peptide. However, in other embodiments, a cargo does not comprise a peptide, polypeptide, or protein.

[0109] Linkers In some embodiments, a cargo and / or a self-assembling peptide comprises a linker. In some embodiments, a linker comprises at least one monomer in length. In some embodiments, a linker comprises a polymeric arrangement of monomers (e.g., amino acids) that does not perturb a peptide assembly and / or cargo molecule activity. In some embodiments, a linker connects a first sequence and a second sequence, wherein the first sequence and the second sequence each comprise a plurality of self-assembly repeat units. In some embodiments, a linker connects a cargo to a sequence comprising a plurality of self-assembly repeat units in a self-assembling peptide.

[0110] In some embodiments, a linker comprises one or more monomers (e.g., amino acids) in length. In some embodiments, a linker comprises 1-100 monomers (e.g., amino acids) in length. In some embodiments, a linker comprises 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 monomers in length. In some embodiments, a linker comprises 2-25 monomers (e.g., amino acids) in length. In some embodiments, a linker comprises 2-10 monomers (e.g., amino acids) in length.

[0111] In some embodiments, a linker comprises any combination of the 20 naturally occurring amino acids. In some embodiments, an amino acid sequence comprises in a linker is a flexible linker, such as a hydrophilic linker. In some embodiments, a linker comprises one or more hydroxyl-bearing amino acids. In some embodiments, the one or more hydroxyl-bearing amino acids comprises one or more serines and / or one or more threonines. In some embodiments, a linker comprises one or more glycine amino acid residues. In some embodiments, a linker comprises an amino acid sequence, wherein approximately 1-5%, 5-10%, 10-20%, 20%-50%, or more of the amino acid sequence comprises hydroxyl-bearing amino acids. In some embodiments, a linker comprises an amino acid sequence, wherein approximately 1-5%, 5-10%, 10-20%, 20%-50%, or more of the amino acid sequence comprises glycines. In some embodiments, a linker comprises an amino acid sequence, wherein the amino acid sequence comprises glycines and hydroxyl-bearing amino acids. In some embodiments, a linker comprises an amino acid sequence comprises one or more glycines and one or more serines.

[0112] In some embodiments, a linker comprises a sequence comprising (XiXiXiX2)y, wherein Xi and X2 each correspond to respective amino acids. In some embodiments, Xi is glycine and X2 is serine. In some embodiments, Xi is serine and X2 is glycine. In some embodiments, a linker comprises the sequence (XiXiXiX2)y, wherein the sequence comprising X1X1X1X2 is present a “y” number of times in the linker, wherein “y” is 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, etc.). In some embodiments, one or more amino acids residues are located in the N-terminal and / or C-terminal direction relative to the repeat sequence in the linker. In some embodiments, the one or more amino acids that are located in the N-terminal and / or C- terminal direction relative to the repeat sequence in the linker comprise one or more glycine amino acids and / or one or more cysteine amino acids.

[0113] In some embodiments, a self-assembling peptide comprises a synthetic linker, such as a polyethylene glycol (PEG) linker.

[0114] Nucleic Acids

[0115] Aspects of the present disclosure also relate to nucleic acids. In some embodiments, a nucleic acid comprises a sequence encoding a polymer described herein. In some embodiments, a nucleic acid comprises a plurality of sequences, wherein each sequence of the plurality encodes a different polymer. In some embodiments, a nucleic acid comprises a plurality of sequences, wherein each sequence of the plurality encodes the same polymer. In some embodiments, a polymer encoded by a nucleic acid sequence is a polymer comprising a cargo and a self-assembling peptide described herein.

[0116] In some embodiments, a nucleic acid comprising a sequence encoding a polymer further comprises one or more sequences that do not encode a polymer. In some embodiments, the one or more sequences comprises one or more regulatory sequences. As used herein, a “regulatory sequence” is a DNA sequence which modulates the expression, stability, and / or levels of an RNA when operably linked to a gene sequence. As used herein, a nucleic acid sequence and regulatory sequences may be referred to as being “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences.

[0117] In some embodiments, a regulatory sequence is a transcriptional regulatory sequence, a post-transcriptional regulatory sequence (e.g., a splicing regulatory sequence), or a translation regulatory sequence. In some embodiments, a nucleic acid comprises one or more regulatory sequences, such as a promoter, an enhancer, a silencer, a transcription factor binding sequence, a 5’ UTR, a 3’ UTR, a translation initiation signal (e.g., a Shine-Dalgarno sequence or a Kozack sequence), a transcriptional start sequence, a transcription terminator sequence, an acceptor / donor splicing site, a mRNA degradation or decay signal, a polyadenylation signal, a start codon, a ribosome binding site, a ribozyme, an intron, and / or a stop codon. In some embodiments, a nucleic acid comprises a promoter. In some embodiments, a promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, a synthetic promoter, or a native promoter. In some embodiments, a constitutive promoter maintains constant expression of RNAs regardless of the conditions or physiological state of a host cell. In some embodiments, a tissue- specific promoter binds tissue- specific transcription factors that induce transcription in a tissue specific manner. In some embodiments, a native promoter is native to a gene which is endogenous to a cell comprising a nucleic acid described herein. In some embodiments, a native promoter may be preferred when it is desired that expression of the polynucleotide should mimic the native expression of a gene of interest. In some embodiments, a native promoter may be used when expression of the polynucleotide must be regulated temporally, developmentally, in a tissue-specific manner, or in response to specific transcriptional stimuli. In some embodiments, a sequence encoding a polymer is linked (e.g., operably linked) to an inducible promoter. In some embodiments, an inducible promoter allows regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state. In some embodiments, a sequence encoding a polymer is operably linked to an inducible promoter that is responsive to an inducing agent. In some embodiments, a nucleic acid comprising a sequence encoding a polymer comprises a sequence encoding an inducible polymerase that is operably linked to an inducible promoter that is responsive to an inducing agent. In some embodiments, an inducing agent is IPTG. In some embodiments, an inducible polymerase is a T7 polymerase.

[0118] In some embodiments, a nucleic acid is a vector. In some embodiments, vectors comprise deoxyribonucleotides. In some embodiments, vectors comprise ribonucleotides. In some embodiments, vectors comprise both deoxyribonucleotides and ribonucleotides. In some embodiments, vectors are single-stranded. In some embodiments, vectors are double- stranded. In some embodiments, vectors are circular (e.g., plasmids, such as circular plasmids, nanoplasmids, and minicircle plasmids). In some embodiments, vectors are linear. In some embodiments, a vector may be maintained in high levels in a cell using a selection method, such as one involving an antibiotic resistance gene. In some embodiments, a vector may comprise a partitioning sequence which ensures stable inheritance of the vector. In some embodiments, a vector is a high copy number vector. In some embodiments, a vector, or a fragment thereof (e.g., the heterologous nucleic acid), becomes integrated into the genome of a cell. Polymer Assemblies and Semi-Solid Materials Thereof

[0119] Aspects of the present disclosure also relate to polymer assemblies. Without wishing to be bound by any particular theory or belief, self-assembly repeat units described herein may form non-covalent intramolecular and / or intermolecular interactions under certain conditions that result in phase-separation and assembly of the interacting polymers (e.g., polymers comprising self-assembling peptides) into larger scale structures.

[0120] In some embodiments, self-assembling peptides comprising self-assembly repeat units adopt ordered structures as a result of forming intramolecular and / or intermolecular interactions (e.g., non-covalent interactions, such as via electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, pi-pi stacking, etc). In some embodiments, selfassembling peptides assemble into structures comprising beta strands, such as parallel and / or anti-parallel sheets (see, e.g., FIGs. 2A-2B). In some embodiments, amino acids comprised in self-assembly repeat units described herein are located in beta strands of parallel and / or antiparallel beta sheets. In some embodiments, self-assembling peptides form structures comprising beta meanders, beta-alpha beta motifs, beta hairpins, Greek key motifs, beta solenoids (e.g., structures comprising beta rolls and / or beta helices, such as parallel beta helices), and / or psi- loop motifs (see, as non-limiting examples, FIGs. 2A-2B). In some embodiments, amino acids falling outside of a self-assembly repeat unit form unstructured portions of the self-assembling peptides (e.g., intrinsically disorders structures) and / or other structures, such as beta turns. However, in some embodiments, amino acids falling outside of a self-assembly repeat unit may also form structures, such as beta strands.

[0121] In some embodiments, self-assembly repeat units comprise an arrangement of amino acids that adopt ordered structures which are stabilized by interactions involving the side chains of amino acids in the self-assembling peptide. In some embodiments, self-assembly repeat units drive folding events such that the self-assembling peptide adopts a three-dimensional conformation comprising an interior and / or exterior aspect (or face) of the molecule which is substantially hydrophobic and a separate interior and / or exterior aspect (or face) of the molecule which is substantially hydrophilic. In some embodiments, the three-dimensional conformation of a self-assembling peptide is such that it can interact intermolecularly with one or more selfassembling peptides in a face-face manner (e.g., wherein beta-sheets stably associate with each and / or wherein flexible linkers, such as those which are serine- and / or glycine-rich, interact with each other) and / or in an end-end manner. Non-limiting examples of tools which are useful for prediction of protein structure and may be used to model peptides, polypeptides, and / or proteins comprising amino acid sequences described herein include AlphaFold, Rossetta, I-TASSER, Robetta, Phyre2, RaptorX, and SWISS-MODEL.

[0122] In some embodiments, a polymer assembly is comprised in a semi-solid material. In some embodiments, the semi-solid material comprises a gel. As used herein, the term “gel” refers to a polymer network that comprises a fluid throughout its whole volume, wherein regions of local order comprising interacting polymers acts as network junction points. In some embodiments, a semi-solid material is a hydrogel. As used herein, the term “hydrogel” refers to a gel, in which the fluid is water (e.g., a gel comprising an aqueous buffer, such as a pharmaceutically acceptable buffer). In some embodiments, a gel (e.g., a hydrogel) comprises a covalent polymer network, such as a network formed by crosslinking polymers or by nonlinear polymerization. In some embodiments, a gel (e.g., a hydrogel) comprises a polymer network formed through non-covalent aggregation of polymers (e.g., caused by complexation, such as coordination bond formation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, pi-pi stacking, or a combination thereof).

[0123] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least O.OOlpM in the semi-solid material. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 0.001-0.002 pM, 0.002-0.003 pM, 0.003-0.004 pM, 0.004-0.005 pM, 0.005-0.006 pM, 0.006-0.007 pM, 0.007-0.008 pM, 0.009-0.01 pM, 0.01-0.02 pM, 0.02-0.03 pM, 0.03-0.04 pM, 0.04-0.05 pM, 0.05-0.06 pM, 0.06-0.07 pM, 0.07-0.08 pM, 0.09-0.1 pM, 0.1-0.2 pM, 0.2-0.3 pM, 0.3-0.4 pM, 0.4-0.5 pM, 0.5-0.6 pM, 0.6-0.7 pM, 0.7-0.8 pM, 0.8-0.9 pM, or 0.9-1.0 pM. In some embodiments, a semisolid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 1.0 pM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 1-5 pM, 5-10 pM, 10-20 pM, 20-30 pM, 30-40 pM, 40-50 pM, 50-100 pM, 100-150 pM, 150-200 pM, 200-250 pM, 250-500 pM, 500-750 pM, or 750 pM-1,000 pM. In some embodiments, a semisolid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 1.0 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 1.0- 1.1 mM, 1.1-1.2 mM, 1.2-1.3 mM, 1.3-1.4 mM, 1.4-1.5 mM, 1.5-1.6 mM, 1.6-1.7 mM, 1.7-1.8 mM, 1.8- 1.9 mM, 1.9-2.0 mM, 2.0-2.25 mM, 2.25-2.5 mM, 2.5-2.75 mM, 2.75-3.0 mM, 3.0-4.0 mM, 4.0-5.0 mM, 5.0-6.0 mM, 6.0-7.0 mM, 7.0-8.0 mM, 8.0-9.0 mM, or 9.0-10.0 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 2 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, or

[0124] 100 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of at least 100-120 mM, 120-140 mM, 140-160 mM, 160-180 mM, 180-200 mM, 200-250 mM, 250-300 mM, 300-400 mM, or 400- 500 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a plurality of self-assembling peptides which are at a concentration of more than 500 mM.

[0125] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a cargo. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a cargo which is at a concentration of at least 0.001 pM in the semi-solid material. In some embodiments, a semisolid material (e.g., a hydrogel) comprises a cargo at a concentration of 0.001-0.002 pM, 0.002- 0.003 pM, 0.003-0.004 pM, 0.004-0.005 pM, 0.005-0.006 pM, 0.006-0.007 pM, 0.007-0.008 pM, 0.009-0.01 pM, 0.01-0.02 pM, 0.02-0.03 pM, 0.03-0.04 pM, 0.04-0.05 pM, 0.05-0.06 pM, 0.06-0.07 pM, 0.07-0.08 pM, 0.09-0.1 pM, 0.1-0.2 pM, 0.2-0.3 pM, 0.3-0.4 pM, 0.4-0.5 pM, 0.5-0.6 pM, 0.6-0.7 pM, 0.7-0.8 pM, 0.8-0.9 pM, 0.9-1.0 pM, 1-5 pM, 5-10 pM, 10-20 pM, 20- 30 pM, 30-40 pM, 40-50 pM, 50-100 pM, 100-150 pM, 150-200 pM, 200-250 pM, 250-500 pM, 500-750 pM, 750 pM-1,000 pM, 1.0-1.1 mM, 1.1-1.2 mM, 1.2-1.3 mM, 1.3-1.4 mM, 1.4-

[0126] 1.5 mM, 1.5-1.6 mM, 1.6-1.7 mM, 1.7-1.8 mM, 1.8-1.9 mM, 1.9-2.0 mM, 2.0-2.25 mM, 2.25-

[0127] 2.5 mM, 2.5-2.75 mM, 2.75-3.0 mM, 3.0-4.0 mM, 4.0-5.0 mM, 5.0-6.0 mM, 6.0-7.0 mM, 7.0- 8.0 mM, 8.0-9.0 mM, or 9.0-10.0 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a cargo at a concentration of at least 10 mM. In some embodiments, a semisolid material (e.g., a hydrogel) comprises a cargo at a concentration of at least 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, or 100 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a cargo at a concentration of at least 100-120 mM, 120-140 mM, 140-160 mM, 160-180 mM, 180-200 mM, 200-250 mM, 250-300 mM, 300-400 mM, or 400-500 mM. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a cargo at a concentration of more than 500 mM.

[0128] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a 1:1 molar ratio of self-assembling peptide to cargo. In some embodiments, a semi-solid material (e.g., a hydrogel) comprising a 1:1 molar ratio of self-assembling peptide to cargo is a hydrogel comprising a plurality of polymers, wherein each polymer of the plurality comprises one cargo which is fused to one self-assembling peptide.

[0129] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a non-equal molar ratio of self-assembling peptide to cargo. In some embodiments, a semi-solid material (e.g., a hydrogel) comprising a non-equal molar ratio of self-assembling peptides to cargo comprises a first plurality of self-assembling peptides and a second plurality of self-assembling peptides, wherein each self-assembling peptide of the first plurality comprises a cargo and each self-assembling peptide of the second plurality does comprise a cargo. In some embodiments, a semi-solid material (e.g., a hydrogel) comprising a non-equal molar ratio of self-assembling peptide to cargo, wherein 100% of the self-assembling peptides in the semi-solid material (e.g., a hydrogel) is made up of the first plurality of self-assembling peptides and the second plurality of self-assembling peptides , wherein “x%” of the self-assembling peptides in the semi-solid material (e.g., a hydrogel) correspond to the amount of the first plurality of self-assembling peptides and 100%-“x%” correspond to the amount of the second plurality of self-assembling peptides. In some embodiments, the “x%” is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

[0130] 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,

[0131] 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

[0132] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0133] 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0134] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a neutral or near physiological pH. In some embodiments, the pH of a semi-solid material (e.g., a hydrogel) can range from about 6.5 to about 8.5. In some embodiments, the pH of a semi- solid material (e.g., a hydrogel) is 6.6-6.7, 6.7-6.8, 6.8-6.9, 6.9-7.0, 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9, 7.9-8.0, 8.0-8.1, 8.1-8.2, 8.2-8.3, 8.3-8.4, or 8.4-8.5. In some embodiments, the pH of a semi-solid material (e.g., a hydrogel) is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of a semi-solid material (e.g., a hydrogel) comprises a pH of about 7.0 to about 8.0 (e.g., 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8).

[0135] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a water content of 25% of more. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a water content of 50% or more. In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a water content that is 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or more than 95%. In some embodiments, a semi- solid- material (e.g., a hydrogel) comprises a water content that is approximately 80-90%.

[0136] In some embodiments, a semi-solid material (e.g., a hydrogel) comprises a viscosity in the range of 50-150 Pa*s at 37 °C. In some embodiments, a semi-solid material comprises a viscosity that is in the range of 85-115 Pa*s at 37 °C. In some embodiments, a semi-solid material comprises a viscosity that is 95-105 Pa*s at 37 °C (e.g., about 100 Pa*s at 37 °C).

[0137] Methods

[0138] Methods of Assembling Polymers

[0139] The present disclosure also relates to methods of assembling polymers comprising selfassembling peptides. In some embodiments, a method comprises generating a mixture comprising a buffer (e.g., an aqueous buffer, such as a pharmaceutically acceptable buffer) and a self-assembling peptide fused to a cargo, thereby forming a polymer assembly. In some embodiments, a method of assembling polymers described herein is useful for producing a polymer assembly described herein (e.g., a hydrogel).

[0140] In some embodiments, producing the polymer assembly comprises incubating the mixture at a temperature of about 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C. In some embodiments, producing the polymer assembly comprises incubating the mixture at a temperature of about 4 °C to about 37 °C. In some embodiments, producing the polymer assembly comprises incubating the mixture at a temperature of less than 0°C (e.g., at a temperature of about -80 °C, -60 °C, -40 °C, -20 °C, -10 °C, etc.) for at least one hour (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 8-12 hours, 12-24 hours, 24- 48 hours, etc.) and then incubating the mixture at a temperature of about 4 °C to about 37 °C (e.g., in order to thaw the mixture). In some embodiments, the thawed mixture is centrifuged (e.g., at 14,000-15,000 x g, such as 14,000 x g, 14,100 x g, 14,200 x g, 14,300 x g, 14,400 x g, 14,500 x g, 14,600 x g, 14,700 x g, 14,800 x g, 14,900 x g, or 15,000 x g) to pack the polymer into a semi-solid material (e.g., hydrogel). In some embodiments, a mixture comprising a polymer and a buffer comprises the polymer at a concentration of at least 0.1% (weight / volume). In some embodiments, the mixture comprises the polymer at a concentration of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, or 0.99% (weight / volume). In some embodiments, the mixture comprises the polymer at a concentration of about 1.0% to 2.0% (weight / volume). In some embodiments, the mixture comprises the polymer at a concentration of 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% (weight / volume). In some embodiments, the mixture comprises the polymer at a concentration of 1.5-1.75% (e.g., 1.5%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.63%, 1.64%, 1.65%, 1.66%, 1.67%, 1.68%, 1.69%, 1.70%, 1.71%, 1.72%, 1.73%, 1.74%, or 1.75%) (weight / volume). In some embodiments, the mixture comprises the polymer at a concentration of greater than 2.0% (weight / volume). In some embodiments, the buffer and / or the mixture is at neutral or near physiological pH.

[0141] In some embodiments, the pH of the buffer and / or the mixture can range from about 6.5 to about 8.5. In some embodiments, the pH of the buffer and / or the mixture is 6.6-6.7, 6.7-6.8, 6.8-6.9, 6.9-7.0, 7.0-7.1, 7.1-7.2, 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.6-7.7, 7.7-7.8, 7.8-7.9, 7.9- 8.0, 8.0-8.1, 8.1-8.2, 8.2-8.3, 8.3-8.4, or 8.4-8.5. In some embodiments, the pH of the buffer and / or the mixture is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the buffer and / or the mixture comprises a pH of about 7.0 to about 8.0 (e.g., 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8).

[0142] In some embodiments, producing the polymer assembly further comprises contacting a nucleic acid described herein with one or more cells. In some embodiments, producing the polymer assembly further comprises obtaining the plurality of polymers from the one or more cells before generating the mixture. In some embodiments, the one or more cells are contacted with an inducing agent capable of inducing expression of the polymer from the nucleic acid. In some embodiments, the one or more cells comprise bacterium or a bacterial cell population thereof. In some embodiments, the one or more cells comprise E. coli (e.g., BL121s). In some embodiments, the one or more cells comprise mammalian cells or a mammalian cell population thereof (e.g., mammalian cells that are ex vivo, such as cells of a commercially available cell line). In some embodiments, a cell population comprises 2-100, 100-500, 500-1,000, 1,000- 5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000, 1,000,000-5,000,000, 5,000-10,000,000 or more cells. However, such disclosures should not be considered limiting as, in some embodiments, polymers of the present disclosure may be produced using methods which are not cell-based (e.g., via synthetic methods, such as using solid-phase peptide synthesis (SPSS)).

[0143] In some embodiments, polymers are isolated and / or purified prior to being used to produce a polymer assembly (e.g., a semi-solid material, such as a hydrogel). In some embodiments, isolating and / or purifying the polymers comprises lysing a cell or cell population comprising a nucleic acid encoding the polymer and harvesting lysate comprising the polymers. In some embodiments, polymers are isolated and / or purified using one or more chromatography methods, such as size-exclusion chromatography, affinity chromatography (e.g., metal affinity chromatography), ion exchange chromatography, high-performance liquid chromatography, etc. In some embodiments, polymers are isolated and / or purified by concentrating the polymers, such as via dialyzing polymers (e.g., overnight at 4 °C).

[0144] Methods of Administration

[0145] In some embodiments, a method described herein comprises administering a polymer assembly (e.g., one comprised in a semi-solid material, such as a hydrogel) to a subject. In some embodiments, the polymer assembly comprises a cargo, such as a therapeutic cargo described herein. In some embodiments, a method comprises administering a therapeutic cargo to a subject in need thereof, wherein the subject has or is suspected of having a disease, disorder, or condition for which the therapeutic cargo is capable of treating. In some embodiments, one or more of each therapeutic cargo in a polymer assembly or a semi-solid material thereof (e.g., a hydrogel) is fused to a self-assembling peptide.

[0146] A “subject” to which administration of a polymer assembly is contemplated refers to a human (e.g., a human of any age group, including a pediatric subject, such as an infant, child, or adolescent, or adult subject, such as a young adult, middle-aged adult, or senior adult) or nonhuman animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. In some embodiments, a subject is a “subject in need thereof’ which refers to a subject (e.g., a human subject) having, at risk of having, previously had, or is suspected of having a disease, disorder, or condition.

[0147] In some embodiments, administration of a polymer assembly is used to treat a subject. The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease, disorder, or condition. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0148] In some embodiments, administration of a polymer assembly achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease, disorder, or condition or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease, disorder, or condition; (iii) protection against the progression of a disease or disorder or symptom associated therewith; (iv) regression of a disease, disorder, or condition or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease, disorder, or condition; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease, disorder, or condition; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0149] In some embodiments, administration of a polymer assembly is performed intravenously, subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intra- cerebroventricularly, intramuscularly, intracranially, intrathecally, orally, intraperitoneally, or by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs. In some embodiments, direct injection is performed concurrently with a surgical procedure or interventional procedure. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the cargo (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration, injection, etc.). In some embodiments, a polymer assembly is administered to a subject through only one administration route. In some embodiments, multiple administration routes may be exploited (e.g., serially, or simultaneously) for administration of a polymer assembly to a subject. In some embodiments, administering a polymer assembly comprises injecting a subject with a hydrogel.

[0150] In some embodiments, a polymer assembly is administered to a subject in an effective amount. In some embodiments, an effective amount is a “therapeutically effective amount” which refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. In some embodiments, a therapeutically effective amount means an amount of therapeutic cargo, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder, or condition. In some embodiments, a therapeutically effective amount can be an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the disease, disorder, or condition, and / or enhances the therapeutic efficacy of another therapeutic cargo. In some embodiments, an effective amount is an amount effective for producing an immunogenic response against an antigen comprised in a cargo in the polymer assembly.

[0151] Detection Methods

[0152] In some embodiments, a detection method described herein is useful in treating a subject having or suspected of having a disease, disorder, or condition when the detection method is performed before and / or after administering a therapeutic cargo to the subject. In some embodiments, a method described herein comprises obtaining a biological sample from a subject. In some embodiments, a method described herein comprises obtaining a biological sample from a subject before and / or after administration of a polymer assembly (e.g., one comprised in a semi-solid material, such as a hydrogel comprising a therapeutic cargo).

[0153] As used herein, a “biological sample” may refer to any specimen derived or obtained from a subject having or suspected of having a disease, disorder, or condition. In some embodiments, a biological sample is a tissue sample. In some embodiments, a biological sample comprises one or more cells from a subject. In some embodiments, a biological sample is blood sample (e.g., a sample of whole blood, serum, or plasma), a urine sample, a sputum sample, a stool sample, or a biopsy. In some embodiments, a biological sample comprises or is suspecting of comprising the analyte. In some embodiments, an analyte is a peptide, polypeptide, or protein. In some embodiments, an analyte comprises an antibody or an antigen-binding fragment thereof. In some embodiments, an analyte is a nucleic acid.

[0154] In some embodiments, a biological sample has been subjected to one or more processing steps prior to being used in a detection method. In some embodiments, a biological sample has been subjected to one or more of enzymatic digestion (e.g., with a nuclease and / or a protease), contacted with a chemical (e.g., for the purposes of cell permeabilization, cell lysis, and / or for improving sample stability), or storage for a given time period (e.g., about 6, 5, 4, 3, 2, or 1 weeks or 6, 5, 4, 3, 2, or 1 days) and / or at a given temperature (e.g., at 25 °C, 4 °C, -20 °C or lower). In some embodiments, a biological sample has been subjected to one or more steps that removes and / or enriches for one or more cell types in the biological sample. In some embodiments, a method comprises isolating and / or purifying an analyte from a biological sample.

[0155] In some embodiments, a method described herein comprises contacting a biological sample with one or more detection agents. In some embodiments, a detection agent is capable of binding to an analyte. In some embodiments, a detection agent comprises an antibody or an antigen-binding fragment thereof. In some embodiments, a detection agent comprises a polymer described herein or a fragment thereof (e.g., a fragment comprising a cargo described herein).

[0156] In some embodiments, a method of detecting an analyte in a biological sample comprises an assay which is capable of detecting a peptide, polypeptide, or protein. In some embodiments, a method of detecting an analyte in a biological sample comprises performing an immunoassay comprising contacting a biological sample with a detection agent to identify the presence of an analyte. In some embodiments, a method of detecting an analyte in a biological sample comprises immunoblot (e.g., dot blot, 2-D gel electrophoresis, Western Blot, etc.), electrochemiluminescence immunoassay (e.g., Meso-Scale Detection (MSD)), immunohistochemistry (IHC), ELISA (e.g., RCA-based ELISA or RT-PCR-based ELISA), label free immunoassays such as surface plasmon resonance bio layer interferometry, immunoquantitative PCR, bead-based immunoassays, immunoprecipitation, immunostaining, or immunoelectrophoresis. However, in some embodiments, methods of detecting an analyte in a biological sample comprises mass spectrometry such as GC-MS, LC-MS, MALDI-TOF-MS.

[0157] In some embodiments, a method of detecting an analyte in a biological sample comprises an assay which is capable of detecting a nucleic acid (e.g., DNA or RNA). In some embodiments, a method of detecting an analyte in a biological sample comprises polymerase chain reaction (PCR), such as quantitative PCR or real-time qPCR (RT-qPCR). In some embodiments, a method of detecting an analyte in a biological sample comprises fluorescence in situ hybridization (FISH), microarray, or RNA-seq. In some embodiments, a method of detecting an analyte in a biological sample comprises DNA sequencing (e.g., next-generation sequencing). In some embodiments, a method of detecting an analyte comprises DNA gel electrophoresis.

[0158] Compositions In some aspects, the disclosure relates to compositions. In some embodiments, a composition comprises a self-assembling peptide described herein. In some embodiments, a composition comprises a polymer described herein. In some embodiments, a composition comprises a nucleic acid described herein. In some embodiments, a composition comprises a cell or cell population described herein. In some embodiments, a composition comprises a polymer assembly described herein. In some embodiments, a composition comprises a semi-solid material, such as a hydrogel, described herein. In some embodiments, a composition is useful in a method of treating a subject described herein (e.g., a mammalian subject, such as a human subject). In some embodiments, a composition comprising a semi-solid material (e.g., a semisolid materials comprising a polymer assembly, such as a hydrogel) is administered to a subject (e.g., wherein the administration is used to treat the subject), wherein the semi-solid material comprises a cargo at a concentration of at least 10 nM (e.g., 10 nM-10 mM, such as 10 nM-20 nM, 20-50 nM, 50 nM-100 nM, 100 nM-250 nM, 250 nM-500 nM, 500 nM-1 mM, 1 mM-2 mM, 2 mM-5 mM, or 5 mM-10 mM, or more than 10 mM).

[0159] In some embodiments, a composition comprises a pharmaceutical excipient. Pharmaceutically acceptable excipients (excipients) are substances other than a therapeutic agent (e.g., a therapeutic cargo) that are intentionally included in a delivery system (e.g., a hydrogel). In some embodiments, excipients do not exert or are not intended to exert a therapeutic effect. In some embodiments, excipients may act to a) aid in processing of a polymer and / or polymer assembly delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, or delivery of a polymer and / or polymer assembly during storage or use. In some embodiments, a pharmaceutically acceptable excipient may be an inert substance. In some embodiments, a pharmaceutically acceptable excipient may not be an inert substance. Excipients include, but are not limited to, absorption enhancers, anti- adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. In some embodiments, a composition further comprises additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to: anti-pruritic s, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine).

[0160] Pharmaceutical compositions of the present disclosure may be suitable for treatment regimens and thereby administered to a subject via a variety of methods described herein. Such compositions may be formulated for use in a variety of therapies, such as, in the amelioration, prevention, and / or treatment of conditions for which the cargo comprised in the composition is therapeutic. Accordingly, compositions described herein may be administered to a subject such as human or non-human subjects, a cell in situ in a subject, a cell ex vivo, a cell derived from a subject, or a biological sample (e.g., one derived from a subject).

[0161] For administration of an injectable aqueous solution, the composition may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline, polyalcohols, or glucose. For example, one dosage of a therapeutic agent may be dissolved in an isotonic NaCl solution and optionally added to a larger volume of hypodermoclysis fluid prior to being injected at the proposed site of infusion. In some embodiments, the composition is provided in a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0162] Kits

[0163] In some aspects, the disclosure relates to kits. In some embodiments, a kit comprises a self-assembling peptide described herein. In some embodiments, a kit comprises a polymer described herein. In some embodiments, a kit comprises a nucleic acid described herein. In some embodiments, a kit comprises a cell or cell population described herein. In some embodiments, a kit comprises a polymer assembly described herein. In some embodiments, a kit comprises a semi-solid material, such as a hydrogel, described herein. Accordingly, kits provided by the present disclosure may comprise any or all of the materials necessary for producing a polymer, polymer assembly, and / or semi-solid material (e.g., a hydrogel) and / or contacting said polymer, polymer assembly, and / or semi-solid material with one or more cells (e.g., one or more cells in a subject that has been administered a polymer assembly).

[0164] In some embodiments, the kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use. In some embodiments, the components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, in some embodiments, they may be housed in a vial or other container for storage. In some embodiments, a second container may have other components prepared sterilely. Alternatively, in some embodiments, the kits may include the active agents premixed and shipped in a vial, tube, or other container. In some embodiments, the kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.

[0165] In some embodiments, any of the kits described herein may further comprise components needed for inducing uptake of a polymer assembly into a cell. In some embodiments, each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In some embodiments, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0166] In some embodiments, a kit further comprises a set of instructions for carrying out the methods described herein. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of this disclosure. In some embodiments, instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. In some embodiments, the written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with this disclosure. Additionally, in some embodiments, the kits may include other components depending on the specific application, as described herein. In some embodiments, the kits may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. In some embodiments, the kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. In some embodiments, the kits, or any of its components, can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.

[0167] In some embodiments, a kit comprises a polymer assembly or a semi-solid material thereof (e.g., a hydrogel) described herein which is present in a device. In some embodiments, the device is one that is suitable for administering polymers to a subject, such as a syringe for injection of a subject.

[0168] General Techniques

[0169] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as the following references which are incorporated by reference herein for their disclosures related to methods: Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Lreshney, ed. (1986; Immobilized Cells and Enzymes (IRL Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); L.M. Ausubel et al. (eds.).

[0170] EXAMPLES

[0171] Example 1. N-GlycoTag (NGT) and NGT-protein fusion molecules.

[0172] N-GlycoTag (NGT) was designed with two repeating sequences: a segment comprising the amino acid sequence GGNWTT (SEQ ID NO:1) and a glycine / serine linker comprising the amino acid sequence GGGSGGGS (SEQ ID NO: 2) which separated each of the segments. The full-length sequence was a 10-repeat sequence of the asparagine / tryptophan motif (or selfassembly repeat unit) flanked by two cytosines: CGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGGNW TTGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGG NWTTGGGSGGGSGGNWTTGGGSGGGSGGNWTTRC (SEQ ID NO: 3). The structure of the NGT was modeled using Alpha-Fold (FIGs. 2A-2B).

[0173] DNA encoding for NGT was fused to a target protein and inserted into a plasmid, which was subsequently transformed into BL21(DE3)-competent Escherichia coli for expression and purification using established microbiological methods. Briefly, transformed E. coli BL21(DE3) cells were grown in media and purified using immobilized metal affinity chromatography to obtain NGT-protein fusion molecules. Using this method, three NGT-fusion proteins were synthesized: NGT fused to superfolder Green Fluorescent Protein, NGT fused to nanoluciferase, and NGT fused to indoleamine-2,3-dioxygenase. Measurement of the molecular mass of these three NGT fusion proteins by MALDI-TOF showed agreement with the theoretical molecular mass (FIG. 3). The ability of the NGT-fusion proteins to phase separate and pack into a gel state were analyzed. The purified protein was first dialyzed in PBS at 4°C overnight. The turbid, dialyzed protein was then aliquoted, frozen at -80°C for at least 4 hours, and then thawed at 4°C. The NGT-fusion proteins showed phase separation after thawing at 4°C and could be packed into a gel state after centrifugation. Optical density measurements showed that the packed gels comprised a high load of fusion protein (e.g., > ImM) (FIGs. 1N-1P).

[0174] To analyze the dynamic viscosity and viscoelasticity of the NGT-fusion proteins, the gels were measured using a stress-control-type rheometer. The dynamic viscoelastic measurements were carried out at 37°C with varying frequencies between 10 rad / s and 0.1 rad / s. FIG. 4 shows the storage modulus (G’) and the loss modulus (G”) of NGT-sfGFP and NGT-nL at 37°C. For both NGT-fusion proteins, the storage modulus was greater than the loss modulus throughout the frequency range, indicating a viscoelastic gel at 37°C.

[0175] Additionally, the NGT-fusion proteins were shown to be capable of recovering from high shear rate disruption and high strain disruption. The viscosity of NGT-sfGFP and NGT-nL was measured over time as the fusion proteins were subjected to 0.5 s'1shear rate and three, evenly spaced bursts of 100 s'1shear rate (FIG. 5A). Both fusion proteins demonstrated recovery of viscoelasticity after high shear rate disruption and shear thinning through a syringe. This result demonstrated that, in some embodiments, the NGT-fusion proteins are injectable (FIG. 5B). Further, storage modulus (G’) and loss modulus (G”) measurements of NGT-sfGFP and NGT- nL showed that the NGT-fusion proteins were capable of recovering from high strain disruption and exhibited self-healing after being subject to 1000% strain (FIG. 6). the enzymatic activity of NGT-fusion proteins in the gel and non-gel states were analyzed. NGT-nL was used to analyze enzymatic activity. Nnanoluciferase (nL) enzymatically converts furamizine to furimamide, giving off light. A droplet of furimazine was contacted with an NGT-nL gel sample, NGT-nL non-gel sample, and to a PBS sample, and the peak relative fluorescence intensity was quantified. The results demonstrated that NGT-nL in both the gel and non-gel states retained enzymatic activity (FIGs. 7A-7B).

[0176] Example 2. Post-Translational Glycosylation of Polypeptide Tags for Modification of Protein Assembly.

[0177] Protein glycosylation can affect all levels of protein functionality, from assembly to binding properties. However, difficulties exist in precisely modifying carbohydrate type, density, and valency to govern these effects. Here, recombinant polypeptide tags amenable to post-translational glycosylation were used to exert user-defined control over protein glycosylation states. The tags were then used to control phase behavior.

[0178] An N-linked glycosylation sequence (GGNWTT) (SEQ ID NO: 1) fused to sfGFP was inserted into a pET-21d(+) plasmid and transformed into E. coli as previously described. MALDI-TOF mass spectrometry showed that the N-glycotag-sfGFP was glycosylated by A. pleuroneumoniae N-glycosyltransferse (ApNGT) (FIG. 8).

[0179] The effect of glycosylation on the phase transition of the N-glycotag was analyzed. The relationship between the viscosity reduction of the physical gel and the cloud point of the polymer solution during thermal cycling was first measured from the light beam transmittance change (FIG. 9). The results indicated that N-glycotag-sfGFP undergoes sol-gel transition at 4°C due to distributed hydrogen bonding in the glycotag backbone. With the introduction of increasing concentration of sodium thiocyanate (0.1 M NaSCN and 1 M NaSCN), a chaotropic agent that disrupts hydrogen bonding networks, the phase separation temperature decreased. This gelation transition was abrogated upon glycosylation: unlike N-glycotag-sfGFP, glucose- modifed N-glycotag-sfGFP (Glu-N-glycotag-sfGFP) remained liquid at 4°C (FIG. 10A) and did not exhibit thermally reversible behavior (FIG. 10B).

[0180] Example 3

[0181] This Example relates to self-assembling peptides comprising one or more self-assembly repeat units and methods utilizing the self-assembling peptides. FIGs. 11A-11B show NGT- based fusion proteins were recovered as pure, full-length proteins. MAEDI-TOF m / z measurement was consistent with the expected molecular weight. FIGs. 12A-12B show (NGT)iosfGFP formed assemblies, whereas (NGT)2sfGFP, (NGT)ssfGFP, and (QGT)iosfGFP did not. The (NGT)2sfGFP, (NGT)ssfGFP, (QGT)iosfGFP did not form assemblies based on turbidimetry. No large assemblies were detected in (NGT)2sfGFP or (NGT)ssfGFP via fluorescence microscopy.

[0182] FIG. 13 shows 30 pM of (NGT)iosfGFP (left), sfGFP (middle), and (QGT)iosfGFP (right) after freeze-thaw. Turbidity in the (NGT)iosfGFP sample is visible. FIGs.l4A-14B show analyses of polymers subjected to freeze-thaw conditions. FIG. 14A shows 500 pM (NGT)iosfGFP processed under different temperature conditions. When frozen at -80 °C and thawed at 4 °C, or kept cool at 4 °C, (NGT)iosfGFP formed a self-supporting material that did not flow due to gravity. When incubated at 37 °C with no exposure to cold temperature, 500 pM (NGT)iosfGFP flowed due to gravity when inverted. FIG. 14B shows 500 pM (QGT)iosfGFP after being frozen at -80 °C and thawed at 4 °C. Vial inversion demonstrated that (QGT)iosfGFP under these conditions flowed due to gravity and did not form a gel.

[0183] FIG. 15 shows (NGT)iosfGFP tryptophan fluorescence emission peak red-shifted with increasing temperature. A solution of 30 pM (NGT)iosfGFP containing visible assemblies was used to measure tryptophan fluorescence with increasing temperature. Red-shift of tryptophan fluorescence km ax is indicative of a more polar environment, suggesting disassembly and solvent exposure of the tryptophan residues in the polypeptide sequence.

[0184] FIGs. 16A-16F show no Trp Fl. shift was observed in any other group w.r.t temperature. Tryptophan fluorescence provided information about the local environment of the tryptophan residues. Here, change in tryptophan km ax is used to roughly correlate to the change in solvent exposure of tryptophan residues. No shift in kmax indicates no change in tryptophan local environment with respect to temperature. A red shift in tryptophan kmax is associated with increased exposure to solvent. Increasing the temperature from 4 °C to 60 °C caused a red shift in the tryptophan fluorescence of (NGT)iosfGFP, suggesting that the tryptophan residues were more exposed to solvent. As assembly of (NGT)iosfGFP is sensitive to increases in temperature, this suggests that the (NGT)iosfGFP is assembled at 4 °C and is disassembling with increasing temperature. (NGT)2sfGFP, (NGT)ssfGFP, (QGT)iosfGFP, sfGFP, and L-tryptophan, which did not show assembly, did not have a shift in tryptophan fluorescence with increasing temperature.

[0185] FIG. 17 shows CR staining absorbance comparison. Binding of Congo Red (CR) causes characteristic red shift in the absorbance from 490 nm to a maximum of -540 nm. Small redshift in (NGT)iosfGFP and (NFT)iosfGFP absorbance away from sfGFP peak (-485-488 nm). (NGT)iosfGFP and (NFT)iosfGFP without CR do not have CR peak absorbance. Absorbance peak at 540 nm is present for (NGT)iosfGFP, (NFT)iosfGFP, and CATCH 4K6E in the presence of CR. CATCH 4K6E forms P-sheet fibrils when assembled and is used as a positive control for staining here. Proteins at 30 pM, CATCH 4K6E at 1 mM. Offset, normalized spectra. FIG. 18 shows (NGT)2sfGFP, (NGT)ssfGFP do not show 540 nm peak with Congo Red. (NGT)2sfGFP at 150 pM and (NGT)ssfGFP at 60 pM in the presence of CR display slight 540 nm peak, similar to sfGFP. (NGT)iosfGFP and sfGFP at 30 pM. The PBS + CR signal was subtracted from these spectra. FIGs. 19A-19B show Congo Red Staining of (NGT)iosfGFP. FIGs. 20A-20B show Congo Red Staining of (NFT)iosfGFP. (NFT)iosfGFP did form assemblies after freeze-thaw processing that stained positive for Congo Red, suggesting that the sequence (GGGSGGGSGGNXTT) (SEQ ID NO: 14) can form assemblies if X is an aromatic amino acid.

[0186] FIG. 21 shows (NGT)lOnL binds Thioflavin T. Thioflavin T (ThT) enhances fluorescence when bound to cross-P sheet. ThT fluorescence was enhanced in the presence of (NGT)ionL gel. FIGs. 22A-22B show Ac-GGNWTT-Am (SEQ ID NO: 1) is stained by CR. Ac- GGNWTT-Am (SEQ ID NO: 1), a peptide variant of the core NGT sequence, induces characteristic red shift of bound CR FIG. 23 shows Ac-GGNWTT-Am binds ThT. ThT fluorescence intensity is enhanced in the presence of the peptide Ac-GGNWTT-Am (SEQ ID NO: 1), suggesting that Ac-GGNWTT-Am (SEQ ID NO: 1) is forming P-sheet rich structure

[0187] FIG. 24 shows NGT-Ovalbumin creates sub-micron size particles, when suspended in PBS. This measurement was taken using dynamic light scattering (DLS). This peak persists following sterile filtration with a 0.22 um filte.

[0188] FIG. 25 shows data obtained from analyzing control samples used in analyses of cross presentation on a dendritic cell line (DC2.4s): experimental design and controls. On day 1, Plate DC2.4s was seeded at 100,000 cells per well in a 24 well plate. On day 2, NGT-Ovalbumin in PBS was added to treatment groups. On day 3, pulse control with SIINFEKL peptide was performed. Flow cytometry was completed 2 hours later. The stains was anti-SIINFEKL-MHC-I (PE-Cy7). SIINFEKL is a MHC Class I restricted peptide of Ovalbumin. FIG. 26 shows NGT- Ovalbumin induces a concentration dependent increase in SIINFEKL-MHCI expression on DC2.4s FIGs. 27A-27C show PEG interacts with NGT-Ovalbumin to form sub-micron peaks. The Z- Average increases with increasing PEG chain length. FIG. 28 shows NGT-OVA + PEG formulations do not yield SIINFEKL-MHCI presentation on DC2.4s.

[0189] INCORPORATION BY REFERENCE

[0190] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0191] EQUIVALENTS While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0192] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0193] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0194] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0195] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0196] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0197] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0198] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMSWhat is claimed is:

1. A polymer comprising a self-assembling peptide fused to a cargo, wherein the selfassembling peptide comprises a plurality of self-assembly repeat units, wherein each selfassembly repeat unit of the plurality comprises an amide-bearing amino acid and an aromatic amino acid.

2. The polymer of claim 1, wherein the cargo comprises a peptide, a polypeptide, or a protein.

3. The polymer of claim 1 or 2, wherein the amide-bearing amino acid and the aromatic amino acid are contiguous in at least one self-assembly repeat unit of the plurality.

4. The polymer of any one of claims 1-3, wherein the amide-bearing amino acid is N- terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality.

5. The polymer of any one of claims 1-3, wherein the amide-bearing amino acid is C- terminal relative to the aromatic amino acid in one or more of the self-assembly repeat units of the plurality.

6. The polymer of any one of claims 1-5, wherein the amide-bearing amino acid in at least one self-assembly repeat unit of the plurality is asparagine (N).

7. The polymer of any one of claims 1-6, wherein the amide-bearing amino acid in at least one self-assembly repeat unit of the plurality is glutamine (Q).

8. The polymer of any one of claims 1-7, wherein the aromatic amino acid in at least one self-assembly repeat unit of the plurality is tryptophan (W).

9. The polymer of any one of claims 1-8, wherein the plurality of self-assembly repeat units comprises 2-10 self-assembly repeat units.

10. The polymer of any one of claims 1-9, wherein the polymer comprises at least one of: (i) a first sequence comprising one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality;(ii) a second sequence comprising one or more amino acids that are C-terminal to at least one self-assembly repeat unit of the plurality; and(iii) a third sequence comprising one or more amino acids that are N-terminal to at least one self-assembly repeat unit of the plurality and one or more amino acids that are C-terminal to the at least one self-assembly repeat unit.

11. The polymer of claim 10, wherein the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence of (i) and / or the third sequence of (iii) comprises 2-10 amino acids.

12. The polymer of claim 10 or 11, wherein the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence of (i) and / or the third sequence of (iii) comprises at least one glycine (G) amino acid.

13. The polymer of any one of claims 10-12, wherein the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence of (i) and / or the third sequence (iii) comprises at least one hydroxyl-bearing amino acid.

14. The polymer of any one of claims 10-13, wherein the one or more amino acids that are N-terminal to the at least one self-assembly repeat unit in the first sequence of (i) and / or the third sequence of (iii) comprises an amino acid sequence of GGGSGGGSGG (SEQ ID NO: 15).

15. The polymer of any one of claims 10-13, wherein the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit in the second sequence of (ii) and / or the third sequence of (iii) comprises 2 amino acids.

16. The polymer of any one of claims 10-15, wherein the one or more amino acids that are C-terminal to the at least one self-assembly repeat unit in the second sequence of (ii) and / or the third sequence of (iii) comprises at least one hydroxyl-bearing amino acid.

17. The polymer of any one of claims 13-16, wherein each of the at least one hydroxylbearing amino acid that are N-terminal to the at least one self-assembly repeat unit in the first sequence of (i) and the third sequence of (iii) is serine (S) and / or wherein each of the at least one hydroxyl-bearing amino acid that are C-terminal to the at least one self-assembly repeat unit in the second sequence of (ii) and the third sequence of (iii) is threonine (T).

18. The polymer of any one of claims 10-17, wherein the third sequence of (iii) comprises GGGSGGGSGGNWTT (SEQ ID NO: 11).

19. The polymer of any one of claims 10-18, wherein the first sequence of (i), the second sequence of (ii), and / or the third sequence of (iii) is repeated one or more times.

20. The polymer of any one of claims 10-19, wherein the first sequence of (i), the second sequence of (ii), and / or the third sequence of (iii) is repeated nine times.

21. The polymer of any one of claims 1-20, wherein a linker connects the cargo to the selfassembling peptide.

22. The polymer of any one of claims 1-21, wherein a cysteine (C) is N-terminal and / or C- terminal to a sequence comprising the plurality of self-assembly repeat units.

23. A nucleic acid comprising a sequence encoding the polymer of any one of claims 1-22.

24. The nucleic acid of claim 23, wherein the sequence encoding the polymer is operably linked to at least one regulatory sequence.

25. The nucleic acid of claim 23 or 24, wherein the at least one regulatory sequence comprises an inducible regulatory sequence.

26. The nucleic acid of any one of claims 23-25, wherein the nucleic acid is a vector.

27. A cell or cell population thereof comprising the nucleic acid of any one of claims 23-26.

28. A polymer assembly comprising a plurality of the polymer of any one of claims 1-22.

29. The polymer assembly of claim 28, wherein the polymer is present at a concentration of at least O.lmM.

30. The polymer assembly of claim 28 or 29, wherein the polymer is present at a concentration greater than or equal to l.OmM.

31. The polymer assembly of any one of claims 28-30, wherein the polymer assembly is comprised in a semi-solid material.

32. The polymer assembly of claim 31, wherein the semi-solid material comprises a hydrogel.

33. A method of producing the polymer assembly of any one of claims 28-32, wherein the method comprises generating a mixture comprising the polymer and a buffer, thereby assembling the polymers.

34. The method of claim 33, wherein producing the polymer assembly comprises incubating the mixture at a temperature of about 4 °C to about 37 °C.

35. The method of claim 33 or 34, wherein the mixture comprises the polymer at a concentration of about 1.0% to 2.0% (weight / volume).

36. The method of any one of claims 33-35, wherein the buffer comprises a pH of about 7.0 to about 8.0.

37. The method of any one of claims 33-36, wherein the method further comprises contacting a nucleic acid comprising a sequence encoding the polymer with one or more cells and obtaining the plurality of polymers from the one or more cells before generating the mixture.

38. The method of claim any one of claims 33-37, wherein the method further comprises contacting the one or more cells with an inducing agent which is capable of inducing expression of the polymer from the nucleic acid.

39. An ex vivo method comprising contacting the polymer assembly of any one of claims 28- 32 with at least one cell.

40. The method of claim 39, wherein the method further comprises obtaining a biological sample, wherein the biological sample comprises the at least one cell or a descendant cell thereof.

41. A method comprising administering the polymer assembly of any one of claims 28-32 to a subject.

42. The method of claim 41, wherein administering the polymer assembly comprises injecting the subject with the polymer assembly.

43. The method of claim 41 or 42, wherein the method further comprises obtaining a biological sample from the subject after administration of the hydrogel.

44. The method of claim 43, wherein the biological sample comprises one or more cells from the subject.

45. The method of claim 43 or 44, wherein the biological sample is a blood sample or a urine sample.

46. The method of 40 or 43-45, wherein the method further comprises contacting the biological sample with one or more detection agents capable of binding to an analyte, wherein the biological sample comprises or is suspecting of comprising the analyte.

47. The method of claim 46, wherein the analyte comprises an antibody or an antigenbinding fragment thereof.

48. The method of claim 47, wherein the one or more detection agents comprises the polymer or a fragment thereof.

49. The method of claim 47 or 48, wherein the one or more detection agents comprises an antibody or an antigen-binding fragment capable of binding to the analyte.

50. The method of any one of claims 46-49, wherein the method further comprises detecting the analyte in the biological sample.

51. The method of any one of claims 46-50, wherein the method further comprises isolating and / or purifying the analyte from the biological sample.

52. A composition comprising the polymer of any one of claims 1-22, the nucleic acid of any one of claims 23-26, the cell or cell population of claim 27, and / or the polymer assembly of any one of claims 28-32.

53. A kit comprising the polymer of any one of claims 1-22, the nucleic acid of any one of claims 23-26, the cell or cell population of claim 27, and / or the polymer assembly of any one of claims 28-32, and / or the composition of claim 52.

54. The kit of claim 53, wherein the polymer assembly is comprised in a syringe.

55. The kit of claim 54, wherein the syringe further comprises a pharmaceutically acceptable buffer.

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