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WO2025106582A1 - Ensembles polymères pour l'administration de charges - Google Patents

Ensembles polymères pour l'administration de charges Download PDF

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Publication number
WO2025106582A1
WO2025106582A1 PCT/US2024/055781 US2024055781W WO2025106582A1 WO 2025106582 A1 WO2025106582 A1 WO 2025106582A1 US 2024055781 W US2024055781 W US 2024055781W WO 2025106582 A1 WO2025106582 A1 WO 2025106582A1
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WIPO (PCT)
Prior art keywords
self
polymer
assembly
sequence
amino acid
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Eric D. HILL
Gregory Allan Hudalla
Benjamin G. Keselowsky
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C07K2319/715—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16 containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance.
  • Bioly 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.
  • 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.
  • 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.
  • polymers described herein can be useful for producing delivery systems comprising cargos that retain their function following polymer selfassembly.
  • polymers described herein may be expressed in cellular systems which make production of said polymers efficient and scalable.
  • 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.
  • the cargo comprises a peptide, a polypeptide, or a protein.
  • a linker connects the cargo to the selfassembling peptide.
  • the amide-bearing amino acid and the aromatic amino acid are contiguous in at least one self-assembly repeat unit of the plurality.
  • 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.
  • 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.
  • the amide-bearing amino acid in at least one selfassembly repeat unit of the plurality is asparagine (N).
  • the amide-bearing amino acid in at least one self-assembly repeat unit of the plurality is glutamine (Q).
  • the aromatic amino acid in at least one self-assembly repeat unit of the plurality is tryptophan (W).
  • the plurality of self-assembly repeat units comprises 2- 10 self-assembly repeat units.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • the third sequence of (iii) comprises GGGSGGGSGGNWTT (SEQ ID NO: 11).
  • the first sequence, the second sequence, and/or the third sequence is repeated one or more times.
  • the first sequence, the second sequence, and/or the third sequence is repeated nine times.
  • a cysteine (C) is N-terminal and/or C-terminal to a sequence comprising the plurality of self-assembly repeat units.
  • the disclosure relates to a nucleic acid comprising a sequence encoding a polymer described herein.
  • the sequence encoding the polymer is operably linked to at least one regulatory sequence.
  • the at least one regulatory sequence comprises an inducible regulatory sequence.
  • the nucleic acid is a vector.
  • the disclosure relates to a cell or cell population thereof comprising a nucleic acid described herein.
  • the disclosure relates to a polymer assembly comprising a plurality of a polymer described herein.
  • the polymer is present at a concentration of at least O.lmM in the polymer assembly.
  • the polymer is present at a concentration greater than or equal to l.OmM in the polymer assembly.
  • the polymer assembly is comprised in a semi-solid material.
  • the semisolid material comprises a hydrogel.
  • 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.
  • 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.
  • 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.
  • a method is an ex vivo method which comprises contacting a polymer assembly with at least one cell.
  • the method further comprises obtaining a biological sample, wherein the biological sample comprises the at least one cell or a descendant cell thereof.
  • a method comprises administering a polymer assembly to a subject.
  • administering the polymer assembly comprises injecting the subject with the polymer assembly.
  • the method further comprises obtaining a biological sample from the subject after administration of the hydrogel.
  • the biological sample comprises one or more cells from the subject.
  • the biological sample is a blood sample or a urine sample.
  • 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.
  • the analyte comprises an antibody or an antigen-binding fragment thereof.
  • the one or more detection agents comprises the polymer or a fragment thereof.
  • the one or more detection agents comprises an antibody or an antigen-binding fragment capable of binding to the analyte.
  • the method further comprises detecting the analyte in the biological sample.
  • the method further comprises isolating and/or purifying the analyte from the biological sample.
  • the disclosure relates to a composition comprising a polymer, a nucleic acid, a cell or cell population, and/or a polymer assembly described herein.
  • 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.
  • the polymer assembly is comprised in a syringe.
  • the syringe further comprises a pharmaceutically acceptable buffer.
  • 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. 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
  • 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. 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. 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.
  • 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. 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.
  • 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.
  • 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.
  • 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.
  • 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' 1 between dotted lines and the shear rate was measured at 0.5 s' 1 shear rate before and after dotted lines.
  • FIG. 5B shows time-lapse images of a representative NGT-fusion protein being extruded from a 27G needle.
  • 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.
  • 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.
  • RLU peak relative light units
  • 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).
  • FIG. 9 shows a transmittance chart representation of representative results from analyses of N-glycotag-sfGFP during thermal cycling with sodium thiocynate.
  • 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.
  • FIGs. 11A-11B show NGT-based fusion proteins were recovered as pure, full-length proteins.
  • FIGs. 12A-12B show (NGT)iosfGFP formed assemblies, whereas (NGT)2sfGFP, (NGT)ssfGFP, and (QGT)iosfGFP did not.
  • FIG. 13 shows 30 pM of (NGT)iosfGFP (left), sfGFP (middle), and (QGT)iosfGFP (right) after freeze-thaw.
  • 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.
  • FIG. 15 shows (NGT)iosfGFP tryptophan fluorescence emission peak red-shifted with increasing temperature.
  • 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.
  • FIG. 18 shows (NGT)2sfGFP, (NGT)ssfGFP do not show 540 nm peak with Congo Red.
  • FIGs. 19A-19B show Congo Red Staining of (NGT)iosfGFP.
  • FIGs. 20A-20B show Congo Red Staining of (NFT)iosfGFP.
  • FIG. 21 shows (NGT)lOnL binds Thioflavin T.
  • FIGs. 22A-22B show Ac-GGNWTT-Am (SEQ ID NO: 1) is stained by CR.
  • FIG. 23 shows Ac-GGNWTT-Am (SEQ ID NO: 1) binds ThT.
  • FIG. 24 shows NGT-Ovalbumin creates sub-micron size particles, when suspended in PBS.
  • FIG. 25 shows data obtained from analyzing control samples used in analyses of cross presentation on a dendritic cell line (DC2.4s).
  • 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.
  • FIG. 28 shows NGT-OVA + PEG formulations do not yield SIINFEKL-MHCI presentation on DC2.4s.
  • polymers which are useful for delivering biological active molecules to cells.
  • 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.
  • 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).
  • 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.
  • 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.
  • polymer assemblies are comprised within and/or form semi-solid materials described herein, such as hydrogels.
  • 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.
  • 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.
  • 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).
  • a polymer comprises a self-assembling peptide which is fused to a cargo via a linker (see, e.g., FIGs. 1C-1D and 1G).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • a nucleic acid comprises an inducible-regulatory sequence which is operably linked to a sequence encoding a polymer (see, e.g., FIG. 10).
  • 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).
  • 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).
  • a method comprises administering polymers to a subject (e.g., via injection).
  • 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).
  • a method comprises assaying one or more analytes in a biological sample.
  • a polymer e.g., polypeptide
  • 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)).
  • amide-bearing amino acid e.g., asparagine (N) or glutamine (Q)
  • an aromatic amino acid e.g., tryptophan (W) or phenylalanine (F)
  • 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).
  • a linker e.g., a linker described herein
  • 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
  • amino acids
  • a polymer (e.g., a polypeptide) comprises a plurality of monomers, wherein each monomer of the plurality is linked by covalent bonds.
  • a polymer comprises at least 3 monomers.
  • 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,
  • a polymer comprises a molecular weight of at least 300 g/mol.
  • 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
  • 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.
  • a polymer (e.g., a polypeptide) comprises naturally occurring (e.g., amino acids), recombinant, and/or non-naturally occurring monomers (e.g., synthetic monomers).
  • a polymer comprises a peptide (e.g., a self-assembling peptide), polypeptide, or protein, wherein the constituent monomers comprise amino acids.
  • amino acids may be naturally-occurring amino acids.
  • Non-limiting examples of naturally occurring amino acids include hydrophilic and hydrophobic amino acids.
  • 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)).
  • an amide-bearing amino acid e.g., asparagine (N) and glutamine (Q)
  • a hydroxyl-bearing amino acid e.g., serine (S) and threonine (T)
  • a thiol-bearing amino acid e.g., cysteine (C)
  • 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)).
  • an “amide-bearing amino acid” is asparagine (N) or glutamine (G).
  • 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).
  • G glycine
  • A alanine
  • V valine
  • L leucine
  • M methionine
  • I phenylalanine
  • F tyrosine
  • tryptophan Y
  • proline proline
  • an “aromatic amino acid” is tryptophan (W), phenylalanine (F), or tyrosine (Y).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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)).
  • 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)).
  • a polymer comprising an amino acid sequence linked to a polynucleotide (e.g., a single- and/or doublestranded nucleic
  • a polymer e.g., a polypeptide
  • a plurality of components e.g., one or more self-assembling peptides and one or more cargos
  • the plurality of components are configured in the polymer according to any of the following:
  • 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.
  • 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.
  • the polymer 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.
  • 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.
  • self-assembling peptides described herein comprise a plurality of self-assembly repeat units.
  • a self-assembly repeat unit comprises at least two amino acids in length.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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%,
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • the one or more amino acids found between each self-assembly repeat unit of the plurality are equal in length (see, e.g., “1 st sequence” in FIG. IK).
  • 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).
  • 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.
  • 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.
  • 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).
  • amino acids comprised in self-assembly repeat units form beta strands that interact to form parallel and/or anti-parallel beta sheets.
  • 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).
  • beta solenoids e.g., structures comprising beta rolls and/or beta helices, such as parallel beta helices
  • psi-loop motifs see, as a non-limiting example, FIGs. 2A-2B.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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., “3 rd self-assembly repeat unit configuration” in FIG. IK).
  • 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., “1 st self-assembly repeat unit configuration” and “2 nd self-assembly repeat unit configuration” in FIG. IK).
  • 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., “1 st self-assembly repeat unit configuration” in FIG. II).
  • 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., “2 nd self-assembly repeat unit configuration” in FIG. II).
  • a self-assembly repeat unit comprises an arrangement of amino acids that have a repeating or alternating chemistry.
  • 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.)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • a self-assembly repeat unit comprises a sequence comprising X1X2X3, wherein Xi, X2, and X3 each correspond to respective amino acids.
  • 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).
  • Xi is an aromatic amino acid (e.g., F or W)
  • X2 is an amide-bearing amino acid (e.g., N or Q)
  • X3 is a hydrophobic amino acid (e.g., G).
  • Xi is an amide-bearing amino acid (e.g., N or Q)
  • X2 is an aromatic amino acid (e.g., F or W)
  • X3 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • 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).
  • a self-assembly repeat unit comprises a sequence comprising X1X1X2X3, wherein Xi, X2, and X3 each correspond to respective amino acids.
  • 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).
  • 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).
  • 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).
  • a self-assembly repeat unit comprises a sequence comprising X1X2X3X3, wherein Xi, X2, and X3 each correspond to respective amino acids.
  • Xi is an amide-bearing amino acid (e.g., N or Q)
  • X2 is an aromatic amino acid (e.g., F or W)
  • each X3 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • Xi is an aromatic amino acid (e.g., F or W)
  • X2 is an amide- bearing amino acid (e.g., N or Q)
  • each X3 is a hydrophobic amino acid (e.g., G).
  • a self-assembly repeat unit comprises a sequence comprising X1X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids.
  • 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)
  • X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • 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)
  • X4 is a hydrophobic amino acid (e.g., G).
  • a self-assembly repeat unit comprises a sequence comprising X1X1X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids.
  • 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)
  • each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • 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
  • each X4 is a hydrophobic amino acid (e.g., G).
  • a self-assembly repeat unit comprises a sequence comprising X1X2X3X4X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids.
  • 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)
  • each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • 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
  • each X4 is a hydrophobic amino acid (e.g., G).
  • a self-assembly repeat unit comprises a sequence comprising X1X1X2X3X4X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids.
  • 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)
  • each X4 is a hydrophilic amino acid (e.g., a hydroxyl-bearing amino acid, such as T).
  • each Xi is a hydroxyl-bearing amino acid (e.g., T)
  • X2 is an aromatic amino acid
  • X3 is an amide-bearing amino acid
  • each X4 is a hydrophobic amino acid (e.g., G).
  • a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X2X3X4, wherein Xi, X2, X3, and X4 each correspond to respective amino acids.
  • each Xi is a hydroxyl-bearing amino acid and each X2 is a hydrophobic amino acid.
  • each Xi is a serine.
  • each X2 is glycine.
  • each Xi is a hydrophobic amino acid and each X2 is a hydroxyl-bearing amino acid.
  • each Xi is glycine and each X2 is serine.
  • 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.
  • 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 X1X1X2X1X1X1X2X2X3X4, 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.
  • a self-assembly repeat unit comprises a sequence comprising X1X1X2X2X2X3X2X2X2X3X3X4X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids.
  • each Xi and X2 is a hydroxyl-bearing amino acid and each X3 is hydrophobic amino acid.
  • each Xi is threonine.
  • each X2 is serine.
  • each X3 is glycine.
  • X4 is an amide-bearing amino acid and X5 is an aromatic amino acid.
  • X4 is an aromatic amino acid and X5 is an amide-bearing amino acid. In some embodiments, X4 is tryptophan and X5 is asparagine.
  • 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 X1X1X2X2X2X3X2X2X2X3X4X5, wherein X1X1X2X2X2X3X2X2X2X3X4X5 is repeated in the self-assembling peptide 1, 2, 3, 4, 5, 6, 7, 8, or 9 times. In some embodiments, X1X1X2X2X2X3X2X2X2X3X4X5 is repeated in the self-assembling peptide 10 times or more.
  • a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X2X3X4X5X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids.
  • each Xi is a hydroxyl-bearing amino acid
  • each X2 is a hydrophobic amino acid
  • each X5 is a hydroxyl-bearing amino acid.
  • each Xi is serine.
  • each X2 is glycine.
  • each Xi is a hydrophobic amino acid
  • each X2 is a hydroxyl-bearing amino acid
  • each X5 is a hydroxylbearing amino acid.
  • each Xi is glycine and each X2 is serine.
  • X3 is an amide-bearing amino acid and X4 is an aromatic amino acid.
  • X3 is asparagine and X4 is tryptophan.
  • X3 is an aromatic amino acid and X4 is an amide-bearing amino acid.
  • X3 is tryptophan and X4 is asparagine.
  • each X5 is threonine.
  • 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.
  • X1X1X1X2X1X1X1X2X2X3X4X5X5 is repeated in the self-assembling peptide 10 times or more.
  • a self-assembly repeat unit comprises a sequence comprising X1X1X1X2X1X1X1X2X1X1X3X4X5X5, wherein Xi, X2, X3, X4, and X5 each correspond to respective amino acids.
  • each Xi is a hydroxyl-bearing amino acid
  • each X2 is a hydrophobic amino acid
  • each X5 is a hydroxyl-bearing amino acid.
  • each Xi is serine and each X2 is glycine.
  • each Xi is a hydrophobic amino acid
  • each X2 is a hydroxyl-bearing amino acid
  • each X5 is a hydroxylbearing amino acid.
  • each Xi is glycine and each X2 is serine.
  • X3 is an amide-bearing amino acid and X4 is an aromatic amino acid.
  • X3 is asparagine.
  • X3 is glutamine.
  • X4 is phenylalanine.
  • X4 is tryptophan.
  • X3 is asparagine and X4 is tryptophan.
  • X3 is an aromatic amino acid and X4 is an amide-bearing amino acid.
  • X3 is tryptophan and X4 is asparagine.
  • each X5 is threonine.
  • 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, X1X1X2X1X1X1X2X1X1X3X4X5X5 is repeated in the self-assembling peptide 10 times or more.
  • 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).
  • the at least one amino acid is a cysteine (C).
  • 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.
  • the at least one amino acid is comprised in a linker described herein.
  • 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).
  • the at least one amino acid is a cysteine (C).
  • 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.
  • the at least one amino acid is comprised in a linker described herein.
  • a sequence comprising a plurality of self-assembly repeat units comprises an amino acid sequence set forth in SEQ ID NO: 3.
  • a polymer comprises one or more amino acid sequences described in Table 1 below.
  • 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.
  • a comprises a cargo comprises molecular weight of at least 1 g/mol.
  • 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
  • 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.
  • a cargo may comprise a molecular weight of that is greater than 5,000,000 g/mol.
  • a cargo (e.g., a therapeutic cargo) comprises a peptide, polypeptide, or a protein.
  • 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.).
  • 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.).
  • 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 enzyme (e
  • a cargo is selected for the purposes of vaccine production against a pathogen.
  • 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.
  • the pathogen is pathogenic (e.g., infects and/or causes one or more symptoms of a disease, disorder, or condition) to humans.
  • 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.
  • 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
  • a cargo comprises one or more moieties and/or molecules which are not a peptide, polypeptide, or protein.
  • cargos may comprise one or more synthetic moieties and/or molecules (e.g., a small molecule or a polynucleotide).
  • 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.).
  • a cargo comprises a nucleic acid.
  • a nucleic acid comprised in a cargo comprises approximately 2-10,000 nucleotides.
  • 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.
  • 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.).
  • 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.
  • a cargo comprises an antibody-drug conjugate or an antigen-binding fragment-drug conjugate.
  • a cargo comprises a conjugate, such as a peptide-oligonucleotide (e.g., a single- stranded oligonucleotide) conjugate.
  • a cargo comprises a compound (e.g., small molecules, such as a drug molecules) that is therapeutic for a disease, disorder, or condition.
  • 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.
  • the compound is conjugated to a peptide, polypeptide, or protein which is linked to the self-assembling peptide.
  • a cargo does not comprise a peptide, polypeptide, or protein.
  • a cargo and/or a self-assembling peptide comprises a linker.
  • a linker comprises at least one monomer in length.
  • a linker comprises a polymeric arrangement of monomers (e.g., amino acids) that does not perturb a peptide assembly and/or cargo molecule activity.
  • 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.
  • a linker connects a cargo to a sequence comprising a plurality of self-assembly repeat units in a self-assembling peptide.
  • 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.
  • a linker comprises any combination of the 20 naturally occurring amino acids.
  • an amino acid sequence comprises in a linker is a flexible linker, such as a hydrophilic linker.
  • a linker comprises one or more hydroxyl-bearing amino acids.
  • the one or more hydroxyl-bearing amino acids comprises one or more serines and/or one or more threonines.
  • a linker comprises one or more glycine amino acid residues.
  • 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.
  • 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.
  • a linker comprises a sequence comprising (XiXiXiX2) y , wherein Xi and X2 each correspond to respective amino acids.
  • Xi is glycine and X2 is serine.
  • Xi is serine and X2 is glycine.
  • 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.).
  • 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.
  • 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.
  • a self-assembling peptide comprises a synthetic linker, such as a polyethylene glycol (PEG) linker.
  • a synthetic linker such as a polyethylene glycol (PEG) linker.
  • 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.
  • a nucleic acid comprising a sequence encoding a polymer further comprises one or more sequences that do not encode a polymer.
  • the one or more sequences comprises one or more regulatory sequences.
  • 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.
  • 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.
  • a regulatory sequence is a transcriptional regulatory sequence, a post-transcriptional regulatory sequence (e.g., a splicing regulatory sequence), or a translation regulatory sequence.
  • 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.
  • a promoter such as a promoter, an enhancer, a silencer, a transcription factor binding sequence, a 5’ UTR, a 3’ UTR, a translation
  • a nucleic acid comprises a promoter.
  • a promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, a synthetic promoter, or a native promoter.
  • a constitutive promoter maintains constant expression of RNAs regardless of the conditions or physiological state of a host cell.
  • a tissue- specific promoter binds tissue- specific transcription factors that induce transcription in a tissue specific manner.
  • a native promoter is native to a gene which is endogenous to a cell comprising a nucleic acid described herein.
  • 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.
  • 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.
  • a sequence encoding a polymer is linked (e.g., operably linked) to an inducible promoter.
  • 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.
  • a sequence encoding a polymer is operably linked to an inducible promoter that is responsive to an inducing agent.
  • 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.
  • an inducing agent is IPTG.
  • an inducible polymerase is a T7 polymerase.
  • a nucleic acid is a vector.
  • vectors comprise deoxyribonucleotides.
  • vectors comprise ribonucleotides.
  • vectors comprise both deoxyribonucleotides and ribonucleotides.
  • vectors are single-stranded.
  • vectors are double- stranded.
  • vectors are circular (e.g., plasmids, such as circular plasmids, nanoplasmids, and minicircle plasmids).
  • vectors are linear.
  • a vector may be maintained in high levels in a cell using a selection method, such as one involving an antibiotic resistance gene.
  • a vector may comprise a partitioning sequence which ensures stable inheritance of the vector.
  • a vector is a high copy number vector.
  • a vector, or a fragment thereof e.g., the heterologous nucleic acid
  • aspects of the present disclosure also relate to polymer assemblies.
  • 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.
  • 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).
  • selfassembling peptides assemble into structures comprising beta strands, such as parallel and/or anti-parallel sheets (see, e.g., FIGs. 2A-2B).
  • amino acids comprised in self-assembly repeat units described herein are located in beta strands of parallel and/or antiparallel beta sheets.
  • 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).
  • 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.
  • amino acids falling outside of a self-assembly repeat unit may also form structures, such as beta strands.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a polymer assembly is comprised in a semi-solid material.
  • the semi-solid material comprises a gel.
  • 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.
  • a semi-solid material is a hydrogel.
  • 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).
  • a gel (e.g., a hydrogel) comprises a covalent polymer network, such as a network formed by crosslinking polymers or by nonlinear polymerization.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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
  • 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.
  • 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.
  • a semi-solid material (e.g., a hydrogel) comprises a cargo.
  • 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.
  • 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
  • a semi-solid material (e.g., a hydrogel) comprises a cargo at a concentration of at least 10 mM.
  • 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,
  • 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.
  • a semi-solid material (e.g., a hydrogel) comprises a cargo at a concentration of more than 500 mM.
  • a semi-solid material (e.g., a hydrogel) comprises a 1:1 molar ratio of self-assembling peptide to cargo.
  • 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.
  • a semi-solid material (e.g., a hydrogel) comprises a non-equal molar ratio of self-assembling peptide to cargo.
  • 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.
  • a semi-solid material 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.
  • a semi-solid material e.g., a hydrogel
  • 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%,
  • a semi-solid material (e.g., a hydrogel) comprises a neutral or near physiological pH.
  • the pH of a semi-solid material can range from about 6.5 to about 8.5.
  • 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.
  • the pH of a semi-solid material 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.
  • 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.
  • 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%.
  • 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).
  • 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.
  • a method of assembling polymers described herein is useful for producing a polymer assembly described herein (e.g., a hydrogel).
  • 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.
  • 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).
  • 0°C e.g., at a temperature of about -80 °C, -60 °C, -40 °C, -20 °C, -10 °C, etc.
  • at least one hour e.g., 1
  • 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).
  • a mixture comprising a polymer and a buffer comprises the polymer at a concentration of at least 0.1% (weight/volume).
  • 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%, 0.6
  • 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).
  • 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).
  • the mixture comprises the polymer at a concentration of greater than 2.0% (weight/volume).
  • the buffer and/or the mixture is at neutral or near physiological pH.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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)).
  • SPSS solid-phase peptide synthesis
  • 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).
  • 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.
  • 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.
  • polymers are isolated and/or purified by concentrating the polymers, such as via dialyzing polymers (e.g., overnight at 4 °C).
  • 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.
  • the polymer assembly comprises a cargo, such as a therapeutic cargo described herein.
  • 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.
  • one or more of each therapeutic cargo in a polymer assembly or a semi-solid material thereof is fused to a self-assembling peptide.
  • 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.
  • 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)).
  • primate e.g., cynomolgus monkey or rhesus monkey
  • commercially relevant mammal e.g., cattle, pig, horse, sheep, goat, cat, or dog
  • bird e.g., commercially relevant bird, such as chicken,
  • 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.
  • 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.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease, disorder, or condition.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • 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.
  • 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
  • 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.
  • direct injection is performed concurrently with a surgical procedure or interventional procedure.
  • 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.).
  • 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.
  • a polymer assembly is administered to a subject in an effective amount.
  • 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.
  • 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.
  • 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.
  • an effective amount is an amount effective for producing an immunogenic response against an antigen comprised in a cargo in the polymer assembly.
  • 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.
  • a method described herein comprises obtaining a biological sample from a subject.
  • 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).
  • a “biological sample” may refer to any specimen derived or obtained from a subject having or suspected of having a disease, disorder, or condition.
  • a biological sample is a tissue sample.
  • a biological sample comprises one or more cells from a subject.
  • 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.
  • a biological sample comprises or is suspecting of comprising the analyte.
  • an analyte is a peptide, polypeptide, or protein.
  • an analyte comprises an antibody or an antigen-binding fragment thereof.
  • an analyte is a nucleic acid.
  • a biological sample has been subjected to one or more processing steps prior to being used in a detection method.
  • 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).
  • enzymatic digestion e.g., with a nuclease and/or a protease
  • a chemical e.g., for the purposes of cell permeabilization, cell lysis, and/or for improving sample stability
  • storage e.g., about 6, 5, 4, 3, 2, or 1 weeks or 6, 5, 4, 3, 2,
  • 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.
  • a method comprises isolating and/or purifying an analyte from a biological sample.
  • a method described herein comprises contacting a biological sample with one or more detection agents.
  • a detection agent is capable of binding to an analyte.
  • a detection agent comprises an antibody or an antigen-binding fragment thereof.
  • a detection agent comprises a polymer described herein or a fragment thereof (e.g., a fragment comprising a cargo described herein).
  • 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.
  • 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.
  • methods of detecting an analyte in a biological sample comprises mass spectrometry such as GC-MS, LC-MS, MALDI-TOF-MS.
  • 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).
  • 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).
  • PCR polymerase chain reaction
  • RT-qPCR real-time qPCR
  • a method of detecting an analyte in a biological sample comprises fluorescence in situ hybridization (FISH), microarray, or RNA-seq.
  • a method of detecting an analyte in a biological sample comprises DNA sequencing (e.g., next-generation sequencing).
  • a method of detecting an analyte comprises DNA gel electrophoresis.
  • compositions in some aspects, the disclosure relates to compositions.
  • a composition comprises a self-assembling peptide described herein.
  • a composition comprises a polymer described herein.
  • a composition comprises a nucleic acid described herein.
  • a composition comprises a cell or cell population described herein.
  • a composition comprises a polymer assembly described herein.
  • a composition comprises a semi-solid material, such as a hydrogel, described herein.
  • a composition is useful in a method of treating a subject described herein (e.g., a mammalian subject, such as a human subject).
  • a composition comprising a semi-solid material 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).
  • 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
  • a composition comprises a pharmaceutical excipient.
  • Pharmaceutically acceptable 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.
  • 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.
  • Excipients include, but are not limited to, absorption enhancers, anti- adherents, anti-foaming agents, anti-oxidants
  • a composition further comprises additional components commonly found in pharmaceutical compositions.
  • additional components can include, but are not limited to: anti-pruritic s, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine).
  • 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).
  • the composition may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline, polyalcohols, or glucose.
  • 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.
  • 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.
  • kits In some aspects, 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.
  • 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).
  • semi-solid material e.g., a hydrogel
  • kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use.
  • 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.
  • the kits may include the active agents premixed and shipped in a vial, tube, or other container.
  • 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.
  • other components 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.
  • any of the kits described herein may further comprise components needed for inducing uptake of a polymer assembly into a cell.
  • each component of the kits may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder).
  • 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.
  • a kit further comprises a set of instructions for carrying out the methods described herein.
  • “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of this disclosure.
  • 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.
  • 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.
  • kits 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.
  • the kits may include other components depending on the specific application, as described herein.
  • 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.
  • kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
  • 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.
  • a kit comprises a polymer assembly or a semi-solid material thereof (e.g., a hydrogel) described herein which is present in a device.
  • the device is one that is suitable for administering polymers to a subject, such as a syringe for injection of a subject.
  • NGT N-GlycoTag
  • NGT-protein fusion molecules NGT-GlycoTag
  • N-GlycoTag 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 TTGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGGNWTTGGGSGGGSGGGSGG NWTTGGGSGGGSGGNWTTGGGSGGGSGGNWTTRC (SEQ ID NO: 3).
  • the structure of the NGT was modeled using Alpha-Fold (FIGs. 2A-2B).
  • NGT 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.
  • FIG. 4 shows the storage modulus (G’) and the loss modulus (G”) of NGT-sfGFP and NGT-nL at 37°C.
  • G storage modulus
  • G loss modulus
  • 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' 1 shear rate and three, evenly spaced bursts of 100 s' 1 shear 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).
  • NGT-nL 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).
  • 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.
  • 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.
  • 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.
  • GGNWTT N-linked glycosylation sequence 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).
  • 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).
  • sodium thiocyanate 0.1 M NaSCN and 1 M NaSCN
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NTT tryptophan fluorescence
  • (NGT)iosfGFP 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.
  • 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.
  • FIGs. 19A-19B show Congo Red Staining of (NGT)iosfGFP.
  • FIGs. 20A-20B show Congo Red Staining of (NFT)iosfGFP.
  • NFT Congo Red Staining of (NFT)iosfGFP.
  • NFTiosfGFP 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.
  • 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
  • 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.
  • DLS dynamic light scattering
  • 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.
  • DC2.4s dendritic cell line
  • Plate DC2.4s was seeded at 100,000 cells per well in a 24 well plate.
  • NGT-Ovalbumin in PBS was added to treatment groups.
  • 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.
  • FIG. 28 shows NGT-OVA + PEG formulations do not yield SIINFEKL-MHCI presentation on DC2.4s.
  • inventive 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.
  • 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.
  • 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.
  • “at least one of A and 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.
  • 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.

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Abstract

La présente divulgation concerne, au moins en partie, des peptides à autoassemblage comprenant une pluralité d'unités de répétition à autoassemblage qui favorisent la formation de structures à plus grande échelle, telles qu'un hydrogel. Dans certains modes de réalisation, un polymère décrit dans la présente invention comprend un autoassemblage et une molécule cargo, telle qu'un peptide, un polypeptide ou une protéine. Dans certains modes de réalisation, une charge est une charge thérapeutique. Les ensembles polymères décrits dans la présente invention, tels que ceux compris dans les hydrogels, sont utiles pour administrer des charges thérapeutiques à des cellules et peuvent être utilisés pour traiter une maladie, un trouble ou un état pathologique chez un sujet. La présente divulgation concerne en outre des procédés de production d'ensembles polymères et l'administration d'ensembles polymères à un sujet en ayant besoin.
PCT/US2024/055781 2023-11-13 2024-11-13 Ensembles polymères pour l'administration de charges Pending WO2025106582A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200009214A1 (en) * 2018-07-03 2020-01-09 3-D Matrix, Ltd. Ionic Self-Assembling Peptides
US10906939B2 (en) * 2016-08-09 2021-02-02 University Of Florida Research Foundation, Incorporated Co-assembly peptides, nanostructures, and methods of making and using the same
WO2022081774A1 (fr) * 2020-10-13 2022-04-21 University Of Florida Research Foundation, Incorporated Peptides de fusion uricase à autoassemblage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10906939B2 (en) * 2016-08-09 2021-02-02 University Of Florida Research Foundation, Incorporated Co-assembly peptides, nanostructures, and methods of making and using the same
US20200009214A1 (en) * 2018-07-03 2020-01-09 3-D Matrix, Ltd. Ionic Self-Assembling Peptides
WO2022081774A1 (fr) * 2020-10-13 2022-04-21 University Of Florida Research Foundation, Incorporated Peptides de fusion uricase à autoassemblage

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