Compositions and methods for nerve repair

Abstract

Disclosed herein are methods and recombinant partially ordered polypeptides for repairing nerve injuries, such as a peripheral nerve injury. The methods and polypeptides can take advantage of temperature-dependent phase transition behavior of the polypeptide to provide biocompatible, porous scaffolds for beneficial outcomes in nerve repair. An example method includes administering a recombinant partially ordered polypeptide to a location of a nerve injury in a subject. An example recombinant partially ordered polypeptide includes a plurality of disordered domains, a plurality of structured domains, and an optional neuroregenerative domain.

Description

Attorney Docket No.028193-0029-WO01 COMPOSITIONS AND METHODS FOR NERVE REPAIR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 566,032 filed on March 15, 2024, which is incorporated fully herein by reference. TECHNICAL FIELD

[0002] This disclosure relates to recombinant partially ordered polypeptides (POPs) and their use in nerve repair. INTRODUCTION

[0003] Peripheral nerve injuries impact millions of people across the globe, resulting in billions of healthcare costs annually. These types of injures can cause functional disability and chronic pain in those affected. And, despite available advanced microsurgical techniques, patients are often left with lifelong disability. SUMMARY

[0004] In one aspect, disclosed are methods for repairing a nerve in a subject, the method comprising: administering a recombinant partially ordered polypeptide to a location of a nerve injury in the subject, the recombinant partially ordered polypeptide comprising a plurality of disordered domains and a plurality of structured domains, wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

[0005] In another aspect, disclosed are recombinant partially ordered polypeptides comprising: a plurality of disordered domains; a plurality of structured domains; and a neuroregenerative domain, wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature. BRIEF DESCRIPTION OF THE DRAWINGSAttorney Docket No.028193-0029-WO01

[0006] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] FIG.1A shows a graphical depiction of POP usage in peripheral nerve repair. POPs can include an intrinsically disordered protein (IDP) backbone with periodically inserted ordered domains. Elastin-like polypeptides (ELPs) are an example type of IDP, which are inspired by tropoelastin, and have a consensus sequence of VPGXG where X is any amino acid other than proline. ELPs can be used for the POPs backbone due to their lower critical solution temperature (LCST) phase transition behavior and tunability. The ordered domains can include polyalanines, as they are important structural elements of native tropoelastin. The insertion of ordered domains does not disrupt the LCST properties of ELPs, which can allow POPs to be a low viscosity liquid below a critical tunable transition temperature (Tt) (left insert, left image) while above they transition to a stable solid scaffold (left insert, right image) with properties that can be tuned by the ratio of ordered : disordered domains (helicity), guest residue of the ELP, molecular weight of the polymer chain, and / or concentration of the protein in solution. After phase transition, microporous protein networks can form with fractal dimensions reminiscent of native tropoelastin. A POP can be applied during a peripheral nerve surgery to the site of injury to create a healing microenvironment, which can permit neovascularization and axonal migration through the material, which can allow for improved neuroaxonal regeneration. Following injection, POPs can permit neovascularization (right insert, hematoxylin and eosin (H&E) staining 10 days after subcutaneous POP injection showing extensive neovascularization). The POP can also integrate with the native tissue, which can allow it to provide continued mechanical support at the nerve coaptation site.

[0008] FIG.1B shows a graphical representation of an example POP structure. POPs can have tunable mechanical and material properties based on helicity, ELP guest residue, molecular weight of the polypeptide chain, and / or concentration in solution.

[0009] FIG.2A shows POPs can have lower critical solution temperature behavior, where phase separation occurs above a critical temperature (e.g., transition temperature). The LCST phase behavior can be precisely controlled, which can allow for temperature-driven self- assembly. The transition temperature of a POP can be tunable based on helicity, molecular weight of the polymer chain, ELP guest residue, and / or concentration.

[0010] FIG.2B shows a plot of transition temperatures for example POPs. Transition temperatures were determined through optical turbidity measurements across a range of temperatures using a Cary UV-Vis Spectrophotometer. E4 POPs have an ELP guest residue ofAttorney Docket No.028193-0029-WO01 100% valine. E36 POPs have ELP guest residues of 20% alanine and 80% valine.80mers refer to the ELP repeats, which have a molecular weight of each polypeptide chain of 33 kDa. 120mers have a molecular weight of 50 kDa. The 12.5%H means the POP is made of 12.5% polyalanine repeats and 87.5% ELP backbone. Likewise for 25%H, this refers to the POP being 25% polyalanine and 75% ELP.

[0011] FIG.3 shows secondary structure of example POPs. POPs can include a backbone of disordered ELP and ordered polyalanine domains. Polyalanines can form alpha-helical secondary structures. Therefore, POPs can show alpha-helical secondary structure with circular dichroism (CD) analysis. With CD, organized secondary structure elements have a much stronger signal than disordered proteins, so if there is a secondary structure, a trace will show the characteristic findings of the organized secondary structural elements (e.g. alpha helices, beta sheets, etc.) and minimal characteristic findings for a disordered protein. CD data indicates that shown POPs have a secondary structure consistent with 100% alpha-helical elements and 0% beta sheet elements, consistent with secondary structure predictions. The strength of the signal is related to the ratio of ordered : disordered domains, with 25%H POPs showing a stronger signal than 12.5%H POPs. ELP (no polyalanines present) shows a CD pattern consistent with a disordered protein.

[0012] FIG.4 shows microarchitecture of example POPs. POPs labeled with AF-647 fluorophore were transitioned to 37°C and allowed to stabilize at least two minutes before imaging using confocal microscopy. POPs are shown to have a regular fractal microstructure with a pore size that is controllable, e.g., via concentration in solution and / or molecular weight of the polymer chain. Scale bar = 50 μm.

[0013] FIG.5A shows microarchitecture of example POPs using scanning electron microscopy (SEM). POPs were transitioned at 37°C, fixed, exchanged into ethanol, and then critical point drying was used to remove the ethanol, allowing for gentler drying less disruptive to the scaffold microstructure. SEM imaging shows POPs have a regular fractal microstructure with a tunable pore size. Left: 1.5 mM, scale bar = 40 μm.

[0014] FIG.5B shows the microarchitecture of FIG.5A at a higher magnification (2.5 mM, scale bar = 2 μm).

[0015] FIG.6 shows mechanical properties of example POPs. POPs can have tunable mechanical properties based on concentration in solution, molecular weight of the polypeptide chain, helicity, and / or whether additional chemical crosslinking is used. POPs were transitioned to 37°C, allowed to stabilize for at least two minutes, and the storage (G’) and loss modulus (G’’) were measured using a 40 mm parallel plate on a TA rheometer. For all POP samples, theAttorney Docket No.028193-0029-WO01 storage modulus was greater than the loss modulus, indicating solid-like behavior. However, most mechanical properties of biological systems are described using Young’s Modulus (E); therefore, all G’ were converted to E. The gray bar indicates the range of materials biomechanically favorable for Schwann cells, ~7-20 kPa. Several POP formulations are in this range.

[0016] FIG.7A shows that recombinantly produced E480 H 25% POP (SEQ ID NO:37) can be synthesized at the predicted molecular weight and is pure. POPs can be produced at high purity, and, as recombinant proteins, POPs can be produced with nearly complete monodispersity. MALDI-TOF mass spectrometry was used to analyze produced POP proteins to ensure expected molecular weight here and in FIG.7B-7D below.

[0017] FIG.7B shows that recombinantly produced E3612025%H POP (SEQ ID NO:41) can be synthesized at the predicted molecular weight and is pure.

[0018] FIG.7C shows that recombinantly produced E368025%H POP (SEQ ID NO:39) can be synthesized at the predicted molecular weight and is pure.

[0019] FIG.7D shows that recombinantly produced E368012.5%H POP (SEQ ID NO:43) can be synthesized at the predicted molecular weight and is pure.

[0020] FIG.7E shows that recombinantly produced E4120 POP (SEQ ID NO:36) can be synthesized at the predicted molecular weight and is pure.

[0021] FIG.8A shows that POPs can improve Schwann cell proliferation and neurite outgrowth in vitro via fluorescence microscopy. A POP with beneficial properties shows promise in enhancing neurite outgrowth and Schwann cell proliferation in in vitro dorsal root ganglion cultures. Dorsal root ganglions (DRGs), a collection of nerve sensory cell bodies abutting the vertebrae and containing a collection of Schwann cells, neurons, and fibroblasts, are a commonly used in vitro model to investigate nerve repair. DRGs were harvested, dissociated, and then either seeded onto poly-D-lysine (PDL) and laminin-coated plates or encapsulated within POPs networks. DRGs were allowed to culture for 3 days, then stained for TUJ1 (neurons), VIME (intermediate filaments), DAPI (nuclei), and S100 (Schwann cells).

[0022] FIG.8B shows that POPs can improve Schwann cell proliferation and neurite outgrowth in vitro. Schwann cell proliferation (right bar graph) was determined by counting cells per viewing field and compared by one-way analysis of variation (ANOVA). Neurite outgrowth (left bar graph) was measured with ImageJ, compared by one-way ANOVA. DRGs cultured with a POP with beneficial mechanical properties (E=8.5 kPa) showed significant increases in neurite outgrowth and Schwann cell proliferation relative to culture in basal media or NGF supplemented media. Initial results with DRGs showcase POPs as promising for improvingAttorney Docket No.028193-0029-WO01 peripheral nerve repair, given increases in neurite outgrowth and increased Schwann cell proliferation.

[0023] FIG.9A shows that POPs can improve peripheral nerve repair outcomes in rodents. To show that POPs can have an impact on neurorepair, a POP with beneficial microarchitecture (e.g., 67 μm pore size) was piloted in transection sciatic nerve injury in Lewis rats and then analyzed one-month after injury. Immediately after injury, rats either received no further intervention (no repair), primary neurorrhaphy using 1-210-0 interpositional microsutures for tension-free apposition of the epineurium (primary repair – left image), POPs, or POPs with primary repair (right image). Both POPs treatment groups resulted in grossly more vascular, healthier appearing nerves.

[0024] FIG.9B shows that POPs can improve peripheral nerve repair outcomes in rodents via H&E section. Microscopically, POPs and primary repairs (left image) had similar morphology at the proximal nerve site, however, at the distal nerve site, POPs (right image) showed more microvasculature formation, less vacuolation (indicating less overall cellular stress), and were healthier-appearing with more aligned nerve fibers. Arrows point out some microvasculature present.

[0025] FIG.9C shows that POPs can improve peripheral nerve repair outcomes in rodents via functional analysis. Functionally, rats treated with POPs also had significantly improved sciatic nerve function at one month (left bar graph), quantified by sciatic function index (SFI) and compared by one-way ANOVA. Additionally, rats had trends towards decreased denervation and atrophy of the gastrocnemius (right bar graph), a muscle innervated by the sciatic nerve, evaluated by comparing wet weight of the muscle by one-way ANOVA. Overall, treatment with POPs resulted in qualitative improvements in vascularization, alignment of nerve fibers at the distal nerve site, and significantly improved recovery of sciatic nerve function at early timepoints, showcasing POPs’ promise in peripheral nerve repair.

[0026] FIG.10 is supplementary to FIG.9B and shows the H&E section of the full nerve that was used to make region of interests (ROIs). Arrows point out some microvasculature present. Boxes pick out ROI in the proximal and distal part of the nerve, relative to the injury.

[0027] FIG.11 shows that POPs can be fused with various neurotrophic, angiogenic, or neuroangiogenic sequences and maintain LCST phase behavior. To illustrate this, POPs were fused with various neuroprotective, neuroproliferative or neuroangiogenic motifs. Selected sequences fused to POPs include the first domain of NGF, which signals through the TrkA pathway on neurons with neurotrophic effects (termed Nerve Signaling Peptide (NSP POPs)), Vascular Endothelial Growth Factor (isoform 121), a neuroangiogen with neurotrophic andAttorney Docket No.028193-0029-WO01 angiogenic effects (VEGF-121 POPs), and IKVAV peptide derived from laminin, an extracellular matrix protein important in neurorepair. Fusion POPs maintain the LCST phase transition behavior of the parent POP sequence, allowing them to transition from a low viscosity injectable liquid to a stable solid scaffold at a tunable transition temperature. Transition temperatures can be altered by the additional sequences, but general trends and behavior are conserved.

[0028] FIG.12 shows that fusion POPs maintain similar microarchitecture to the parent POP sequence. POPs can be fused with various neurotrophic, angiogenic, or neuroangiogenic sequences. Pictured here are confocal images analyzing scaffold microarchitecture for two fusion POPs compared with their parent POP sequence. All POPs were labeled with AF-647 fluorophore and transitioned to 37°C, then allowed to stabilize at least two minutes before imaging using confocal microscopy. Shown POPs have a regular fractal microstructure with a pore size tunable controlled by the concentration in solution and molecular weight of the polymer chain. Fusion POPs have similar microarchitecture, pore size, and behavior to the parent POP. Scale bar = 50 μm.

[0029] FIG.13 shows that fusion POPs maintain similar mechanical properties to the parent POP sequence. POPs can be fused with various neurotrophic, angiogenic, or neuroangiogenic sequences. The addition of these sequences does not significantly impact the mechanical strength of the POP scaffold. POPs were transitioned to 37°C, allowed to stabilize for at least two minutes, and the storage (G’) and loss modulus (G’’) measured using a 40 mm parallel plate on a TA rheometer. For all shown POP samples, the storage modulus was greater than the loss modulus, indicating solid-like behavior. However, most mechanical properties of biological systems are described using Young’s Modulus (E); therefore, all G’ were converted to E. The gray bar indicates the range of materials biomechanically favorable for Schwann cells, ~7-20 kPa. Several POP formulations are in this range.

[0030] FIG.14A shows that VEGF-121 POPs maintain angiogenic activity, as assessed using a 2D tube angiogenesis assay in human umbilical vein endothelial cells (HUVECs). Treatment with VEGF-121 POPs results in robust and branching tube formation, with significant increases by one-way ANOVA in tube length (FIG.14A) and branching (FIG.14C) compared to basal media, with similar or greater angiogenic signaling to media supplemented with angiogenesis promoters (VEGF-121, low serum growth supplement (LSGS)), as analyzed using the ImageJ angiogenesis analyzer plugin. Scale bars = 300 μm.

[0031] FIG.14B shows the tube length characterization referenced in FIG.14A.

[0032] FIG.14C shows the branch number characterization referenced in FIG.14A.Attorney Docket No.028193-0029-WO01

[0033] FIG.15A shows that IKVAV POP and NSP POPs both have neurotrophic activity in PC12 cells. PC12 cells are a rat pheochromocytoma cell line commonly used for neuronal studies, which, upon the addition of neurotrophic signals will differentiate into neuron-like cells. Treatment with IKVAV POPs resulted in trends to mild increases in neurite outgrowth, similar to a commercial IKVAV hydrogel. NSP POPs also had trends towards increased neurite outgrowth. Neurite outgrowth measured by ImageJ. Scale bars = 100 μm.

[0034] FIG.15B shows neurite outgrowth length quantified as referenced in FIG.15A. IKVAV POPs + NSP POPs resulted in significant increases in neurite outgrowth, at a level similar to NGF (positive control). Analyzed by one-way ANOVA.

[0035] FIG.16 shows that mesenchymal stem cells can be encapsulated into POPs and co- cultured. Mesenchymal stem cells (MSCs) can be injected to the site of nerve injury as a strategy to improve peripheral nerve repair. For MSCs, spheroids within the shown scaffold generally have improved survival and benefits to neurorepair than cells suspended within a scaffold. MSCs encapsulated in POP scaffolds have the ability to self-organize into spheroids and be maintained in culture for at least 7 days. Encapsulation within the POP scaffold is also shown not to be cytotoxic. Blue = DAPI, green = calcein, red = SYTOX. Scale bars = 200 μm. For TC plate and Matrigel groups, cells were seeded on top.

[0036] FIG.17 shows a graphical summary of example advantages of POPs (referred to here as a “Fractomer”) vs. other materials used for nerve repair. DETAILED DESCRIPTION

[0037] Disclosed herein are partially ordered polypeptides (POPs) that can enhance nerve repair, such as peripheral nerve repair, by addressing unmet needs in current nerve repair methods. The POPs can exhibit temperature-dependent phase transition to form a biomaterial scaffold that can provide (i) microstructure that supports vascularization, cell migration, and axonal growth; (ii) biocompatibility; (iii) molecular tunability to modulate bioactive signaling; (iv) ease of use in the operating room; and (v) affordability and scalability for clinical translation. 1. Definitions

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materialsAttorney Docket No.028193-0029-WO01 similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0039] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0040] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0042] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, or U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0043] “Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUBAttorney Docket No.028193-0029-WO01 Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0044] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an agent, composition, or recombinant partially ordered polypeptide or network thereof being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of a damaged nerve being experienced or that the subject is susceptible to. The result can be the reduction or alleviation of the signs, symptoms, or causes of a damaged nerve. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of the nerve injury, route of administration, the type or extent of supplemental therapy used, ongoing nerve injury, and type of treatment desired. In reference to nerve repair, an effective or therapeutically effective amount can include an amount sufficient to, among other things, support axonal growth and / or axonal regeneration.

[0045] The term “expression vector” indicates a plasmid, a virus or another medium, known in the art, into which a nucleic acid sequence for encoding a desired protein can be inserted or introduced.

[0046] The term “host cell” is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector. Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc. In some embodiments, the host cell includes Escherichia coli.

[0047] “Nerve injury” as used herein refers to damage to any nerve, where a nerve refers to tissue bundles that can be composed of neurons surrounded by stromal and glial cells that can be organized into endoneurium, perineurium, and epineurium with associated penetrating supportive microvasculature. Nerve injury can include any degree of injury as defined by the Sunderland (Grade I to Grade V inclusive) and Seddon (neurapraxia, axonotmesis, neurotmesis) classification schemes. Nerve injuries can be caused by any etiology including, but not limited to, compression, crush, and transection injuries.

[0048] “Polynucleotide” as used herein can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.Attorney Docket No.028193-0029-WO01

[0049] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids in length. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif” is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of motifs, which may be the same or different.

[0050] “Recombinant” when used with reference to, e.g., a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

[0051] “Subject” as used herein can mean a mammal that wants or is in need of the herein described methods and polypeptides. The subject may be a patient. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, or guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, or gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant.Attorney Docket No.028193-0029-WO01

[0052] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0053] “Substantially identical” can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical over a region of 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 amino acids.

[0054] As used herein, “repairing,” “treatment,” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of a nerve injury. A treatment may be either performed in an acute or chronic way. The term “repairing” also refers to reducing the severity of symptoms associated with a nerve injury prior to affliction with the injury. “Repressing” or “ameliorating” a nerve injury, or the symptoms thereof involves administering a recombinant partially ordered polypeptide or network thereof as described herein to a subject after clinical appearance of the nerve injury or its symptoms. “Prophylaxis of” or “preventing” a nerve injury or the symptoms thereof involves administering a recombinant partially ordered polypeptide or network thereof as described herein to a subject prior to onset of the nerve injury or the symptoms thereof. “Suppressing” a nerve injury involves administering a recombinant partially ordered polypeptide or network thereof as described herein to a subject after induction of the nerve injury but before its clinical appearance or symptoms thereof have manifest.

[0055] “Variant” as used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequence substantially identical thereto.

[0056] A “variant” can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a substantially identical sequence. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem orAttorney Docket No.028193-0029-WO01 separated by a linker. Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids. See Kyte et al., J. Mol. Biol.1982, 157, 105-132. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indices of ±2 are substituted. The hydrophobicity of amino acids can also be used to reveal substitutions that would result in polypeptides retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity, as discussed in U.S. Patent No.4,554,101, which is fully incorporated herein by reference. Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0057] A variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof. 2. MethodsAttorney Docket No.028193-0029-WO01

[0058] Disclosed herein are methods of repairing a nerve in a subject (e.g., in need thereof). The method can include administering a recombinant partially ordered polypeptide to a location of a nerve injury in the subject. The recombinant partially ordered polypeptide can include a plurality of disordered domains and a plurality of structured domains. The recombinant partially ordered polypeptide can also exhibit phase transition behavior that can be defined by a transition temperature of the recombinant partially ordered polypeptide. The temperature- dependent phase transition behavior can allow the recombinant partially ordered polypeptide to form a porous network above the transition temperature.

[0059] The disclosed methods can be used in a number of different situations where nerve repair would be beneficial for a subject. For example, the nerve injury can be a gapless nerve injury or a gap nerve injury. In some embodiments, the nerve injury is a peripheral nerve injury, a neurotmesis nerve injury, or an axonotmesis nerve injury. In some embodiments, the nerve injury is a peripheral nerve injury.

[0060] The method can provide advantageous benefits to the subject. For example, the porous network that can be formed from the recombinant partially ordered polypeptide can provide a biocompatible scaffold-like structure at the location of the nerve injury. The porous network can provide an anti-inflammatory environment that can be supportive of repair, neovascularization to support the increased metabolic demands of nerve repair (e.g., nascent axon), and mechanical support that can promote Schwann cell differentiation. All of these foregoing properties can enhance neuroregeneration. In some embodiments, the porous network supports cell infiltration, vascularization, axonal growth, axonal regeneration, axonal migration, reinnervation of an organ, promotion of myelination, or a combination thereof. Example cells that can infiltrate the porous network include, but are not limited to, Schwann cells, endothelial cells, macrophages, fibroblasts, and / or a combination thereof. A. Recombinant Partially Ordered Polypeptides

[0061] The recombinant partially ordered polypeptide is an artificial protein designed to mimic native elastin and can be thermally responsive. The thermal responsiveness of the recombinant partially ordered polypeptide can allow the polypeptide to have temperature- dependent phase transition behavior. This phase transition property can allow the peptides to be injected as a liquid, yet rapidly form a porous network at a transition temperature, such as that of the subject’s body temperature. The recombinant partially ordered polypeptide may also be referred to as a “partially ordered polypeptide (POP)” or “Fractomer” herein.

[0062] The recombinant partially ordered polypeptide can include a plurality of disordered domains and a plurality of structured domains. The disordered domains and the structuredAttorney Docket No.028193-0029-WO01 domains can be arranged in any number of possible ways. In some embodiments, one or more disordered domains are positioned between at least two adjacent structured domains. In some embodiments, the recombinant partially ordered polypeptide includes alternating disordered domains and structured domains. In some embodiments, the recombinant partially ordered polypeptide includes a plurality of structured domains repeated in tandem and a plurality of disordered domains repeated in tandem. In some embodiments, the plurality of structured domains repeated in tandem are positioned C-terminal to the plurality of disordered domains repeated in tandem. In some embodiments, the plurality of structured domains repeated in tandem are positioned N-terminal to the plurality of disordered domains repeated in tandem. In some embodiments, the recombinant partially ordered polypeptide is arranged as [disordered domain]q-[structured domain]r-[disordered domain]s-[ structured domain]t, wherein q, r, s, and t are independently an integer from 0 to 100, such as from 1 to 100, from 2 to 100, from 1 to 50, or from 2 to 50. In some embodiments, the recombinant partially ordered polypeptide is arranged as [disordered domain]q-[structured domain]r, wherein q and r are independently an integer from 1 to 100. In some embodiments, the recombinant partially ordered polypeptide is arranged as ([disordered domain]q-[structured domain]r)1-50, wherein q and r are independently an integer from 1 to 100. In some embodiments, q, r, s, and t are each independently an integer from 0 to 10, from 0 to 20, from 0 to 30, from 0 to 40, from 0 to 50, from 0 to 60, from 0 to 70, from 0 to 80, from 0 to 90, from 0 to 100, from 1 to 10, from 1 to 20, from 1 to 30, from 1 to 40, from 1 to 150, from 1 to 60, from 1 to 70, from 1 to 80, from 1 to 90, or from 1 to 100.

[0063] The recombinant partially ordered polypeptide can have a varying molecular weight. For example, the recombinant partially ordered polypeptide can have a molecular weight of about 10 kilodaltons (kDa) to about 80 kDa, such as about 12 kDa to about 80 kDa, about 15 kDa to about 70 kDa, about 20 kDa to about 70 kDa, about 25 kDa to about 65 kDa, or about 30 kDa to about 60 kDa. In some embodiments, the recombinant partially ordered polypeptide has a molecular weight of greater than 10 kDa, greater than 20 kDa, or greater than 30 kDa. In some embodiments, the recombinant partially ordered polypeptide has a molecular weight of less than 80 kDa, less than 70 kDa, or less than 60 kDa. Molecular weight of the recombinant partially ordered polypeptide can be measured by techniques known within the art such as, but not limited to, matrix assisted laser desorption / ionization (MALDI) mass spectrometry. i. Disordered Domains

[0064] The recombinant partially ordered polypeptide may include a plurality of disordered domains. The disordered domain may comprise any polypeptide that has minimal or no secondary structure as observed by CD, and that can have phase transition behavior. TheAttorney Docket No.028193-0029-WO01 disordered domain may include an amino acid sequence of repeated amino acids, non-repeated amino acids, or a combination thereof.

[0065] The recombinant partially ordered polypeptide can include a varying amount of the disordered domain. For example, the recombinant partially ordered polypeptide can include the plurality of disordered domains at about 20% to about 99%, such as about 25% to about 97%, about 35% to about 95%, or about 50% to about 94% by the total amount of amino acids in the recombinant partially ordered polypeptide. In some embodiments, at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the recombinant partially ordered polypeptide includes disordered domains (based on the total amount of amino acids in the recombinant partially ordered polypeptide). In some embodiments, at most 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, or 2% of the recombinant partially ordered polypeptide includes disordered domains (based on the total amount of amino acids in the recombinant partially ordered polypeptide).

[0066] The disordered domain can include a PG motif or a GP motif. In some embodiments, each disordered domain includes a PG motif. The PG motif may include an amino acidsequence selected from PG, P(X)nG (SEQ ID NO:3), and (B)mP(X)nG(Z)p(SEQ ID NO:4), or acombination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein B, X, and Z are independently any amino acid. In some embodiments, m, n, and p are independently an integer less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m, n, and p are independently an integer greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. P(X)nG (SEQ ID NO:3) may include PXG, PXXG (SEQ ID NO:5), PXXXG (SEQ ID NO:6), PXXXXG (SEQ ID NO:7), PXXXXXG (SEQ ID NO:8), PXXXXXXG (SEQ ID NO:9), PXXXXXXXG (SEQ ID NO:10), PXXXXXXXXG (SEQ ID NO:11), PXXXXXXXXXG (SEQ ID NO:12), PXXXXXXXXXXG (SEQ ID NO:13), PXXXXXXXXXXXG (SEQ ID NO:14), PXXXXXXXXXXXXG (SEQ ID NO:15), PXXXXXXXXXXXXXG (SEQ ID NO:16), PXXXXXXXXXXXXXXG (SEQ ID NO:17), PXXXXXXXXXXXXXXXG (SEQ ID NO:18), or a combination thereof. The disordered domain may further include additional amino acids at the C-terminal and / or N-terminal end of the PG motif. These amino acids surrounding the PG motif may also be part of the overall repeated motif. The amino acids that surround the PG motif may balance the overall hydrophobicity and / or charge so as to control the phase transition behavior of the disordered domain.Attorney Docket No.028193-0029-WO01

[0067] In some embodiments, each disordered domain includes an amino acid sequence of (GXGVP)n(SEQ ID NO:1), wherein X is any amino acid and n is an integer greater than or equal to 1. In some embodiments, n is an integer from 1 to 500. In some embodiments, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500. In some embodiments, n may be less than 500, less than 400, less than 300, less than 200, or less than 100. In some embodiments, n is from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, from 5 to 40, from 10 to 40, from 15 to 35, from 10 to 30, or from 60 to 180. In some embodiments, n is 10, 15, 30, 35, 60, 120, or 180.

[0068] In some embodiments, X is any amino acid except proline. In some embodiments, X is Val, Ala, or a mixture of Ala and Val. In some embodiments, X is Val. In some embodiments, X is Ala. In some embodiments, X is a mixture of Ala and Val. In some embodiments, X is a mixture of Ala and Val in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, X is a mixture of Ala and Val in a ratio of 1:1 or 1:4. In some embodiments, X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val), such as from 5:1 to 1:5 or from 1:1 to 1:4.

[0069] In some embodiments, each disordered domain includes an amino acid sequence of (GXGVP)n(SEQ ID NO:2), wherein X is Val or Ala, or a mixture of Ala and Val, and wherein n is an integer from 1 to 30. In some embodiments, X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val), and wherein n is an integer from 1 to 30.

[0070] In some embodiments, the disordered domain includes a non-repetitive polypeptide. The non-repetitive polypeptide may include a sequence of at least 60 amino acids, wherein at least about 10% of the amino acids are proline (P), and wherein at least about 20% of the amino acids are glycine (G). The non-repetitive polypeptide may include a sequence of at least 60 amino acids, wherein at least about 40% of the amino acids are selected from the group consisting of valine (V), alanine (A), leucine (L), lysine (K), threonine (T), isoleucine (I), tyrosine (Y), serine (S), and phenylalanine (F). The non-repetitive polypeptide may include a sequence of at least 60 amino acids, wherein the sequence does not contain three contiguous identical amino acids, wherein any 5-10 amino acid subsequence does not occur more than once in the non-repetitive polypeptide, and wherein when the non-repetitive polypeptide includes aAttorney Docket No.028193-0029-WO01 subsequence starting and ending with proline (P), the subsequence further includes at least one glycine (G). The non-repetitive polypeptide may include less than about 50, 100, 200, 300, or 400 amino acids. The non-repetitive polypeptide may include at least about 50, 60, 70, 80, 90, or 100 amino acids. ii. Structured Domains

[0071] The recombinant partially ordered polypeptide may include a plurality of structured domains. The structured domain may have a secondary structure as observed by CD, such as, e.g., an alpha helix. The structured domain may include at least one of a polyproline domain and a polyalanine domain. In some embodiments, the structured domain includes only polyalanine domains. In some embodiments, the structured domain includes only polyproline domains.

[0072] The recombinant partially ordered polypeptide can include a varying amount of the structured domain. For example, the recombinant partially ordered polypeptide can include the plurality of structured domains at about 4% to about 75%, such as about 5% to about 70%, about 6% to about 60%, or about 7% to about 50% by the total amount of amino acids in the recombinant partially ordered polypeptide. In some embodiments, at least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the recombinant partially ordered polypeptide includes structured domains (based on the total amount of amino acids in the recombinant partially ordered polypeptide). In some embodiments, at most 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, or 2% of the recombinant partially ordered polypeptide includes structured domains (based on the total amount of amino acids in the recombinant partially ordered polypeptide).

[0073] In some embodiments, each structured domain includes a polyalanine domain. Each polyalanine domain may include at least 4 alanine residues, at least 5 alanine residues, at least 6 alanine residues, at least 7 alanine residues, at least 8 alanine residues, at least 9 alanine residues, or at least 10 alanine residues. In some embodiments, each polyalanine domain includes at least 4 consecutive alanine residues, at least 5 consecutive alanine residues, at least 6 consecutive alanine residues, at least 7 consecutive alanine residues, at least 8 consecutive alanine residues, at least 9 consecutive alanine residues, or at least 10 consecutive alanine residues.

[0074] In some embodiments, at least 25% of the amino acids in the polyalanine domain are alanine residues, such as at least 30% of the amino acids in the polyalanine domain are alanine residues, at least 40% of the amino acids in the polyalanine domain are alanine residues, atAttorney Docket No.028193-0029-WO01 least 50% of the amino acids in the polyalanine domain are alanine residues, at least 60% of the amino acids in the polyalanine domain are alanine residues, at least 70% of the amino acids in the polyalanine domain are alanine residues, at least 80% of the amino acids in the polyalanine domain are alanine residues, at least 90% of the amino acids in the polyalanine domain are alanine residues, or at least 95% of the amino acids in the polyalanine domain are alanine residues. In some embodiments, about 25% to about 100% of the amino acids in the polyalanine domain are alanine residues, such as about 30% to about 100%, about 50% to about 100%, about 75% to about 100%, about 25% to about 50%, or about 40% to about 80%. In some embodiments, all of the amino acids in the polyalanine domain are alanine residues.

[0075] The structured domain may have at least 50% of the amino acids in an alpha-helical conformation, such as at least 55% of the amino acids in an alpha-helical conformation, at least 60% of the amino acids in an alpha-helical conformation, or at least 65% of the amino acids in an alpha-helical conformation.

[0076] In some embodiments, each structured domain includes an amino acid sequence of [Bp(A)qZr]n(SEQ ID NO:19) or [(BAs)tZr]n(SEQ ID NO:20), or a combination thereof, wherein B is Lys, Arg, Asp, or Glu; A is Ala; Z is Lys, Arg, Asp, or Glu; n is an integer from 1 to 50; p is an integer from 0 to 2; q is an integer from 1 to 50; r is an integer from 0 to 2; s is an integer from 1 to 5; and t is an integer from 1 to 50.

[0077] In some embodiments, each structured domain includes an amino acid sequence of (A)m(SEQ ID NO:21), wherein m is an integer from 4 to 50, such as from 10 to 40 or from 15 to30. In some embodiments, each structured domain includes an amino acid sequence of (A)25(SEQ ID NO:22), K(A)25K (SEQ ID NO:23), (KAAAA)5K (SEQ ID NO:24), D(A)25K (SEQ ID NO:25), GK(A)25K (SEQ ID NO:26), GD(A)25K (SEQ ID NO:27), or a combination thereof. Insome embodiments, each structured domain includes an amino acid sequence of (A)25(SEQ IDNO:22).

[0078] In some embodiments, the structured domain includes a polyproline domain. Each polyproline domain may include at least 5 proline resides. Each polyproline domain may have at least about 50% of the amino acids in a Polyproline Helix I (PPI) polyproline helical conformation or a Polyproline Helix II (PPII) polyproline helical conformation, or a combination thereof. PPI includes a helical conformation with backbone dihedral angles of roughly [-75,160] and having cis isomers of the peptide bonds. PPII includes a helical conformation with backbone dihedral angles of roughly [-75,150] and having trans isomers of the peptide bonds. At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the amino acids inAttorney Docket No.028193-0029-WO01 each polyproline domain may be in a helical conformation. In some embodiments, about 50% to about 95% of the amino acids in each polyproline domain are in helical conformation. iii. Neuroregenerative Domains

[0079] The recombinant partially ordered polypeptide can also include a domain that promotes nerve repair (referred to as a neuroregenerative domain). The recombinant partially ordered polypeptide can include at least one neuroregenerative domain or more than one neuroregenerative domain. Promoting nerve repair can include, but is not limited to, promoting cell infiltration, vascularization, axonal growth, axonal regeneration, axonal migration, reinnervation of an organ, and / or myelination. Example neuroregenerative domains include, but are not limited to, a vascular endothelial growth factor (VEGF) domain, a nerve growth factor (NGF) domain, an extracellular matrix (ECM) domain, and combinations thereof.

[0080] The neuroregenerative domain can be located at different positions on the recombinant partially ordered polypeptide. For example, the neuroregenerative domain can be present at the N-terminus or the C-terminus of the recombinant partially ordered polypeptide. In some embodiments, the neuroregenerative domain is inserted within the recombinant partially ordered polypeptide. In some embodiments, the neuroregenerative domain is branched off the recombinant partially ordered polypeptide. In some embodiments, the neuroregenerative domain is positioned at the N-terminus, the C-terminus, branched off, or within the recombinant partially ordered polypeptide.

[0081] In some embodiments, the VEGF domain includes an amino acid sequence of MAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCN DEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENCDKPRR (SEQ ID NO:28), QK, KLT, MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIF QEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCD KPRR (SEQ ID NO:29), or a combination thereof.

[0082] In some embodiments, the VEGF domain includes an amino acid sequence of MAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCN DEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENCDKPRR (SEQ ID NO:28), QK, KLT, or MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIF QEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNAttorney Docket No.028193-0029-WO01 KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCD KPRR (SEQ ID NO:29).

[0083] In some embodiments, the NGF domain includes an amino acid sequence of SSSHPIFHRGEFSV (SEQ ID NO:30), MSALLHRVLACGRAVQGAGWHAGPKLTSVSGPNKGFAKDAAFYPGHSEVHSVMSMLFYTLIT AFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRRARSAPAEPIAARVTGQTRNITVDP KLFKKRRLRSPRVLFSTQPPPTSSDTLDLDFQAHGTISFNRTHRSKRSSTHPVFHMGEFSVCD SVSVWVGDKTTATDIKGKEVTVLGEVNINNSVFKQYFFETKCRAPNPVESGCRGIDSKHWNSY CTTTHTFVKALTTDDKQAAWRFIRIDTACVCVLSRKAARRG (SEQ ID NO:31), MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAG QTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFH RGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGI DSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO:32), or a combination thereof.

[0084] In some embodiments, the NGF domain includes an amino acid sequence of SSSHPIFHRGEFSV (SEQ ID NO:30), MSALLHRVLACGRAVQGAGWHAGPKLTSVSGPNKGFAKDAAFYPGHSEVHSVMSMLFYTLIT AFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRRARSAPAEPIAARVTGQTRNITVDP KLFKKRRLRSPRVLFSTQPPPTSSDTLDLDFQAHGTISFNRTHRSKRSSTHPVFHMGEFSVCD SVSVWVGDKTTATDIKGKEVTVLGEVNINNSVFKQYFFETKCRAPNPVESGCRGIDSKHWNSY CTTTHTFVKALTTDDKQAAWRFIRIDTACVCVLSRKAARRG (SEQ ID NO:31), or MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAG QTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFH RGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGI DSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO:32).

[0085] In some embodiments, the ECM domain includes an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), RGD, MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:49) or a combination thereof.

[0086] In some embodiments, the ECM domain includes an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), RGD, or MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:49). iv. CrosslinkingAttorney Docket No.028193-0029-WO01

[0087] The recombinant partially ordered polypeptide can include a crosslinking moiety. The crosslinking moiety can be any suitable moiety known within the art that allows the polypeptides to be covalently crosslinked with other polypeptides, within the same polypeptide, or both. Examples of crosslinking moieties include, but are not limited to, unnatural amino acid derivatives (e.g., para-azidophenylalanine), sequences encoded within the polypeptide (e.g., an enzymatic recognition site), and naturally occurring side chains of amino acids within the polypeptide (e.g. using side chain of lysine present within a polypeptide). Accordingly, different crosslinking agents can be used to form the covalent crosslinks. Examples include, but are not limited to, an external stimulus (e.g., UV light), small molecules that can target one or more amino acids (e.g., tetrakis (hydroxylmethyl) phosphonium chloride), and enzymes that can target a specific amino acid(s) or encoded sequence (e.g., lysyl oxidase).

[0088] In some embodiments, the recombinant partially ordered polypeptide may include amino acid derivatives that are not naturally occurring, such as a UV crosslinkable amino acid derivative. The non-native amino acid derivative can be used to introduce covalent crosslinks between different polypeptides and within the same polypeptide. For example, polypeptides that include the UV crosslinkable amino acid derivative can be exposed to UV light, which can result in covalent crosslinks being formed between the amino acid derivative and a side chain of an amino acid of another polypeptide or with a side chain of an amino acid of the same polypeptide (having the amino acid derivative). The UV crosslinkable amino acid derivative may be any amino acid that has been functionalized with an azide group. In some embodiments, the amino acid derivative is para-azidophenylalanine.

[0089] The UV crosslinkable amino acid derivative may be included at varying amounts without affecting the recombinant partially ordered polypeptide’s ability to phase transition at different temperatures. For example, the UV crosslinkable amino acid derivative may be included within the recombinant partially ordered polypeptide at about 0.1% to about 20% of the recombinant partially ordered polypeptide by amino acid, such as about 0.5% to about 15% of the recombinant partially ordered polypeptide by amino acid or about 1% to about 10% of the recombinant partially ordered polypeptide by amino acid. v. Example Recombinant Partially Ordered Polypeptides

[0090] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of [(GVGVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:44), [(G[V4 / A1]GVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:45), and a combination thereof.Attorney Docket No.028193-0029-WO01

[0091] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of [(GVGVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:44) and [(G[V4 / A1]GVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:45).

[0092] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of M-[(GVGVP)15-GD(A)25K]6-GYG (SEQ ID NO:35), M-[(GVGVP)15-GD(A)25K]6-GWP (SEQ ID NO:36), M-[(GVGVP)15-GD(A)25K]4- GWP (SEQ ID NO:37), M-[(GVGVP)15-GD(A)25K]4-GYG (SEQ ID NO:38), M-[(G[V4A1]GVP)15- GD(A)25K]4-GWP (SEQ ID NO:39), M-[(G[V4 / A1]GVP)15-GD(A)25K]4-GYG (SEQ ID NO:40), M- [(G[V4 / A1]GVP)15-GD(A)25K]6-GWP (SEQ ID NO:41), M-[(G[V4 / A1]GVP)15-GD(A)25K]6-GYG (SEQ ID NO:42), M-[(G[V4 / A1]GVP)35-GD(A)25K]2-GWP (SEQ ID NO:43), and a combination thereof.

[0093] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of M-[(GVGVP)15-GD(A)25K]6-GYG (SEQ ID NO:35), M-[(GVGVP)15-GD(A)25K]6-GWP (SEQ ID NO:36), M-[(GVGVP)15-GD(A)25K]4- GWP (SEQ ID NO:37), M-[(GVGVP)15-GD(A)25K]4-GYG (SEQ ID NO:38), M-[(G[V4A1]GVP)15- GD(A)25K]4-GWP (SEQ ID NO:39), M-[(G[V4 / A1]GVP)15-GD(A)25K]4-GYG (SEQ ID NO:40), M- [(G[V4 / A1]GVP)15-GD(A)25K]6-GWP (SEQ ID NO:41), M-[(G[V4 / A1]GVP)15-GD(A)25K]6-GYG (SEQ ID NO:42), and M-[(G[V4 / A1]GVP)35-GD(A)25K]2-GWP (SEQ ID NO:43).

[0094] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of M-[(GVGVP)15-GD(A)25K]4-GWP- MAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCN DEGLECVPTEESNITMQIMRIKPHQGQ HIGEMSFLQHNKCECRPKKDRARQEKCDKPRR. (SEQ ID NO:46), M-[(GVGVP)15-GD(A)25K]4-GWP-SSSHPIFHRGEFSV (SEQ ID NO:47), M- [(GVGVP)15-GD(A)25K]4-GWP- MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:48), and a combination thereof.

[0095] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence selected from the group consisting of M-[(GVGVP)15-GD(A)25K]4-GWP- MAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCN DEGLECVPTEESNITMQIMRIKPHQGQ HIGEMSFLQHNKCECRPKKDRARQEKCDKPRR. (SEQ ID NO:46), M-[(GVGVP)15-GD(A)25K]4-GWP-SSSHPIFHRGEFSV (SEQ ID NO:47), and M-[(GVGVP)15-GD(A)25K]4-GWP- MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:48).

[0096] In some embodiments, the recombinant partially ordered polypeptide includes a first amino acid sequence selected from the group consisting of (SEQ ID NO:35), (SEQ ID NO:36), (SEQ ID NO:37), (SEQ ID NO:38), (SEQ ID NO:39), (SEQ ID NO:40), (SEQ ID NO:41), (SEQAttorney Docket No.028193-0029-WO01 ID NO:42), and (SEQ ID NO:43); and a second amino acid sequence selected from the group consisting of (SEQ ID NO:28), (SEQ ID NO:29), (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), (SEQ ID NO:33), (SEQ ID NO:34), (SEQ ID NO:49), QK, KLT, and RDG.

[0097] In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence at least 95% identical to any one of SEQ ID NO:35 to 43 and SEQ ID NO: 46 to 48. In some embodiments, the recombinant partially ordered polypeptide includes an amino acid sequence at least 99% identical to any one of SEQ ID NO:35 to 43 and SEQ ID NO: 46 to 48. vi. Phase Transition Behavior

[0098] The recombinant partially ordered polypeptide can demonstrate phase transition behavior by changing solubility and network formation with temperature. The phase transition behavior of the recombinant partially ordered polypeptide may be derived from the phase transition behavior of the disordered domains of the recombinant partially ordered polypeptide. “Phase transition” or “transition” may refer to the aggregation of a polypeptide, which occurs sharply at a specific temperature. The phase transition may be reversible, although the specific temperature of dissolution may be the same or different from the specific temperature of aggregation. In some embodiments, the recombinant partially ordered polypeptide exhibits reversible phase transition behavior.

[0099] A transition temperature (Tt) is a temperature at which the recombinant partially ordered polypeptide changes from one state to another. States may include, for example, soluble polypeptides, gels, and aggregates (e.g., porous networks) of varying sizes and dimensions. The recombinant partially ordered polypeptide may have a Ttof about 0 °C to about 100 °C, such as about 10 °C to about 70 °C, about 10 °C to about 45 °C, about 15 °C to about 60 °C, about 20 °C to about 50 °C, or about 20 °C to about 42 °C.

[0100] The recombinant partially ordered polypeptide may have a transition temperature of heating (Tt-heating) and a transition temperature of cooling (Tt-cooling). In some embodiments, the Tt-heatingis concentration-dependent. In some embodiments, the Tt-coolingis concentration- independent. The Tt-heatingmay be primarily determined by the disordered domains. The Tt-coolingmay be primarily determined by the structured domains. The recombinant partially ordered polypeptide may have a Tt-heatingand Tt-cooling, independently, at temperatures as listed above for the Tt. In some embodiments, the Tt-heatingis greater than the Tt-cooling. In embodiments where the recombinant partially ordered polypeptide has a Tt-heatinggreater than the Tt-cooling, the difference between the two transition temperatures may be referred to as a hysteresis. In someAttorney Docket No.028193-0029-WO01 embodiments, the recombinant partially ordered polypeptide has a hysteresis of about 5 °C to about 70 °C, such as about 5 °C to about 60 °C or about 10 °C to about 50 °C.

[0101] The Tt, Tt-heating, and / or the Tt-coolingof the recombinant partially ordered polypeptide can be adjusted by varying the amino acid sequence of the recombinant partially ordered polypeptide (e.g., by varying the amount of the structured domain and / or the X guest residue), by varying the length of the recombinant partially ordered polypeptide (or domains thereof), or a combination thereof. The phase transition behavior may be defined by the Tt, Tt-heating, and / or the Tt-cooling.

[0102] In some embodiments, the recombinant partially ordered polypeptide is soluble below a lower critical solution temperature (LCST). LCST is the temperature below which the polypeptide is miscible. Below the transition temperature (LCST or Tt), the recombinant partially ordered polyprotein may be soluble. Upon heating above the transition temperature, the recombinant partially ordered polypeptide may hydrophobically collapse and aggregate, forming a separate phase.

[0103] The recombinant partially ordered polypeptide may phase transition at various concentrations. For example, the recombinant partially ordered polypeptide may phase transition at a concentration of about 5 M to about 20 mM, such as about 5 M to about 15 mM, about 10 M to about 10 mM, about 10 M to about 5 mM, about 10 μM to about 3.5 mM, about 10 M to about 1 mM, about 25 M to about 800 M, or about 50 M to about 500 M. The recombinant partially ordered polypeptide may phase transition at a concentration that is suitable to form a porous network. In some embodiments, the recombinant partially ordered polypeptide has a transition temperature of about 10 °C to about 45 °C at a concentration of about 10 μM to about 3.5 mM. vii. Porous Networks

[0104] The recombinant partially ordered polypeptide may form a porous network (which can also be referred to as an “aggregate”) when the temperature is greater than the Tt. The porous network may resolubilize when cooled to below a temperature less than the Tt.

[0105] The porous network formed by the recombinant partially ordered polypeptide can have advantageous properties that can arise from the structure of the recombinant partially ordered polypeptide. For example, the porous network may have physical, non-covalent crosslinks. These physical, non-covalent crosslinks may arise from helical bundling of the structured domain(s) interacting with each other. The porous network may also have covalent crosslinks (e.g., chemical crosslinks) in addition to physical, non-covalent crosslinks. Covalent crosslinks can be included in the porous network in order to increase their mechanical stabilityAttorney Docket No.028193-0029-WO01 without altering their porous architecture. In some embodiments, the porous network can be formed from the recombinant partially ordered polypeptide and can then be further stabilized by covalent crosslinking (after the formation of the aggregate).

[0106] The porous network may be a variety of sizes and dimensions. In some embodiments, the porous network is a stable three-dimensional matrix. In some embodiments, the porous network is fractal-like. In some embodiments, the porous network is gel-like. In some embodiments, the porous network has a void volume, e.g., the non-protein rich phase of the network. In some embodiments, the void volume is tunable. For example, the porous network may have a void volume from about 60% to about 90% (of the volume of the network).

[0107] The porous network can include a plurality of pores. The pores can vary in size and dimension. For example, each pore may have a size of about 0.1 μm to about 200 μm, such as about 0.5 μm to about 175 μm, about 1 μm to about 150 μm, about 10 μm to about 120 μm, about 0.1 μm to about 125 μm, or about 10 μm to about 200 μm. In some embodiments, each pore is greater than 0.1 μm, greater than 0.5 μm, greater than 1 μm, or greater than 5 μm. In some embodiments, each pore is less than 200 μm, less than 175 μm, less than 150 μm, or less than 125 μm.

[0108] The porous network may have physical properties that are advantageous for nerve repair. For example, the porous network can have an elastic modulus of about 1 Pa to about 0.1 MPa, such as about 5 Pa to about 0.05 MPa, about 1 Pa to about 0.01 MPa, or about 10 Pa to about 0.1 MPa. In some embodiments, the porous network has an elastic modulus greater than 0.1 Pa. In some embodiments, the porous network has an elastic modulus of less than 0.1 MPa. Modulus can be measured by techniques known within the art such as, but not limited to, a parallel plate rheometer.

[0109] The porous network may also have low immunogenicity or low antigenicity or both. B. Administration

[0110] The disclosed methods can take advantage of the phase transition behavior of the recombinant partially ordered polypeptide in its administration. For example, the recombinant partially ordered polypeptide can be administered to the subject below the transition temperature, and upon exposure to the subject’s body temperature, it can form a porous network at the location of the nerve injury. In other embodiments, the recombinant partially ordered polypeptide can be administered to the subject above the transition temperature at the location of the nerve injury. Thus, in this latter embodiment, the recombinant partially ordered polypeptide is being administered in aggregate form, e.g., as a porous network.Attorney Docket No.028193-0029-WO01

[0111] The recombinant partially ordered polypeptide or network thereof can be administered to contact the nerve injury. A trained physician (e.g., a neurosurgeon) can identify a nerve in need of repair in the subject based on history of the subject, physical exam results of the subject, and / or imaging studies of the subject. The nerve injury can be surgically exposed prior to administration. In other embodiments, the nerve injury is not surgically exposed prior to administration. A trained physician can administer the recombinant partially ordered polypeptide or network thereof in a manner that localizes the recombinant partially ordered polypeptide or network thereof at the site / location of the nerve injury, where the recombinant partially ordered polypeptide or network thereof can contact the nerve injury.

[0112] The recombinant partially ordered polypeptide or network thereof can be administered in combination with a pharmaceutically acceptable excipient. Embodiments that administer the recombinant partially ordered polypeptide or network thereof and a pharmaceutically acceptable excipient in combination can also be referred to as a pharmaceutical composition. An example pharmaceutically acceptable excipient includes, but is not limited to, a buffer. Example buffers include, but are not limited to, phosphate buffered saline and saline. The recombinant partially ordered polypeptide or network thereof can also be administered with other biomolecules. For example, the recombinant partially ordered polypeptide or network thereof can be administered in combination with a stromal vascular fraction, a neuroangiogenic growth factor (e.g., NGF, VEGF, etc.), a stem cell, an extracellular matrix protein (e.g., IKVAV, RGD, YIGSR, etc.), or a combination thereof. Example stem cells include, but are not limited to, a mesenchymal stem cell, an adipose derived stem cell, a bone marrow derived stem cell, a neural stem cell, a neural crest stem cell, and an induced pluripotent stem cell. In some embodiments, the stem cell is a mesenchymal stem cell. In some embodiments, the recombinant partially ordered polypeptide or network thereof is administered without any biomolecule (e.g., as listed above).

[0113] In embodiments that include administration of the recombinant partially ordered polypeptide with a stem cell, the recombinant partially ordered polypeptide can be administered as a porous network where the stem cell is encapsulated therein. In some embodiments, the stem cell can be cultured in the porous network prior to administration. The stem cell can be cultured for any suitable time useful for nerve repair, such as, but not limited to, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or more. The recombinant partially ordered polypeptide and network thereof can be biocompatible with the stem cell (e.g., no significant toxicity). In addition, the stem cell may form or self-organize into different structures in the porous network including, but not limited to, spheroids.Attorney Docket No.028193-0029-WO01

[0114] The recombinant partially ordered polypeptide or network thereof can also be used in combination with other materials used for nerve repair. For example, the recombinant partially ordered polypeptide or network thereof can be applied to the location of the nerve injury in conjunction with an autograft, an allograft, a suture, a tissue adhesive, and combinations thereof. In some embodiments, the recombinant partially ordered polypeptide or network thereof is not used in conjunction with any of the foregoing materials.

[0115] The recombinant partially ordered polypeptide or network thereof can be administered at a varying concentration at the location of the nerve injury. For example, the recombinant partially ordered polypeptide or network thereof, can be administered at a concentration of about 100 μM to about 3 mM, such as about 500 μM to about 2 mM, about 1 mM to about 3 mM, about 100 μM to about 1 mM, or about 100 μM to about 3 mM. In some embodiments, the recombinant partially ordered polypeptide or network thereof is administered at a concentration of greater than 100 μM, greater than 500 μM, or greater than 1 mM. In some embodiments, the recombinant partially ordered polypeptide or network thereof is administered at a concentration of less than 3 mM, less than 2 mM, or less than 1 mM.

[0116] The recombinant partially ordered polypeptide or network thereof can be administered prophylactically or therapeutically. In prophylactic administration, the recombinant partially ordered polypeptide or network thereof can be administered in an amount sufficient to induce a response. In therapeutic applications, the recombinant partially ordered polypeptide or network thereof can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. Amounts effective for this use will depend on, e.g., the particular composition of the recombinant partially ordered polypeptide or network thereof regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0117] Further discussion on the recombinant partially ordered polypeptide can be found in U.S. Patent No.11,680,083; U.S. Patent Application No.17 / 798,868; and U.S. Patent Application No.18 / 007,879 – each of which is incorporated by reference herein in its entirety. 3. Recombinant Partially Ordered Polypeptides with Neuroregenerative Domains

[0118] Also disclosed herein are recombinant partially ordered polypeptides that include a neuroregenerative domain. The recombinant partially ordered polypeptide includes a plurality of disordered domains, a plurality of structured domains, and at least one neuroregenerative domain. The recombinant partially ordered polypeptide can exhibit phase transition behavior, which can be defined by a transition temperature. As described elsewhere herein, this phaseAttorney Docket No.028193-0029-WO01 transition behavior can result in the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

[0119] The recombinant partially ordered polypeptide including neuroregenerative domains is also disclosed herein with respect to the methods of nerve repair. The description above regarding the recombinant partially ordered polypeptide, the disordered domains, the structured domains, the neuroregenerative domains, phase transition behavior, and aggregate formation (e.g., porous networks) can also be applied to the recombinant partially ordered polypeptides with neuroregenerative domains. For the purposes of brevity, they will not be repeated here. 4. Polynucleotides

[0120] Further provided are polynucleotides encoding the recombinant partially ordered polypeptides detailed herein. A vector may include the polynucleotide encoding the recombinant partially ordered polypeptide detailed herein. To obtain expression of a polypeptide, one may subclone the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription / translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. An example of a vector is pet24. Suitable bacterial promoters are well known in the art. Further provided is a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding the recombinant partially ordered polypeptide as detailed herein. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Paiva et al., Gene 1983, 22, 229-235; Mosbach et al., Nature 1983, 302, 543-545 – both of which are incorporated by reference in their entirety herein). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Retroviral expression systems can also be used.

[0121] The recombinant partially ordered polypeptides may be expressed recombinantly in a host cell according to one of skill in the art. The recombinant partially ordered polypeptides may be purified by any means known to one of skill in the art. For example, the recombinant partially ordered polypeptides may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, affinity chromatography, or a combination thereof. In some embodiments, the recombinant partially ordered polypeptides are purified without chromatography. In some embodiments, the recombinant partially ordered polypeptides are purified using inverse transition cycling.Attorney Docket No.028193-0029-WO01

[0122] The present disclosure has multiple aspects, illustrated by the following non-limiting examples. 5. Examples Example 1 POPs have tunable material and mechanical properties.

[0123] Partially Ordered Polypeptides (POPs) are a class of injectable biomaterials including alternating ordered and disordered domains and inspired by native tropoelastin (FIG.1A and FIG.1B). In various natural proteins, ordered and disordered segments can program material properties, including tropoelastin. Elastin is an essential component of the extracellular matrix that provides physical elasticity, durability, and plays a physiological role in regulating the remodeling of the extracellular matrix and in controlling inflammation. For all example POPs presented here, elastin-like polypeptides (ELPs) are used as the disordered segment as its lower critical solution temperature (LCST) phase behavior can be precisely controlled, allowing for temperature-driven self-assembly. For the ordered domain, polyalanine helices are used as they are important structural elements of tropoelastin and these domains can have secondary structure (FIG.3).

[0124] By altering the composition of POPs and their ordered and disordered organization, mechanically stable, interpercolated networks with high surface to volume ratios and fractal dimensions similar to native elastin can be created. These POPs maintain the LCST phase transition behavior of ELPs, rapidly phase transitioning (<30 s) at a tunable threshold temperature (FIG.2A and FIG.2B), from a low-viscosity injectable liquid at room temperature to a stable solid porous scaffold at body temperature. Importantly, the material and mechanical properties of POPs networks can be precisely engineered by modulating variables such as molecular weight, hydrophobicity of the amino acid sequence, and / or its concentration in solution (FIG.4, FIG.5A, FIG.5B, and FIG.6). These different versions of POPs can be produced with high purity (FIG.7A, FIG.7B, FIG.7C, FIG.7D, and FIG.7E). Example 2 When injected in vivo, POPs exhibit exceptional biocompatibility, cellular infiltration, and robust neovascularization, creating a local microenvironment directed towards healing.

[0125] Primary neurorrhaphy, the direct surgical repair of a divided nerve, is the gold- standard treatment for PNI when tension-free repair is feasible (FIG.17). For nerve injuries thatAttorney Docket No.028193-0029-WO01 result in a gap between nerve ends, options such as autografts, allografts, or biocompatible nerve conduits are used to reconstruct the nerve. However, given the need to regenerate across a gap, these alternatives may demonstrate worse outcomes than primary repair (FIG.17). Methods to enhance the local availability of angiogenic factors to enhance the efficacy of nerve repair procedures are being currently explored in clinical and laboratory settings, with leading approaches involving autologous procedures, such as the use of vascularized nerve grafts that provide direct nutritive effects. However, autologous techniques (vascularized or non- vascularized) suffer from significant donor site morbidity and are technically complex, which limits their applicability in routine clinical practice (FIG.17).

[0126] POPs exhibit cellular integration and neovascularization in vivo with minimal immunogenicity, promising for improving peripheral nerve repair. POPs have been subjected to extensive testing to evaluate its capabilities as a tissue support matrix, showing its excellent biocompatibility and tissue integration (see, e.g., Roberts, S. et al. Injectable tissue integrating networks from recombinant polypeptides with tunable order. Nat Mater 17, 1154-1163, (2018) and Roberts, S. et al. Complex microparticle architectures from stimuli-responsive intrinsically disordered proteins. Nat Commun 11, 1342, (2020) – both of which are incorporated by reference herein in their entirety). POPs show rapid mechanical integration into host tissue, as early as 24 h after subcutaneous injection, and retain its initial injected shape even after several months, suggesting long-term matrix stability. Further, POPs show robust vascularization, with branching vessels visible to the unaided eye as early as 10 days following subcutaneous injection, as well as extensive cellular infiltration, encouraging slow replacement with healthy autologous, vascularized tissue over time. That POP’s porous networks encourage cellular migration directed toward healing (not chronic inflammation), is promising for developing a neuroregenerative environment by allowing initial recruitment of appropriate macrophages and Schwann cells and encouraging neuroaxonal growth. Additionally, POP’s ability to rapidly induce neovascularization in vivo can allow it to meet the increased metabolic demand and nutrient requirements for healthy nerve repair, which may potentially eliminate the necessity for more complex procedures (e.g., vascularized nerve grafts, surgical angiogenesis via adipofascial flaps), which are technically challenging and have significant morbidity (FIG.17).

[0127] POPs show promise improving nerve repair in vitro in dorsal root ganglion (DRG) cultures (FIG.8A and FIG.8B). DRGs encapsulated into a POP with optimized mechanical properties (E=8.5 kPa) showed increased neurite outgrowth and Schwann cell proliferation relative to culture in basal media or NGF supplemented media after 3 days of culture.Attorney Docket No.028193-0029-WO01

[0128] To show that POPs can have an impact on neurorepair, a POP with beneficial microarchitecture (~67 μm pore size) was piloted in transection sciatic nerve injury in Lewis rats and then analyzed one-month after injury (FIG.9A, FIG.9B, FIG.9C, and FIG.10). Immediately after injury, rats either received no further intervention (no repair), primary neurorrhaphy using 1-210-0 interpositional microstructures for tension-free apposition of the epineurium (primary repair), POPs, or POPs with primary repair. Both POPs treatment groups resulted in grossly more vascular, healthier appearing nerves. Microscopically, POPs and primary repairs had similar morphology at the proximal nerve site, however, at the distal nerve site, POPs showed more microvasculature formation, less vacuolation (indicating less overall cellular stress), and were healthier appearing with more aligned nerve fibers. Functionally, rats treated with POPs also had significantly improved sciatic nerve function at one month, quantified by sciatic function index (SFI) and compared by one-way ANOVA. Thus, treatment with POPs resulted in clear early qualitative improvements in vascularization, alignment of nerve fibers at the distal nerve site, and significantly improved recovery of sciatic nerve function at early timepoints, showcasing POPs’ promise in peripheral nerve repair. Example 3 Neurotrophic or neuroangiogenic sequences can be fused to POPs to promote specific nerve repair pathways.

[0129] POPs can be fused to various bioactive sequences to specifically target aspects of neurorepair in a facile manner. Through careful manipulation of the material properties of POPs, formulations designed for neuroaxonal migration, vascularization, and Schwann cell interactions can be produced. However, since POPs are genetically encoded and produced recombinantly in E. coli, bioactive sequences capable of enhancing other aspects of neurorepair can also be fused to POPs. Three POPs fusion proteins were made and designed to further enhance neurorepair.

[0130] Vascular Endothelial Growth Factor (VEGF) is an angioneurin – a molecule with both angiogenic and neuroprotective properties - with multiple isoforms. VEGF-121 was fused to POPs to create an injectable VEGF-decorated scaffold that rapidly phase transitions at body temperature (FIG.11) to a stable, solid, porous scaffold (FIG.12 and FIG.13) capable of inducing angiogenesis in vitro (FIG.14A, FIG.14B, and FIG.14C). The sequence used for this example was: M-[(GVGVP)15-GD(A)25K]4-GWP- MAPMAEGGGQNHHEVVKFMDVYQRSYCHPI ETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQ HIGEMSFLQHNKCECRPKKDRARQEKCDKPRR (SEQ ID NO:46).Attorney Docket No.028193-0029-WO01

[0131] Nerve Growth Factor (NGF) is a neurotrophic molecule capable of enhancing nerve repair. Nerve Signaling Peptide (NSP) is a short bioactive peptide sequence composed of the AA 1-14 of NGF that partially recapitulates the activity of NGF, by signaling through the TrkA pathway, and has neuroprotective and neuroproliferative effects. NSP was fused to POPs to create injectable NSP-decorated scaffolds that phase transition to form stable, solid, porous scaffolds at physiologic temperatures (FIG.11, FIG.12, and FIG.13) and show bioactivity. NSP POPs were tested in PC12 cells, a pheochromocytoma line whose cells form neurites when exposed to neurotrophic molecules. Treatment with NSP POPs resulted in trends towards mildly increased neurite outgrowth. The sequence used for this example was: M-[(GVGVP)15- GD(A)25K]4-GWP-SSSHPIFHRGEFSV (SEQ ID NO:47).

[0132] The surrounding extracellular matrix (ECM) is also important for nerve repair, with significant contributions to neurorepair from collagen, laminin, fibronectin, matrillin-2, and others. Laminin is especially important, guiding organization and pro-reparative interactions with proreparative Schwann cells. IKVAV and YIGSR, among others, are short bioactive peptide sequences derived from laminin and promote cellular adhesion. As short peptides, they can be easily fused to POPs to encourage pro-reparative interactions with Schwann cells present in the nerve microenvironment. POPs with IKVAV and YIGSR domains were created and shown to phase transition (FIG.11) to form stable, solid, porous scaffolds (FIG.12 and FIG.13) with bioactivity (FIG.15A and FIG.15B). In PC12 cells, IKVAV / YIGSR / IKVAV POPs (hereafter shortened to IKVAV POPs) resulted in trends towards increased neurite outgrowth. The observed effects were similar to a commercially available IKVAV hydrogel (which also trends to increased neurite outgrowth). Multiple fusion POPs can be combined to simultaneously target multiple specific pathways. Coadministration of IKVAV POPs and NSP POPs was shown to have increased effects compared to IKVAV POPs or NSP POPs alone, with significant increases in neurite outgrowth, similar to administering nerve growth factor. The sequence used for this example was: M-[(GVGVP)15-GD(A)25K]4-GWP- MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:48) Example 4 POPs can be co-cultured then co-injected with stem cells for promoting peripheral nerve repair.

[0133] Stem cells, including mesenchymal stem cells (MSCs), adipose derived stem cells (ADSCs), bone marrow derived stem cells, neural stem cells (NSCs), neural crest stem cells, and induced pluripotent stem cells (iPSCs) have all been shown to enhance peripheral nerveAttorney Docket No.028193-0029-WO01 repair through secretion of growth factors, anti-inflammatory effects, and cellular differentiation into relevant cell types.

[0134] Stem cells can be co-cultured with POPs, then co-delivered to the site of nerve repair. To investigate the feasibility of stem cell POP co-culture, MSCs were encapsulated into POP scaffolds (FIG.16). MSCs encapsulated in POP scaffolds have the ability to self-organize into spheroids and be maintained in culture for at least 7 days without any significant cytotoxicity observed (FIG.16). For MSCs, spheroids within a scaffold generally have improved survival and benefits to neurorepair than cells suspended within a scaffold, promising for peripheral nerve repair applications. The sequence of POPs used in this example was: M-[(GVGVP)15- GD(A)25K]4-GWP (SEQ ID NO:37).

[0135] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure.

[0136] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosed technology, may be made without departing from the spirit and scope thereof.

[0137] For reasons of completeness, the following Embodiments are provided.

[0138] Clause 1. A method for repairing a nerve in a subject, the method comprising: administering a recombinant partially ordered polypeptide to a location of a nerve injury in the subject, the recombinant partially ordered polypeptide comprising a plurality of disordered domains and a plurality of structured domains, wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

[0139] Clause 2. The method of clause 1, wherein each disordered domain comprises a PG motif or a GP motif and each structured domain comprises a polyalanine domain or a polyproline domain.

[0140] Clause 3. The method of clause 2, wherein the PG motif comprises an amino acid sequence selected from the group consisting of PG, P(X)nG (SEQ ID NO:3), (B)mP(X)nG(Z)p(SEQ ID NO:4), and a combination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein B, X, and Z are independently any amino acid.Attorney Docket No.028193-0029-WO01

[0141] Clause 4. The method of any one of clauses 1-3, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1.

[0142] Clause 5. The method of any one of clauses 1-4, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:2), wherein X is Val or Ala, or a mixture of Ala and Val, and wherein n is an integer from 1 to 30.

[0143] Clause 6. The method of clause 4 or 5, wherein X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val).

[0144] Clause 7. The method of any one of clauses 1-6, wherein each structured domain comprises a polyalanine domain, at least 25% of the amino acids in the polyalanine domain being alanine residues.

[0145] Clause 8. The method of any one of clauses 1-7, wherein at least 50% of the amino acids in each structured domain is in an alpha-helical conformation.

[0146] Clause 9. The method of any one of clauses 1-8, wherein each structured domain comprises an amino acid sequence of (A)m(SEQ ID NO:21), wherein m is an integer from 4 to 50.

[0147] Clause 10. The method of any one of clauses 1-9, wherein each structured domain comprises an amino acid sequence of: (A)25(SEQ ID NO:22); K(A)25K (SEQ ID NO:23); (KAAAA)5K (SEQ ID NO:24); D(A)25K (SEQ ID NO:25); GD(A)25K (SEQ ID NO:26); GK(A)25K (SEQ ID NO:27); or a combination thereof.

[0148] Clause 11. The method of any one of clauses 1-10, wherein the recombinant partially ordered polypeptide comprises the plurality of structured domains at about 4% to about 75% by the total amount of amino acids in the recombinant partially ordered polypeptide.

[0149] Clause 12. The method of any one of clauses 1-11, wherein the recombinant partially ordered polypeptide comprises a neuroregenerative domain.

[0150] Clause 13. The method of clause 12, wherein the neuroregenerative domain is present at the N-terminus or the C-terminus of the recombinant partially ordered polypeptide.

[0151] Clause 14. The method of clause 12, wherein the neuroregenerative domain is inserted within the recombinant partially ordered polypeptide.

[0152] Clause 15. The method of any one of clauses 12-14, wherein the neuroregenerative domain is a vascular endothelial growth factor (VEGF) domain, a nerve growth factor (NGF) domain, an extracellular matrix (ECM) domain, or a combination thereof.Attorney Docket No.028193-0029-WO01

[0153] Clause 16. The method of clause 15, wherein the VEGF domain comprises an amino acid sequence of (SEQ ID NO:28), (SEQ ID NO:29), QK, KLT, or a combination thereof.

[0154] Clause 17. The method of clause 15, wherein the NGF domain comprises an amino acid sequence of (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), or a combination thereof.

[0155] Clause 18. The method of clause 15, wherein the ECM domain comprises an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), (SEQ ID NO:49), RGD, or a combination thereof.

[0156] Clause 19. The method of any one of clauses 1-18, wherein the recombinant partially ordered polypeptide has a molecular weight of about 10 kilodaltons (kDa) to about 80 kDa.

[0157] Clause 20. The method of any one of clauses 1-19, wherein the transition temperature is about 10 °C to about 45 °C at a concentration of the recombinant partially ordered polypeptide of about 10 μM to about 3.5 mM.

[0158] Clause 21. The method of any one of clauses 1-20, wherein the recombinant partially ordered polypeptide exhibits reversible phase transition behavior.

[0159] Clause 22. The method of any one of clauses 1-21, wherein the porous network comprises a plurality of pores, each pore having a size of about 0.1 μm to about 200 μm.

[0160] Clause 23. The method of any one of clauses 1-22, wherein the porous network has an elastic modulus of about 1 Pa to about 0.1 MPa.

[0161] Clause 24. The method of any one of clauses 1-23, wherein the recombinant partially ordered polypeptide is administered to the subject below the transition temperature, and upon exposure to the subject's body temperature, forms a porous network at the location of the nerve injury.

[0162] Clause 25. The method of any one of clauses 1-23, wherein the recombinant partially ordered polypeptide is administered to the subject above the transition temperature at the location of the nerve injury.

[0163] Clause 26. The method of any one of clauses 1-25, wherein the porous network supports cell infiltration, vascularization, axonal growth, axonal regeneration, axonal migration, reinnervation of an organ, promotion of myelination, or a combination thereof.

[0164] Clause 27. The method of clause 26, wherein cell infiltration comprises Schwann cells, endothelial cells, macrophages, fibroblasts, or a combination thereof.

[0165] Clause 28. The method of any one of clauses 1-27, wherein the porous network contacts the nerve injury.Attorney Docket No.028193-0029-WO01

[0166] Clause 29. The method of any one of clauses 1-28, wherein the nerve injury is a gapless nerve injury or a gap nerve injury.

[0167] Clause 30. The method of any one of clauses 1-29 wherein the nerve injury is a peripheral nerve injury, a neurotmesis nerve injury, or an axonotmesis nerve injury.

[0168] Clause 31. The method of any one of clauses 1-30 wherein the nerve injury is surgically exposed.

[0169] Clause 32. The method of any one of clauses 1-31, wherein the recombinant partially ordered polypeptide is administered at a concentration of about 100 μM to about 3 mM.

[0170] Clause 33. The method of any one of clauses 1-32, wherein the recombinant partially ordered polypeptide is administered in combination with a pharmaceutically acceptable excipient comprising a buffer.

[0171] Clause 34. The method of any one of clauses 1-33, wherein the recombinant partially ordered polypeptide is administered in combination with a stromal vascular fraction, a neuroangiogenic growth factor, a stem cell, an extracellular matrix protein, or a combination thereof.

[0172] Clause 35. The method of clause 34, wherein the stem cell is a mesenchymal stem cell, an adipose derived stem cell, a bone marrow derived stem cell, a neural stem cell, a neural crest stem cell, or an induced pluripotent stem cell.

[0173] Clause 36. The method of clause 34 or 35, wherein the stem cell is encapsulated in the porous network.

[0174] Clause 37. A recombinant partially ordered polypeptide comprising: a plurality of disordered domains; a plurality of structured domains; and a neuroregenerative domain, wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

[0175] Clause 38. The recombinant partially ordered polypeptide of clause 37, wherein the neuroregenerative domain is present at the N-terminus or the C-terminus of the recombinant partially ordered polypeptide.

[0176] Clause 39. The recombinant partially ordered polypeptide of clause 37, wherein the neuroregenerative domain is inserted within the recombinant partially ordered polypeptide.

[0177] Clause 40. The recombinant partially ordered polypeptide of any one of clauses 37- 39, wherein the neuroregenerative domain is a vascular endothelial growth factor (VEGF)Attorney Docket No.028193-0029-WO01 domain, a nerve growth factor (NGF) domain, an extracellular matrix (ECM) domain, or a combination thereof.

[0178] Clause 41. The recombinant partially ordered polypeptide of clause 40, wherein the VEGF domain comprises an amino acid sequence of (SEQ ID NO:28), (SEQ ID NO:29), QK, KLT, or a combination thereof.

[0179] Clause 42. The recombinant partially ordered polypeptide of clause 40, wherein the NGF domain comprises an amino acid sequence of (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), or a combination thereof.

[0180] Clause 43. The recombinant partially ordered polypeptide of clause 40, wherein the ECM domain comprises an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), (SEQ ID NO:49), RGD, or a combination thereof.

[0181] Clause 44. The recombinant partially ordered polypeptide of any one of clauses 37- 43, wherein each disordered domain comprises a PG motif or a GP motif and each structured domain comprises a polyalanine domain or a polyproline domain.

[0182] Clause 45. The recombinant partially ordered polypeptide of clause 44, wherein the PG motif comprises an amino acid sequence selected from the group consisting of PG, P(X)nG (SEQ ID NO:3), (B)mP(X)nG(Z)p(SEQ ID NO:4), and a combination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein U, X, and Z are independently any amino acid.

[0183] Clause 46. The recombinant partially ordered polypeptide of any one of clauses 37- 45, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1.

[0184] Clause 47. The recombinant partially ordered polypeptide of any one of clauses 37- 46, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:2), wherein X is Val or Ala, or a mixture of Ala and Val, and wherein n is an integer from 1 to 30.

[0185] Clause 48. The recombinant partially ordered polypeptide of clause 46 or 47, wherein X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val).

[0186] Clause 49. The recombinant partially ordered polypeptide of any one of clauses 37- 48, wherein each structured domain comprises a polyalanine domain, at least 25% of the amino acids in the polyalanine domain being alanine residues.Attorney Docket No.028193-0029-WO01

[0187] Clause 50. The recombinant partially ordered polypeptide of any one of clauses 37- 49, wherein at least 50% of the amino acids in each structured domain is in an alpha-helical conformation.

[0188] Clause 51. The recombinant partially ordered polypeptide of any one of clauses 37- 50, wherein each structured domain comprises an amino acid sequence of (A)m(SEQ ID NO:21), wherein m is an integer from 4 to 50.

[0189] Clause 52. The recombinant partially ordered polypeptide of any one of clauses 37- 51, wherein each structured domain comprises an amino acid sequence of: (A)25(SEQ ID NO:22); K(A)25K (SEQ ID NO:23); (KAAAA)5K (SEQ ID NO:24); D(A)25K (SEQ ID NO:25); GD(A)25K (SEQ ID NO:26); GK(A)25K (SEQ ID NO:27); or a combination thereof.

[0190] Clause 53. The recombinant partially ordered polypeptide of any one of clauses 37- 52, wherein the recombinant partially ordered polypeptide comprises the plurality of structured domains at about 4% to about 75% by the total amount of amino acids in the recombinant partially ordered polypeptide. SEQUENCES(GXGVP)n(SEQ ID NO:1)X is any amino acid and n is an integer greater than or equal to 1 (GXGVP)n(SEQ ID NO:2) X is Val or Ala, or a mixture of Ala and Val and n is an integer from 1 to 30 P(X)nG (SEQ ID NO:3)(B)mP(X)nG(Z)p(SEQ ID NO:4)PXXG (SEQ ID NO:5) PXXXG (SEQ ID NO:6) PXXXXG (SEQ ID NO:7) PXXXXXG (SEQ ID NO:8) PXXXXXXG (SEQ ID NO:9) PXXXXXXXG (SEQ ID NO:10) PXXXXXXXXG (SEQ ID NO:11) PXXXXXXXXXG (SEQ ID NO:12) PXXXXXXXXXXG (SEQ ID NO:13) PXXXXXXXXXXXG (SEQ ID NO:14) PXXXXXXXXXXXXG (SEQ ID NO:15)Attorney Docket No.028193-0029-WO01 PXXXXXXXXXXXXXG (SEQ ID NO:16) PXXXXXXXXXXXXXXG (SEQ ID NO:17) PXXXXXXXXXXXXXXXG (SEQ ID NO:18)(A)m(SEQ ID NO:21)(A)25(SEQ ID NO:22)K(A)25K (SEQ ID NO:23) (KAAAA)5K (SEQ ID NO:24) D(A)25K (SEQ ID NO:25) GK(A)25K (SEQ ID NO:26) GD(A)25K (SEQ ID NO:27) MAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCN DEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENCDKPRR (SEQ ID NO:28) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIF QEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCD KPRR (SEQ ID NO:29) SSSHPIFHRGEFSV (SEQ ID NO:30) MSALLHRVLACGRAVQGAGWHAGPKLTSVSGPNKGFAKDAAFYPGHSEVHSVMSMLFYTLIT AFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRRARSAPAEPIAARVTGQTRNITVDP KLFKKRRLRSPRVLFSTQPPPTSSDTLDLDFQAHGTISFNRTHRSKRSSTHPVFHMGEFSVCD SVSVWVGDKTTATDIKGKEVTVLGEVNINNSVFKQYFFETKCRAPNPVESGCRGIDSKHWNSY CTTTHTFVKALTTDDKQAAWRFIRIDTACVCVLSRKAARRG (SEQ ID NO:31) MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAG QTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFH RGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGI DSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO:32) IKVAV (SEQ ID NO:33) YIGSR (SEQ ID NO:34)Attorney Docket No.028193-0029-WO01 M-[(G[V4 / A1]GVP)15-GD(A)25K]4-GYG (SEQ ID NO:40) M-[(G[V4 / A1]GVP)15-GD(A)25K]6-GWP (SEQ ID NO:41) M-[(G[V4 / A1]GVP)15-GD(A)25K]6-GYG (SEQ ID NO:42) M-[(G[V4 / A1]GVP)35-GD(A)25K]2-GWP (SEQ ID NO:43) [(GVGVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:44) [(G[V4 / A1]GVP)5-40-GD(A)10-30K]1-10(SEQ ID NO:45) M-[(GVGVP)15-GD(A)25K]4-GWP- MAPMAEGGGQNHHEVVKFMDVYQRSYCHPI ETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQ HIGEMSFLQHNKCECRPKKDRARQEKCDKPRR. (SEQ ID NO:46) M-[(GVGVP)15-GD(A)25K]4-GWP-SSSHPIFHRGEFSV (SEQ ID NO:47) M-[(GVGVP)15-GD(A)25K]4-GWP- MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:48) MVPGVGYISGRVPGVGIKVAVVPGVGYISGRVPGVGIKVAV (SEQ ID NO:49)

Claims

Attorney Docket No.028193-0029-WO01 CLAIMS What is claimed is:

1. A method for repairing a nerve in a subject, the method comprising: administering a recombinant partially ordered polypeptide to a location of a nerve injury in the subject, the recombinant partially ordered polypeptide comprising a plurality of disordered domains and a plurality of structured domains, wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

2. The method of claim 1, wherein each disordered domain comprises a PG motif or a GP motif and each structured domain comprises a polyalanine domain or a polyproline domain.

3. The method of claim 2, wherein the PG motif comprises an amino acid sequence selected from the group consisting of PG, P(X)nG (SEQ ID NO:3), (B)mP(X)nG(Z)p(SEQ ID NO:4), and a combination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein B, X, and Z are independently any amino acid.

4. The method of claim 1, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1.

5. The method of claim 1, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:2), wherein X is Val or Ala, or a mixture of Ala and Val, and wherein n is an integer from 1 to 30.

6. The method of claim 4, wherein X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val).

7. The method of claim 1, wherein each structured domain comprises a polyalanine domain, at least 25% of the amino acids in the polyalanine domain being alanine residues.Attorney Docket No.028193-0029-WO01 8. The method of claim 1, wherein at least 50% of the amino acids in each structured domain is in an alpha-helical conformation.

9. The method of claim 1, wherein each structured domain comprises an amino acid sequence of (A)m(SEQ ID NO:21), wherein m is an integer from 4 to 50.

10. The method of claim 1, wherein each structured domain comprises an amino acid sequence of: (A)25(SEQ ID NO:22); K(A)25K (SEQ ID NO:23); (KAAAA)5K (SEQ ID NO:24); D(A)25K (SEQ ID NO:25); GD(A)25K (SEQ ID NO:26); GK(A)25K (SEQ ID NO:27); or a combination thereof.

11. The method of claim 1, wherein the recombinant partially ordered polypeptide comprises the plurality of structured domains at about 4% to about 75% by the total amount of amino acids in the recombinant partially ordered polypeptide.

12. The method of claim 1, wherein the recombinant partially ordered polypeptide comprises a neuroregenerative domain.

13. The method of claim 12, wherein the neuroregenerative domain is present at the N- terminus or the C-terminus of the recombinant partially ordered polypeptide.

14. The method of claim 12, wherein the neuroregenerative domain is inserted within the recombinant partially ordered polypeptide.

15. The method of claim 12, wherein the neuroregenerative domain is a vascular endothelial growth factor (VEGF) domain, a nerve growth factor (NGF) domain, an extracellular matrix (ECM) domain, or a combination thereof.

16. The method of claim 15, wherein the VEGF domain comprises an amino acid sequence of (SEQ ID NO:28), (SEQ ID NO:29), QK, KLT, or a combination thereof.Attorney Docket No.028193-0029-WO01 17. The method of claim 15, wherein the NGF domain comprises an amino acid sequence of (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), or a combination thereof.

18. The method of claim 15, wherein the ECM domain comprises an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), (SEQ ID NO:49), RGD, or a combination thereof.

19. The method of claim 1, wherein the recombinant partially ordered polypeptide has a molecular weight of about 10 kilodaltons (kDa) to about 80 kDa.

20. The method of claim 1, wherein the transition temperature is about 10 °C to about 45 °C at a concentration of the recombinant partially ordered polypeptide of about 10 μM to about 3.5 mM.

21. The method of claim 1, wherein the recombinant partially ordered polypeptide exhibits reversible phase transition behavior.

22. The method of claim 1, wherein the porous network comprises a plurality of pores, each pore having a size of about 0.1 μm to about 200 μm.

23. The method of claim 1, wherein the porous network has an elastic modulus of about 1 Pa to about 0.1 MPa.

24. The method of claim 1, wherein the recombinant partially ordered polypeptide is administered to the subject below the transition temperature, and upon exposure to the subject’s body temperature, forms a porous network at the location of the nerve injury.

25. The method of claim 1, wherein the recombinant partially ordered polypeptide is administered to the subject above the transition temperature at the location of the nerve injury.

26. The method of claim 1, wherein the porous network supports cell infiltration, vascularization, axonal growth, axonal regeneration, axonal migration, reinnervation of an organ, promotion of myelination, or a combination thereof.Attorney Docket No.028193-0029-WO01 27. The method of claim 26, wherein cell infiltration comprises Schwann cells, endothelial cells, macrophages, fibroblasts, or a combination thereof.

28. The method of claim 1, wherein the porous network contacts the nerve injury.

29. The method of claim 1, wherein the nerve injury is a gapless nerve injury or a gap nerve injury.

30. The method of claim 1, wherein the nerve injury is a peripheral nerve injury, a neurotmesis nerve injury, or an axonotmesis nerve injury.

31. The method of claim 1, wherein the nerve injury is surgically exposed.

32. The method of claim 1, wherein the recombinant partially ordered polypeptide is administered at a concentration of about 100 μM to about 3 mM.

33. The method of claim 1, wherein the recombinant partially ordered polypeptide is administered in combination with a pharmaceutically acceptable excipient comprising a buffer.

34. The method of claim 1, wherein the recombinant partially ordered polypeptide is administered in combination with a stromal vascular fraction, a neuroangiogenic growth factor, a stem cell, an extracellular matrix protein, or a combination thereof.

35. The method of claim 34, wherein the stem cell is a mesenchymal stem cell, an adipose derived stem cell, a bone marrow derived stem cell, a neural stem cell, a neural crest stem cell, or an induced pluripotent stem cell.

36. The method of claim 34, wherein the stem cell is encapsulated in the porous network.

37. A recombinant partially ordered polypeptide comprising: a plurality of disordered domains; a plurality of structured domains; and a neuroregenerative domain,Attorney Docket No.028193-0029-WO01 wherein the recombinant partially ordered polypeptide exhibits phase transition behavior defined by a transition temperature, the recombinant partially ordered polypeptide forming a porous network above the transition temperature.

38. The recombinant partially ordered polypeptide of claim 37, wherein the neuroregenerative domain is present at the N-terminus or the C-terminus of the recombinant partially ordered polypeptide.

39. The recombinant partially ordered polypeptide of claim 37, wherein the neuroregenerative domain is inserted within the recombinant partially ordered polypeptide.

40. The recombinant partially ordered polypeptide of claim 37, wherein the neuroregenerative domain is a vascular endothelial growth factor (VEGF) domain, a nerve growth factor (NGF) domain, an extracellular matrix (ECM) domain, or a combination thereof.

41. The recombinant partially ordered polypeptide of claim 40, wherein the VEGF domain comprises an amino acid sequence of (SEQ ID NO:28), (SEQ ID NO:29), QK, KLT, or a combination thereof.

42. The recombinant partially ordered polypeptide of claim 40, wherein the NGF domain comprises an amino acid sequence of (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), or a combination thereof.

43. The recombinant partially ordered polypeptide of claim 40, wherein the ECM domain comprises an amino acid sequence of IKVAV (SEQ ID NO:33), YIGSR (SEQ ID NO:34), (SEQ ID NO:49), RGD, or a combination thereof.

44. The recombinant partially ordered polypeptide of claim 37, wherein each disordered domain comprises a PG motif or a GP motif and each structured domain comprises a polyalanine domain or a polyproline domain.

45. The recombinant partially ordered polypeptide of claim 44, wherein the PG motif comprises an amino acid sequence selected from the group consisting of PG, P(X)nG (SEQ ID NO:3), (B)mP(X)nG(Z)p(SEQ ID NO:4), and a combination thereof, wherein m, n, and p areAttorney Docket No.028193-0029-WO01 independently an integer from 1 to 15, and wherein U, X, and Z are independently any amino acid.

46. The recombinant partially ordered polypeptide of claim 37, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1.

47. The recombinant partially ordered polypeptide of claim 37, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO:2), wherein X is Val or Ala, or a mixture of Ala and Val, and wherein n is an integer from 1 to 30.

48. The recombinant partially ordered polypeptide of claim 46, wherein X is a mixture of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val).

49. The recombinant partially ordered polypeptide of claim 37, wherein each structured domain comprises a polyalanine domain, at least 25% of the amino acids in the polyalanine domain being alanine residues.

50. The recombinant partially ordered polypeptide of claim 37, wherein at least 50% of the amino acids in each structured domain is in an alpha-helical conformation.

51. The recombinant partially ordered polypeptide of claim 37, wherein each structured domain comprises an amino acid sequence of (A)m(SEQ ID NO:21), wherein m is an integer from 4 to 50.

52. The recombinant partially ordered polypeptide of claim 37, wherein each structured domain comprises an amino acid sequence of: (A)25(SEQ ID NO:22); K(A)25K (SEQ ID NO:23); (KAAAA)5K (SEQ ID NO:24); D(A)25K (SEQ ID NO:25); GD(A)25K (SEQ ID NO:26); GK(A)25K (SEQ ID NO:27); or a combination thereof.Attorney Docket No.028193-0029-WO01 53. The recombinant partially ordered polypeptide of claim 37, wherein the recombinant partially ordered polypeptide comprises the plurality of structured domains at about 4% to about 75% by the total amount of amino acids in the recombinant partially ordered polypeptide.

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