Spider silk protein-based hydrogels for neuroprotection and axon regeneration capable of injection and sustained delivery of protein therapeutic agents

Abstract

The present invention relates to a novel injectable protein delivery system and a method for delivering one or more therapeutic agents to the central nervous system (CNS) to promote axonal regeneration. The system is based on the use of a recombinant spider silk protein, referred to as spiroin-SpyTag, which undergoes a rapid transition from a sol state to a gel state when exposed to ultrasonic treatment and incubated at body temperature. This unique characteristic allows the material to be easily injected into a specific target tissue. The methods disclosed herein can deliver a protein therapeutic agent covalently conjugated to spiroin-SpyTag to a subject affected by a CNS disorder or injury. In addition, methods of making such injectable protein delivery systems are fast, convenient, and economical and effective.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 637,897, filed April 24, 2024, the entire contents of which (including any tables, figures, or drawings) are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomaterials, and in particular to a spidroin-based hydrogel capable of injecting and continuously delivering protein therapeutic agents for neuroprotection and axon regeneration. Background Art

[0004] Destruction of axonal pathways is a common feature of many central nervous system (CNS) diseases such as spinal cord injury, traumatic brain injury, and neurodegenerative diseases (see references: D. Kacy Cullen, M. D. Tang-Schomer, L. A. Struzyna, A. R. Patel, V. E. Johnson, J. A. Wolf, D. H. Smith, Microtissue engineered constructs with living axons for targeted nervous system reconstruction. Tissue Eng. - Part A 18, 2280-2289 (2012); B. K. Mueller, R. Mueller, H. Schoemaker, Stimulating neuroregeneration as a therapeutic drug approach for traumatic brain injury. Br. J. Pharmacol. 157, 675-685 (2009); R. Hussain, H. Zubair, S. Pursell, M. Shahab, Neurodegenerative diseases: Regenerative mechanisms and novel therapeutic approaches. Brain Sci. 8 (2018)). In the CNS of adult mammals, transected axons are almost incapable of regeneration (see references: M. A. Anderson, J. E. Burda, Y. Ren, Y. Ao, T. M. O’Shea, R. Kawaguchi, G. Coppola, B. S. Khakh, T. J. Deming, M. V. Sofroniew, Astrocyte scar formation AIDS central nervous system axon regeneration. Nature 532, 195-200 (2016); J. W. Fawcett, The Struggle to Make CNS Axons Regenerate: Why Has It Been so Difficult? Neurochem. Res. 45, 144-158 (2020)).Currently, there is no effective treatment that can stimulate the regeneration of human CNS neurons (see literature: S. G. Varadarajan, J. L. Hunyara, N. R. Hamilton, A. L. Kolodkin, A. D. Huberman. Central nervous system regeneration. Cell. 185, 77-94 (2022)). In order to promote axon regeneration, researchers are seeking various methods to enhance the intrinsic growth ability of neurons and / or change external factors to create an environment conducive to axon growth (see literature: F. T. Afshari, S. Kappagantula, J. W. Fawcett, Extrinsic and intrinsic factors controlling axonal regeneration after spinal cord injury. Expert Rev. Mol. Med. 11, 1-19 (2009); F. Sun, K. K. Park, S. Belin, D. Wang, T. Lu, G. Chen, K. Zhang, C. Yeung, G. Feng, B. A. Yankner, Z. He, Sustained axon regeneration induced by co-deletion of PTEN and SOCS3. Nature 480, 372-375 (2011); Z. A. N. Kolberg-Edelbrock, I. R. Sasselli, J. A. Ortega, R. Qiu, Z. Syrgiannis, P. A. Mirau, F. Chen, S. M. Chin, S. Weigand, E. Kiskinis, S. I. Stupp, Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury. Science. 374, 848-856 (2021)). Preclinical studies have shown that signal molecules, such as ciliary neurotrophic factor (CNTF) (see references: M. A. Anderson, T. M. O’Shea, J. E. Burda, Y. Ao, S. L. Barlatey, A. M. Bernstein, J. H. Kim, N. D. James, A. Rogers, B. Kato, A. L. Wollenberg, R. Kawaguchi, G. Coppola, C. Wang, T. J. Deming, Z. He, G. Courtine, M. V. Sofroniew, Required growth facilitators propel axon regeneration across complete spinal cord injury. Nature 561, 396-400 (2018); X. Liu, F. Hao, P. Hao, J. Zhang, L. Wang, S. W. You, N. Wang, Z. Yang, K. F. So, X. Li, Regeneration and functional recovery of the completely transected optic nerve in adult rats by CNTF-chitosan. Signal Transduct. Target. Ther. 8 (2023)), insulin-like growth factor 1 (IGF1) (see references: P. H. J. D. Macklis, IGF-I specifically enhances axon outgrowth of corticospinal motor neurons. Nat. Neurosci. 9, 1371-1381 (2006); A. H. Dyer, C. Vahdatpour, A. Sanfeliu, D. Tropea, The role of Insulin-Like Growth Factor 1 (IGF-1) in brain development, maturation and neuroplasticity. Neuroscience 325, 89-99 (2016)) and human neurotrophin-3 (NT-3) (see references: J. Bloch, E. G. Fine, N. Bouche, A. D. Zurn, P. Aebischer, Nerve growth factor- and neurotrophin-3-releasing guidance channels promote regeneration of the transected rat dorsal root. Exp. Neurol. 172, 425-432 (2001); Z. Yang, H. Duan, L. Mo, H. Qiao, X. Li, The effect of the dosage of NT-3 / chitosan carriers on the proliferation and differentiation of neural stem cells. Biomaterials 31, 4846-4854 (2010)). However, the short half-life of these signaling molecules in vivo, in comparison with the well-known poor and slow capacity of CNS axon regeneration, severely hinders their clinical applications. Therefore, the safe and sustained delivery of these protein drugs presents a major challenge to materials scientists and engineers.While viral delivery systems, such as adeno-associated virus, offer a method for long-term delivery of functional proteins in vivo, their clinical application remains limited due to concerns over cost, complexity, safety, and scalability (see literature: Q. Ling, J. A. Herstine, A. Bradbury, S. J. Gray, AAV-based in vivo gene therapy for neurological disorders. Nat. Rev. Drug Discov. 22, 789-806 (2023); E. A. Chowdhury, G. Meno-Tetang, H. Y. Chang, S. Wu, H. W. Huang, T. Jamier, J. Chandran, D. K. Shah, Current progress and limitations of AAV mediated delivery of protein therapeutic genes and the importance of developing quantitative pharmacokinetic / pharmacodynamic (PK / PD) models. Adv. Drug Deliv. Rev. 170, 214-237 (2021)).

[0005] Injectable hydrogels are often used for soft tissue engineering, offering new hope for addressing this unmet clinical need due to their precise in situ delivery capability, minimally invasive administration, and controlled release properties (see references: B. Jiang, X. Liu, C. Yang, Z. Yang, J. Luo, S. Kou, K. Liu, F. Sun, Injectable, photoresponsive hydrogels for delivering neuroprotective proteins enabled by metal-directed protein assembly. Sci. Adv. 6, eabc4824 (2020); S. Jin, H. Choi, D. Seong, C. L. You, J. S. Kang, S. Rho, W. B. Lee, D. Son, M. Shin, Injectable tissue prosthesis for instantaneous closed-loop rehabilitation. Nature 623, 58–65 (2023); Z. Wang, Y. Zhang, Y. Yin, J. Liu, P. Li, Y. Zhao, D. Bai, H. Zhao, X. Han, Q. Chen, High-Strength and Injectable Supramolecular Hydrogel Self-Assembled by Monomeric Nucleoside for Tooth-Extraction Wound Healing. Adv. Mater. 34, 1–20 (2022)).These hydrogel materials are characterized by their high water content, porous structure, and biocompatibility (see references: D. Seliktar, Designing Cell-Compatible Hydrogels for Biomedical Applications. Science. 336, 1124-1128 (2012); J. J. Green, J. H. Elisseeff, Mimicking biological functionality with polymers for biomedical applications. Nature 540, 386-394 (2016); G. Y. Xu, S. Xu, Y. X. Zhang, Z. Y. Yu, F. Zou, X. S. Ma, X. L. Xia, W. J. Zhang, J. Y. Jiang, J. Song, Cell-Free Extracts from Human Fat Tissue with a Hyaluronan-Based Hydrogel Attenuate Inflammation in a Spinal Cord Injury Model through M2 Microglia / Microphage Polarization. Small 18, 1-14 (2022)). A variety of hydrogel systems have been developed for drug controlled release and tissue engineering applications. However, synthetic hydrogels often require harsh gelation conditions and can produce toxic byproducts, while naturally derived polymers such as collagen, gelatin, and chitosan often exhibit poor mechanical properties and can potentially transmit pathogens. Thus, there is a need for a hydrogel that is easy to manufacture, can be functionalized with a wide variety of biological functions, is non-toxic, biocompatible, and has good mechanical properties, and more importantly, can rapidly undergo sol-gel transition in vivo. These requirements together greatly limit the available selection of suitable candidate materials for therapeutic agent delivery in the nervous system.

[0006] Recombinant silk fibroin hydrogels have emerged as a promising biomaterial to address these challenges. Natural materials such as silks produced by silkworms and spiders are typically the result of a protein molecule transitioning from a liquid to a solid phase. This unique phase change behavior can be responsive to a variety of stimuli readily available under physiological conditions, thus offering the possibility of developing these silk proteins into injectable therapeutic delivery systems. Although previous studies have investigated the feasibility of using Bombyx mori silk fibroin (silk fibroin) to fabricate hydrogels for therapeutic delivery and tissue engineering (see references: F. Feng, X. Song, Z. Tan, Y. Tu, L. Xiao, P. Xie, Y. Ma, X. Sun, J. Ma, L. Rong, L. He, Cooperative assembly of a designer peptide and silk fibroin into hybrid nanofiber gels for neural regeneration after spinal cord injury. Sci. Adv. 9, eadg0234 (2023); S. H. Kim, H. Hong, O. Ajiteru, M. T. Sultan, Y. J. Lee, J. S. Lee, O. J. Lee, H. Lee, H. S. Park, K. Y. Choi, J. S. Lee, H. W. Ju, I.-S. Hong, C. H. Park, 3D bioprinted silk fibroin hydrogels for tissue engineering. Nat. Protoc. 16, 5484-5532 (2021); Z. Zhu, S. Ling, J. Yeo, S. Zhao, L. Tozzi, M. J. Buehler, F. Omenetto, C. Li, D. L. Kaplan, High-Strength, Durable All-Silk Fibroin Hydrogels with Versatile Processability toward Multifunctional Applications. Adv. Funct. Mater. 28, 1-10 (2018); L. Semmler, A. Naghilou, F. Millesi, S. Wolf, A. Mann, S. Stadlmayr, S. Mero, L. Ploszczanski, L. Greutter, A. Woehrer, E. F. Vollrath, T. Weiss, C. Radtke, Silk-in-Silk Nerve Guidance Conduits Enhance Regeneration in a Rat Sciatic Nerve Injury Model. Adv. Healthc. Mater. 12, 1-12 (2023)), but the exploration of spider silk proteins in biomedical applications is insufficient, which can be due to the limited supply of their natural sources. In recent years, several recombinant spider silk proteins with good water solubility and high expression rate have been produced by using heterologous E. coli expression, so that their biomedical applications can be explored (see documents: T. Arndt, U. Chatterjee, O. Shilkova, J. Francis, J. Lundkvist, D. Johansson, B. Schmuck, G. Greco, E. Nordberg, Y. Li, L. U. Wahlberg, M. Langton, J. Johansson, C. A. Rising, Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture. Adv. Funct. Mater., doi:10.1002 / adfm.202303622 (2023); K. Schacht, T. Scheibel, Controlled hydrogel formation of a recombinant spider silk protein. Biomacromolecules 12, 2488-2495 (2011); K. Schacht, T. Jungst, M. Schweinlin, A. Ewald, J. Groll, T. Scheibel, Biofabrication of cell-loaded 3D spider silk constructs. Angew. Chemie-Int. Ed. 54, 2816-2820 (2015); W. W. Song, Z. G. Qian, H. Liu, H. F. Chen, D. L. Kaplan, X. X. Xia, On-Demand Regulation of Dual Thermosensitive Protein Hydrogels. ACS Macro Lett. 10, 395-400 (2021)). Materials derived from natural silk fibroin or spider silk proteins

[0007] have been proven to be biocompatible and non-immunogenic (see literature: A. T. Scheibel, Silk-based materials for biomedical applications. Biotechnol. Appl. Biochem. 55, 155-167 (2010)), but not biofunctional

[0008] sufficient. In contrast to this, materials comprising recombinant proteins are more suitable for biofunctionalization by genetic programming and biomolecular engineering. Although several studies have proven the benefits of natural spider silk protein-based materials for peripheral nerve repair, their efficacy in the central nervous system (CNS) has not been proven. In view of these limitations, there is an urgent need to develop alternative strategies for non-invasive, safe and sustained delivery of proteins into the CNS. SUMMARY

[0009] The present invention addresses the need for effective and minimally invasive protein delivery systems to promote axon regeneration in the CNS. In a first aspect, the present invention discloses a new injectable protein delivery system comprising a material based on the use of a recombinant spider silk protein known as spidroin-SpyTag. In preferred embodiments, the spidroin-SpyTag can be injected into a target tissue. When the spidroin-SpyTag is exposed to brief ultrasound treatment and at a temperature of about 37°C, it undergoes a rapid sol-gel transition, rendering it injectable.

[0010] In some embodiments, the application of SpyTag / SpyCatcher click chemistry allows functionalization of the spidroin-SpyTag with various bioactive motifs, including cell-binding ligands and neurotrophic factors. This versatility makes it suitable for both neuronal culture and tailored therapeutic interventions. In some embodiments, the spidroin-SpyTag is in the form of a hydrogel-injectable and comprises one or more bioactive agents covalently coupled to the spidroin-SpyTag. In some embodiments, the one or more bioactive agents are protein therapeutics, including one or more of ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN).

[0011] In a second aspect, the present invention discloses a method of delivering one or more therapeutic agents to a targeted CNS tissue, comprising administering a spidroin-SpyTag hydrogel covalently coupled to a protein therapeutic agent to a targeted CNS tissue of a subject in need thereof. In some embodiments, the targeted CNS tissue comprises a site affected by a CNS condition or injury. In some embodiments, the CNS condition or injury comprises a spinal cord injury, a traumatic brain injury, a stroke, glaucoma, a muscle dystrophy, muscle hypertrophy, a metabolic myopathy, or muscle paralysis. In some embodiments, the site of injury comprises optic nerve and retinal tissue. In some embodiments, the spidroin-SpyTag protein delivery system provides an injectable and sustained delivery of a protein for neuroprotection and neuroregeneration of CNS tissue affected by a condition or injury. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 A schematic showing the preparation and application of a recombinant Spidroin-SpyTag (S-A) hydrogel is provided. Ultrasound pre-treatment accelerates the heat-induced sol-gel transition of S-A. Through SpyTag / SpyCatcher chemistry, the S-A hydrogel can covalently immobilize various target SpyCatcher-fusion proteins (i.e., B-POI-B), such as B-CNTF-B. The resulting hydrogel

[0013] Suitable for neuronal culture and optic nerve regeneration.

[0014] Figures 2A-2H The promotion of the thermally induced sol-gel transition of S-A by ultrasound is shown. Each graph shows: the change in G' and G" of S-A (4 wt%) at 37 °C as a function of time ( Figure 2A ) The change in G' and G" of S-A (4 wt%) treated with ultrasound at 37 °C as a function of time ( Figure 2B ) Frequency sweep test of S-A hydrogel (4 wt%) with strain fixed at 5%

[0015] ( Figure 2C ) Strain sweep test of S-A hydrogel (4 wt%) with frequency fixed at 1 rad / s ( Figure 2D ) Erosion curves of S-A hydrogel (4 wt%, 30 μΐ) immersed in 0.5 ml of Tris buffer, PBS or water ( Figure 2E ) Data are expressed as mean ± SD (n = 3) CD spectra of S-A before and after ultrasound treatment ( Figure 2F ) TEM images of S-A solution before ultrasound treatment, S-A solution after ultrasound treatment (i.e. pre-gel solution) and resuspended S-A hydrogel ( Figure 2G ) Scale bar: 100 nm. Photographs of S-A hydrogel (4 wt%) with scale bar: 5 mm ( Figure 2H ).

[0016] Figures 3A-3C The biofunctionalization of S-A hydrogels by SpyTag / SpyCatcher chemistry is shown. Each graph shows: SpyTag / SpyCatcher reaction for spontaneous formation of isopeptide bonds ( Figure 3A ) SDS-PAGE analysis of the reaction of S-A and SpyCatcher-fusion protein ( Figure 3B ) Comparison of release profiles of proteins physically entrapped in S-A hydrogels and proteins covalently entrapped in S-A hydrogels ( Figure 3C ) Data are expressed as mean ± SD (n = 3).

[0017] Figures 4A-4C The culture of N2A cells on S-A hydrogels modified with functional proteins is shown. Figure 4A Representative images of N2A cells grown on hydrogels for 3 days are shown. Scale bar: 50 μιη. Figure 4BThe percentage of cell viability of blank S-A hydrogels, B-CNTF-B hydrogels and B-LM-B hydrogels determined after 3 days of culture is shown. One-way ANOVA followed by Tukey's multiple comparison test was performed, *P < 0.05. Data are expressed as mean ± SD (n = 5). Figure 4C The cell density of the three hydrogels is shown, data are expressed as mean ± SD (n = 5).

[0018] Figures 5A-5D Growth of dorsal root ganglion (DRG) neurons on blank or functionalized S-A hydrogels is shown. Figure 5A The effect of immobilized factors on DRG neurons adhesion is shown. Cells were stained with Tuj1 antibody after 16 hours of culture. Scale bar: 100 pm. Figure 5B The cell density on different substrates is shown. One-way ANOVA followed by Dunnet's multiple comparison test was performed. Data are expressed as mean ± SEM (n = 3). Figure 5C The effect of immobilized neurotrophic factors on neurite growth is shown. Representative images of neurons after Tuj1 staining are shown. Scale bar: 25 pm. Figure 5D The average neurite length per neuron on different substrates is shown. One-way ANOVA followed by Dunnet's multiple comparison test was performed. Data are expressed as mean ± SEM (n = 3). ns means not significant difference. *P < 0.05; **P < 0.01. *, ** or ns on the lines means the comparison between the two groups connected by the line, while *, ** or ns elsewhere means the comparison with the PDL / Laminin group by default.

[0019] Figure 6 Degradation of S-A hydrogels after injection into the vitreous is shown. Retinal sections were collected at different time points (1, 5, 14 and 28 days) after intravitreal injection of vehicle control, S-A hydrogels, B-CNTF-B solution (15 mM) or B-CNTF-B hydrogels (15 mM) immediately after optic nerve injury. According to histological analysis, the gel was still present after two weeks but disappeared at four weeks. White arrows indicate the presence of the gel.

[0020] Figures 7A-7D Immunogenic effect of S-A hydrogels is shown. Retinal sections were collected at different time points (1, 5, 14 and 28 days) after intravitreal injection of vehicle control or S-A hydrogels immediately after optic nerve injury. Samples were stained with Iba1( Figure 7A ), CD4( Figure 7B ), CD68( Figure 7C ) or CD45( Figure 7D) and DAPI (blue) staining. Representative images of retinas 5 days after lesion are shown. Scale bar: 50 pm. Bar graphs show the density of microglia (Ibal positive) Figure 7A ), helper T cells (CD4 positive) Figure 7B ), activated macrophages and microglia (CD68 positive) Figure 7C ), or hematopoietic cells (CD45 positive) Figure 7D ) in the retina. ns: not significant. *P < 0.05. Multiple t-tests were performed. Data are expressed as mean ± SEM (n = 3-4 mice).

[0021] Figures 8A-8B B-CNTF-B S-A hydrogels were shown to prolong the activation of STAT3 signaling in vivo. Each panel shows retinal sections collected at different time points after injection (1, 5, 14, or 28 days) from C57BL / 6 mice injected with vehicle, blank gel, B-CNTF-B solution (15 mM), or B-CNTF-B hydrogel (15 mM) immediately after optic nerve lesion Figure 8A ). Samples were stained with Tujl (green) and p-STAT3 (Tyr705) (magenta) antibodies. Scale bar: 20 pm. Quantification of p-STAT3-positive RGCs Figure 8B ). ns: not significant. **P < 0.01. One-way ANOVA followed by Bonferroni or Games-Howell multiple comparison tests were performed. Data are expressed as mean ± SEM (n = 4-6 mice).

[0022] Figures 9A-9D B-CNTF-B hydrogels were shown to improve RGCs survival. Each panel shows retinas collected at 14 days Figure 9A ) or 28 days Figure 9C ) after lesion from C57BL / 6 mice injected with vehicle, blank S-A hydrogel, B-CNTF-B solution (15 mM), or B-CNTF-B hydrogel (15 mM) immediately after optic nerve lesion. RGCs were labeled with Tujl antibody (green). Scale bar: 20 pm. Quantification of viable RGCs 14 days Figure 9B ) or 28 days Figure 9D ) after lesion. ns: not significant. *P < 0.05. **P < 0.01. One-way ANOVA followed by Bonferroni multiple comparison tests were performed. Data are expressed as mean ± SEM (n = 5-6 mice).

[0023] Figures 10A-10Dshowing that B-CNTF-B hydrogels promote optic nerve axon regeneration. Each panel shows: C57BL / 6 mice were injected with vehicle, blank gel, B-CNTF-B solution (15 mM) or B-CNTF-B hydrogel (15 mM) by intravitreal injection immediately after optic nerve injury and retinal sections were collected at 14 days post-injury Figure 10A ) and 28 days post-injury Figure 10C ). Two days before sacrificing the animals, axons were labeled by intravitreal injection of CTB-FITC. Samples were stained with FITC (red) antibody. Scale bar: 200 pm. Quantification of regenerated axons at a distance greater than the indicated distance from the lesion site at 14 days post-injury Figure 10B ) and 28 days post-injury Figure 10D ). ns: not significant. *P < 0.05. **P < 0.01. One-way ANOVA followed by Bonferroni or Games-Howell multiple comparison test was performed. Data are expressed as mean ± SEM (n = 5-6 mice).

[0024] Figures 11A-11F showing that covalent immobilization is crucial for the functional duration of CNTF in vivo. Figure 11A showing that C57BL / 6 mice were administered physically mixed CNTF hydrogel (S-A + 15 mM CNTF) or covalently bound B-CNTF-B hydrogel (S-A + 15 mM B-CNTF-B) by intravitreal injection immediately after optic nerve injury and retinal sections were collected at different time points (14 days and 28 days) post-injury. Samples were stained with Tujl (green) and p-STAT3 (magenta) antibodies. Scale bar: 20 pm. Figure 11B showing quantification of p-STAT3 -positive RGCs at different time points (1, 5, 14 or 28 days). ns: not significant; **P < 0.01. Two-way ANOVA followed by Bonferroni multiple comparison test was performed. Data are expressed as mean ± SEM (n = 3 mice). Figure 11C showing that C57BL / 6 mice were administered physically mixed CNTF hydrogel or covalently bound B-CNTF-B hydrogel by intravitreal injection immediately after optic nerve injury and whole-mount retinas were collected at 14 days post-injury. RGCs were labeled with Tujl antibody (green). Scale bar: 20 pm. Figure 11D showing percentage of surviving RGCs at 14 days and 28 days post-injury. *P < 0.05; **P < 0.01. Unpaired t-test was performed. Data are expressed as mean ± SEM (n = 3 mice). Figure 11EOptic nerve sections of mice injected with physically mixed CNTF hydrogel or covalently bound B-CNTF-B hydrogel after optic nerve injury are shown. Samples were collected 14 days after injury and stained with FITC (red) antibody to detect CTB-FITC traced axons. Scale bar: 200 pm. Figure 11F Quantification of regenerated axons beyond the indicated distance from the lesion site at 14 days after injury and 28 days after injury is shown.

[0025] **P < 0.01. Two-way ANOVA followed by Bonferroni’s multiple comparison test was performed. Data are expressed as mean ± SEM (n = 3 mice).

[0026] Figure 12 Table 1 in the Appendix shows the gene sequences used in the present study.

[0027] Figure 13 Table 2 in the Appendix shows the amino acid sequences of the proteins used in the present application. DETAILED DESCRIPTION

[0028] SELECTED DEFINITIONS

[0029] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, the use of the terms "including," "includes," "having," "has," "with," or "contains" or variations thereof does not limit the item, quantity, property, component or process to the inclusion of only the recited item, quantity, property, component or process, but rather allows for the inclusion of other items, quantities, properties, components or processes not expressly recited. The transitional terms / phrases “comprising,” “comprises,” “comprised of,” “consisting essentially of,” and “consisting of” can be used interchangeably.

[0030] The phrase “consisting essentially of’ means that the claim encompasses embodiments having the specified materials or steps in addition to those that do not materially affect the basic and novel characteristics of the claim.

[0031] The term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured, i.e., the limitations of the measurement system. When the amount of an ingredient contained in a composition is stated to be about a certain value, the compositions contain the stated amount of the ingredient, with variations of the stated amount by about 0% to about 10% (X ± 10%) above and below the value. In other instances, the term“about” provides variations of the stated value by 0% to 10% (X ± 10%) above and below the given value. Obviously, such variations are meant to be within skilled in the art ranges for how the value is measured, i.e., the limitations of the measurement system. For example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10% are all within the scope of“about” X.

[0032] In the present disclosure, ranges are stated in shorthand fashion, to avoid having to list and describe each value within the range. Where appropriate, any suitable value within the range can be selected as the upper value, lower value, or terminal value of the range. For example, a range of 0.1 to 1.0 indicates that the terminal values are 0.1 and 1.0, and that intermediate values such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, and 0.7 to 1.0, etc. are contemplated as values within the range having at least two significant digits, e.g., a range of 5 to 10 indicates all values between 5.0 and 10.0, and between 5.00 and 10.00, including the terminal values. When ranges are used herein, combinations and subcombinations of the ranges are expressly included (e.g., subranges of the disclosed ranges) and specific embodiments within the ranges.

[0033] As used herein, the terms“therapeutically effective amount,”“therapeutically effective dose,”“effective amount,” and“effective dose” are used in reference to an amount or dose of a compound or composition that, when administered to a subject, is effective to treat, prevent, or ameliorate a condition, disease, or disorder in the subject. That is, the amount is“therapeutically effective” when administered to the subject. The actual amount will vary depending on factors such as the particular condition, disease, or disorder being treated, prevented, or ameliorated; the severity of the illness; the size, weight, age and health of the patient; and the route of administration.

[0034] As used herein, the term "treatment" refers to: eradication; reduction; amelioration; elimination; alleviation; lessening of symptoms or delay in onset of symptoms; slowing of the rate of worsening or deterioration; less debilitating at the endpoint of worsening; and / or improvement in the physical or mental well-being of the subject, or reversal of signs or symptoms of a health condition, disease, or disorder to any extent, including but not requiring complete cure of the condition, disease, or disorder. Treatment can be curative, ameliorative, or partially ameliorative of a disorder. "Treatment" can also include improving or enhancing a condition or characteristic, for example, bringing a particular system of the body to a higher state of health or homeostasis in the body.

[0035] As used herein, "subject" refers to an animal, such as a mammal, for example, a human. The methods described herein can be used for both human and non-human animals. In some embodiments, the subject is a mammal (such as an animal model of a disease), and in some embodiments, the subject is a human. The terms "subject" and "patient" can be used interchangeably. The animal can be, for example, a human, pig, horse, goat, cat, mouse, rat, dog, ape, fish, chimpanzee, orangutan, guinea pig, hamster, cow, sheep, bird, chicken, and any other vertebrate or invertebrate. In the context of the present application, the preferred subject is a human. The subject can be at any age or stage of development, including an infant, a young child, a teenager, an adolescent, an adult, or an elderly person.

[0036] "Decrease" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0037] "Increase" refers to a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0038] These benefits can include, but are not limited to: treatment of a health condition, disease, or disorder; prevention of a health condition, disease, or disorder; immunity to health; enhancement of the function of a bodily organ, tissue, or system.

[0039] Injectable protein delivery systems and methods for delivering therapeutic agents to CNS-targeted tissues

[0040] A new injectable protein delivery system for promoting neuroprotection and axonal regeneration in the central nervous system (CNS) is disclosed. In a first aspect, the present invention comprises an injectable protein delivery system comprising a recombinant spider silk protein known as spidroin-SpyTag. In some embodiments, the spidroin-SpyTag undergoes a rapid transition from a sol state to a gel state when exposed to brief ultrasonic treatment and incubated at body temperature. In preferred embodiments, the spidroin-SpyTag transitions from a sol state to a gel state after being subjected to brief ultrasonic treatment and incubated at a temperature of about 37 °C. This unique property enables the material to be easily injected into a specific targeted tissue. In some embodiments, the spidroin-SpyTag is coupled to one or more biologically active agents or protein therapeutics including, but not limited to, ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN). In preferred embodiments, the present invention comprises spidroin-SpyTag in the form of a hydrogel coupled to a biologically active agent or protein therapeutic.

[0041] In a second aspect, the present invention discloses a method of delivering a biologically active agent or protein therapeutic to a CNS target comprising administering spidroin-SpyTag in the form of a hydrogel coupled to a biologically active agent or protein therapeutic to a targeted CNS tissue of a subject in need thereof. In some embodiments, a therapeutically effective amount of spidroin-SpyTag covalently coupled to one or more protein therapeutics including, but not limited to, ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN) and in the form of a hydrogel is administered to a subject. In some embodiments, the subject is a mammal, which is a mouse or a human. In some embodiments, the administration to the subject is by intravitreal, intrathecal, intramuscular, intradermal, intracranial, intraspinal, or epidural injection. In some embodiments, the hydrogel is administered to a subject affected by a CNS condition or injury. In some embodiments, the subject is affected by a CNS condition or injury including, but not limited to, a spinal cord injury, a traumatic brain injury, a stroke, glaucoma, an optic nerve injury, a retinal tissue injury or disease, a muscle dystrophy, muscle hypertrophy, a metabolic myopathy, or muscle paralysis. In some embodiments, spidroin-SpyTag in the form of a hydrogel covalently coupled to one or more protein therapeutics is injected to a targeted site affected by a CNS condition or injury to promote neuroprotection and axonal regeneration. In one example, the site of injury includes the optic nerve and / or retinal tissue.

[0042] In some embodiments, the stiffness level of the spidroin-SpyTag hydrogel can be tailored by adjusting the protein concentration of the protein therapeutic covalently coupled to the spidroin-SpyTag. In preferred embodiments, the stiffness level of the spidroin-SpyTag hydrogel covalently coupled to the protein therapeutic is comparable to the stiffness of neural tissue.

[0043] In some embodiments, the covalent immobilization or coupling of the protein therapeutic to the spidroin-SpyTag hydrogel allows for slow, sustained release of the protein therapeutic in vitro or in vivo. In some embodiments, the methods of the present application can be used to culture neurons in a cellular system that studies axonal regeneration. In some embodiments, the spidroin-SpyTag hydrogel coupled to the protein therapeutic can be used as a substrate for primary dorsal root ganglion (DRG) neuron attachment. In some examples, CNTF is simultaneously released from the hydrogel to cultured dorsal root (DRG) neurons, promoting neurite growth by triggering the JAK-STAT3 signaling pathway.

[0044] In some embodiments, the spidroin-SpyTag hydrogel is stable at the target site for up to 14 days, preferably up to 30 days or more.

[0045] In some embodiments, the spidroin-SpyTag hydrogel covalently coupled to one or more protein therapeutics is administered to a subject by intravitreal, intrathecal, intramuscular, intradermal, intracranial, intraspinal, or epidural injection.

[0046] In some embodiments of the present application, the method comprises administering multiple doses of the composition of the present application. The method can comprise administering a therapeutically effective dose of a composition comprising a compound of the present application or a composition thereof as described herein once a week, once a month, once a quarter, twice a year, once a year, or less frequently. In addition, treatment of a subject with a therapeutically effective amount of a composition of the present application can comprise a single treatment or can comprise a series of treatments. It is also understood that the effective dose of the compound or composition thereof for treatment can be increased or decreased during the course of a particular treatment. Changes in dosage can be determined by the results of diagnostic analysis to determine whether neurological function has been restored (or is absent), and it is likely that the dosage will be changed significantly, as is known in the art. Specifically, the identification of nerve regeneration includes, for example, motor skill assessment.

[0047] The recitation of a list of chemical groups in any variable definition herein includes defining that variable to be any single group or combination of listed groups. The recitation of embodiments of a variable or aspect herein includes embodiments as any single embodiment or in combination with any other embodiment or portion thereof.

[0048] Any of the compositions or methods provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0049] Other features and advantages of the present application will become apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are incorporated by reference.

[0050] The concentration / amount of active agent in the formulation can vary widely, and is selected primarily based on the activity of the active ingredient, the particular mode of administration selected, and the needs of the patient, in accordance with the ordinary level of skill in the art. However, concentrations will generally be chosen to provide a dosage in the range of about 1-30 uM. It will be appreciated that these dosages can be varied, to optimize the therapeutic and / or prophylactic regimen for a particular subject or group of subjects.

[0051] It will be clear to those of skill in the art that the exact dose and frequency of administration will depend on the particular condition being treated, the severity of the illness being treated, the age, body weight, general health condition of the particular patient, and other medication that the individual can be taking, as is well known by those skilled in the administration of drugs.

[0052] Materials and Methods

[0053] 1. Construction and production of spidroin-spytag

[0054] The gene and amino acid sequence of NTD2 Rep CTD-SpyTag (S-A) are shown in Table 1 and Table 2. The construct was cloned into pET22b(+) vector, followed by introduction into E. coli BL21(DE3) cells (Invitrogen) by transformation. Cells were cultured at 37 °C and 220 rpm in Luria broth (LB) supplemented with 100 mg / L ampicillin until the optical density at 600 nm (OD600) reached the range of 0.6 to 1.0. To induce protein expression, 3.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG, Sangon Biotech) was added to the culture. After induction, the cells were incubated at 16 °C for 20 hours, then harvested and resuspended in 20 mM Tris-HCl (pH 8). To prevent protein degradation, phenylmethylsulfonyl fluoride (1 mM) was added to the resuspended cells, followed by lysis of the cells using a French Press cell crusher. The lysate was centrifuged at 18,000 x g for 45 min at 4 °C, and the resulting supernatant was filtered through a 0.45 μm filter. The filtered supernatant was loaded onto a Ni-NTA column (Cytiva) and washed with a buffer containing 20 mM Tris-HCl and 20 mM imidazole (pH 8). Finally, the target protein was eluted using an AKTA Explorer liquid chromatography system (GE Healthcare) with an elution buffer containing 20 mM Tris-HCl and 500 mM imidazole (pH 8).

[0055] The obtained protein solution was dialyzed against 20 mM Tris-HCl (pH 8) at 4 °C, with a total volume of 5 liters in six cycles. Subsequently, the solution was filtered using a 0.22 μm filter and concentrated to a final concentration of 40 mg / mL using an Amicon Ultra-15 centrifugal filter (Millipore). The protein concentration was determined by measuring the ultraviolet absorbance at 280 nm, while the purity was evaluated using SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. The expression yield of S-A was approximately 40 milligrams per liter of E. coli culture. The S-A solution was stored at 4 °C or -80 °C for later use.

[0056] 2. Construction and production of SpyCatcher-fusion proteins

[0057] We created three expression systems, pQE80l::B-CNTF-B, pQE80l::B-IGF1-B and pQE80l::B-OPN-B, to produce SpyCatcher-fusion proteins including SpyCatcher-ELP-CNTF-ELP-SpyCatcher, SpyCatcher-ELP-osteopontin-ELP-SpyCatcher and SpyCatcher-ELP-IGF1-ELP-SpyCatcher. They were constructed by inserting the corresponding genes encoding CNTF, osteopontin or IGF1 into the previously described plasmid pQE80l::SpyCatcher-ELP-RGD-ELP-SpyCatcher39using SacI and Spel restriction enzymes. E. coli BL21(DE3) was used to express the corresponding proteins, which were subsequently purified by Ni-NTA affinity chromatography. The purified proteins were dialyzed against Milli-Q water (5 liters x 6) at 4°C, and then sterilized with a 0.22 pm filter. Finally, the proteins were lyophilized using a Labconco freeze dryer and stored at -80°C until use.

[0058] 3. Preparation of Spidroin-SpyTag hydrogels

[0059] We used a Branson SFX250 sonicator (250 W, 20 kHz) equipped with a conical microtip with a 3 mm diameter. In a typical procedure, a 0.5 mL S-A solution in a 1.5 mL Eppendorf tube was pulsed sonicated (20 cycles, 1 s on, 5 s off) at 20% amplitude at room temperature. To ensure sterilization and prevent overheating of the protein solution, 75% ethanol was used. The introduction of air bubbles was minimized during sonication. The resulting sonicated solution was called pre-gel solution. To initiate gelation, the sonicated protein solution was placed at 37°C.

[0060] 4. Rheological tests

[0061] An ARES-RFS rheometer (TA Instruments) was used to perform rheological measurements in time, frequency, and strain sweep modes. The rheometer setup consisted of a bottom steel plate with a diameter of 25 mm, on which the sample was placed in the center. There was a steel plate with a diameter of 8 mm on the top, and the distance between the top and bottom plates was fixed at 0.5 mm. All experiments were performed at 37 °C. To reduce water evaporation, the samples were sealed with silicone oil. The gelation kinetics were monitored by time sweep experiments, with the strain and frequency fixed at 5% and 1 rad / s, respectively. Frequency sweep experiments were performed in the frequency range of 0.01–100 rad / s, with the strain fixed at 5%. Strain sweep experiments were performed in the strain range of 1–250% at a constant frequency of 1 rad / s.

[0062] 5. Erosion test

[0063] To evaluate the erosion of SA hydrogels, 30 μl of 4 wt% hydrogel samples were immersed in 0.5 mL of Tris buffer, PBS, or miliQ water. At designated time points, 2 μl aliquots of the supernatant were taken and the absorbance at 280 nm was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific) to determine the protein concentration. The experiment was performed in triplicate. The erosion percentage was calculated as follows:

[0064] Erosion percentage = (protein content in supernatant / total protein in gel) × 100%

[0065] 6. Circular Dichroism (CD) Spectroscopy

[0066] CD measurements were performed using a Jasco-8815 CD spectrophotometer (Jasco Co.) at room temperature. To assess changes in secondary structure, the ellipticity values ​​of the SA solution before and after ultrasonic treatment were recorded. A 0.2 mL aliquot of a 5 μM SA solution in Tris buffer (pH 8) was dispensed into a quartz cuvette. Subsequently, the sample was scanned over a wavelength range of 260 to 190 nm using the following settings: continuous scan mode, a scan speed of 20 nm / min, and a cumulative amount of 1.

[0067] 7. Transmission Electron Microscopy (TEM) Analysis

[0068] Formvar-coated copper grid was negatively charged. Three microliters of sample was gently deposited on the grid and excess sample was carefully removed with a blotting paper. Subsequently, the sample was washed with 2% (w / v) uranyl formate to enhance contrast. After washing, the grid was stained with uranyl formate for 45 seconds. Then, any excess stain was removed with a blotting paper and the grid was allowed to air dry. Imaging was performed using a Talos 120c microscope operating at 120 keV. Images were captured at a magnification of x 22,000 or x 73,000.

[0069] 8. Covalent coupling by Spy chemistry

[0070] For checking covalent coupling by Spy chemistry, spidroin-SpyTag (S-A, 3 pg^l) was mixed with SpyCatcher-POI-SpyCatcher (B-POI-B, 1 pg^l) at a molar ratio of 4: 1 to 6: 1. The mixture was then incubated at 4°C overnight, followed by SDS-PAGE analysis. The use of an excess of spidroin-SpyTag was intended to ensure complete reaction and depletion of the other reactant B-POI-B, thus simplifying the subsequent SDS-PAGE analysis.

[0071] 9. Protein release assay

[0072] The sonicated S-A solution (3 wt%) was mixed with B-GFP-B or A-GFP-A (1 pg^l) and then incubated at 4°C overnight. Then, 60 mΐ aliquots of the reaction product were transferred to 1.5 mL Eppendorf tubes and incubated at 37°C for 1 hour to promote the formation of hydrogel. To assess the release of GFP, 100 mΐ of 20 mM Tris-HCl buffer (pH 8.0) was added to each tube. These tubes were then placed in a humidified incubator at 37°C. After 1 day and 3 days, 100 mΐ aliquots of the supernatant were transferred to a black 96-well plate. The fluorescence intensity of the supernatant was measured using a Varioskan LUX multimode microplate reader (Thermo Science) with an excitation wavelength of 470 nm and an emission wavelength of 510 nm.

[0073] The ratio of released GFP was calculated as follows:

[0074] Ratio of released GFP (%) = (fluorescence intensity of 100 mΐ supernatant / fluorescence intensity of 100 mΐ of the corresponding control) x 100%

[0075] In this equation, "corresponding control" is the dilution of the gelation precursor (60 μΐ) S-A+B-GFP-B (or A-GFP-A) with 100 μΐ of 20 mM Tris-HCl buffer (pH 8.0) which mimics 100% release of GFP from the gel into the supernatant.

[0076] 10. Functionalization of S-A hydrogels

[0077] The sonicated S-A solution was mixed with B-POI-B and the reaction mixture was left at 4°C overnight. Gelation was initiated by either moving the solution to 37°C or by injecting it into mice. In in vitro experiments, the concentration of B-CNTF-B varied, with 4 μg / μΐ (i.e. 60 μΜ) used for N2A cell experiments and 8 μg / μΐ (i.e. 120 μΜ) for DRG neuron studies. This variation can be due to the different sensitivities of N2A and DRG cells to neurotrophic factors. In all in vivo experiments, a consistent concentration of 1 μg / μΐ (i.e. 15 μΜ) of B-CNTF-B was used.

[0078] 11. Culture of N2A cells

[0079] N2A cells (ATCC, Cat# CCL-131, RRID:CVCL_0470) were cultured in high glucose DMEM (Gibco, #12100046) supplemented with 10% (v / v) fetal bovine serum (FBS) (Gibco, #10500064) and 1% (v / v) penicillin-streptomycin (Gibco, #15140122). Cells were cultured in a CelCulture CO2incubator (Esco Micro Pte. Ltd.) at 37°C under 5% CO2atmosphere and were regularly passaged every three days. When cells reached 70-80% confluency, they were detached using 2-3 ml of TrypLE (Gibco, #12604021). Subsequently, 10 ml of complete culture medium was added to neutralize the TrypLE and stop the digestion process. Cells were seeded onto blank S-A hydrogels (4 wt%) or B-CNTF-B hydrogels (S-A + 60 μΜ B-CNTF-B) or B-LM-B hydrogels (S-A + 80 μΜ B-LM-B) coated confocal dishes. To coat confocal dishes (SPL, #100350) with functionalized S-A hydrogels for cell culture purposes, 120 μΐ of sonicated S-A solution with or without B-POI-B was gently dispensed onto the middle region of the dish and then left to solidify at 37°C for 1 h.

[0080] Inoculated cells were cultured in an incubator for 1 day or 3 days. Cell viability was assessed using the Live / Dead Viability Kit (Invitrogen, #L3224) according to the manufacturer’s guidelines. Prior to staining, DMEM was removed and no fixation step was performed to ensure that only cells attached to the gel were stained. To visualize the cells, a confocal microscope (Nikon C2+) was used. In each experiment, we randomly captured and analyzed one image under each condition, and a total of five independent experiments were performed for each group (n=5).

[0081] 12. Culture of primary DRG neurons

[0082] Adult DRG neurons were prepared following previously established protocols (see X. Wang, C. Yang, X. Wang, J. Miao, W. Chen, Y. Zhou, Y. Xu, Y. An, A. Cheng, W. Ye, M. Chen, D. Song, X. Yuan, J. Wang, P. Qian, A. R. Wu, Z. Y. Zhang, K. Liu, Neuron 2023, 111, 236). Briefly, adult mice were euthanized and L4-L6 DRGs were excised bilaterally. These excised DRGs were then digested with 0.5 mg / ml collagenase P (Roche, #11249002001) for 1.5 hours at 37 °C. Subsequently, the collagenase-containing medium was replaced with Neurobasal A and the DRGs were gently dissociated by pipetting 20 times with a 1 ml pipette tip. These cells were plated on hydrogel-coated or PDL / laminin-coated confocal dishes.

[0083] Hydrogel-coated dishes were prepared following the aforementioned method. S-A hydrogels (4 wt%), B-LM-B hydrogels (S-A + 80 mM B-LM-B), B-CNTF-B hydrogels (S-A + 120 mM B-CNTF-B) were coated on the wells of the confocal dishes. As a positive control, confocal dishes were initially treated with a solution of poly-D-lysine (PDL, Sigma, #P7886) at a concentration of 100 pg / ml and incubated overnight at 37°C. The next day, the PDL solution was aspirated and the confocal dishes were washed five times with sterile water. Subsequently, a solution of laminin (Gibco, #23017015) at a concentration of 10 pg / ml was added and incubated for 2 hours at 37°C. Finally, the laminin solution was aspirated and the confocal dishes were rinsed with IX PBS. Neurons on different substrates were maintained at 37°C and 5% C02. The culture medium consisted of Neurobasal-A (Gibco, #21103049) supplemented with 2% B27 (Gibco, #17504044) and 1% L-glutamine (Gibco, #25030081).

[0084] 13. Immunostaining and quantification of primary DRG neurons

[0085] After 16 hours of incubation, cells were fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, #30525-89-4) for 10 minutes at room temperature. Permeabilization and blocking were performed for half an hour at room temperature using a solution containing 0.1% Triton X-100 (Sigma-Aldrich) and 4% normal goat serum (Invitrogen, #50062Z). Subsequently, cells were incubated overnight at 4°C with primary antibody (Rb-TUJ1, Biolegend, #802001) diluted with 4% NGS. Cells were then washed three times with lx PBS and incubated with goat anti-rabbit 488 secondary antibody (Invitrogen #A-11008) for 1.5 hours at room temperature. After three washes with lx PBS, cells were kept in PBS at 4°C until further analysis. Images of stained cells were captured using a laser scanning confocal microscope (Leica SP8).

[0086] To determine cell density, three independent experiments (n=3) were performed using primary DRG neurons from three different donors. In each experiment, at least 256 DRG neurons were counted under each condition to ensure statistical rigor. Neurite growth was measured and quantified using the NeuronJ plugin in ImageJ. Three independent experiments were performed. In each experiment, the average neurite length of each DRG neuron was determined by analyzing at least 15 DRG neurons under each condition, which corresponded to 7 immunofluorescence images per culture dish.

[0087] 14. Animals and surgery

[0088] C57BL / 6J mice (Charles River) aged 6-8 weeks were used in this study, in strict compliance with the guidelines set by the Experimental Animal Facility of the Hong Kong University of Science and Technology, with animal license number Dr. Chao Yang: DH / HT&A / 8 / 2 / 2 Pt.9. Prior to surgery, mice were randomly divided into different groups and anesthetized using a combination of ketamine (80 mg / kg) and xylazine (10 mg / kg). An arterial clip was used to expose the eye, and a fine incision was made on the conjunctiva using scissors to expose the optic nerve. Subsequently, the optic nerve was gently injured using forceps (Dumont #2 and #5, Fine Science Tools). Next, 2 μΐ of vitreous body was removed using a Hamilton syringe, and 2 μΐ of vehicle, blank S-A hydrogel, B-CNTF-B solution (15 μΜ), B-CNTF-B hydrogel (S-A + 15 μΜ B-CNTF-B), or CNTF hydrogel (S-A + 15 μΜ CNTF, purchased from Alomone Labs #C-245) was injected into the eye. Post-surgery, eye ointment was applied to prevent infection, and ketoprofen (0.05 ml / kg) was injected for analgesia. Two days prior to sacrificing the animals, 2 μΐ of CTB-FITC (1 μg / μΐ, Sigma-Aldrich, #C1655) was injected intravitreally to label the axons.

[0089] 15. Immunostaining of the retina and optic nerve

[0090] Mice were anesthetized with a lethal dose of ketamine / xylazine, followed by PBS and 4% paraformaldehyde (PFA) perfusion. Prior to the dissection staining procedure, the eyes and optic nerves were excised and fixed in 4% PFA overnight. Whole-mount Tujl (Biolegend, #801202 and #802001) staining of the retina was performed to assess RGC survival. Cryosections of the optic nerve and retina were then performed followed by immunostaining to detect regenerated axons (FITC antibody, Invitrogen, #71-1900), p-STAT3 positive RGCs (Cell Signaling, #9145), Ibal positive microglia (Wako Chemicals USA, #019-19741), CD4 positive helper T cells (BioLegend, #100506), CD68 positive activated macrophages and microglia (Bio-Rad, #MCA1957), and CD45 positive hematopoietic cells (BioLegend, #103101). Specifically, the retinal and optic nerve sections were first cryoprotected in 30% sucrose overnight, then embedded in OCT compound (SAKURA, #4583). The retinal sections were 25 pm thick and the optic nerve sections were 8 pm thick. Subsequently, the dissected retinal tissues or sectioned samples were blocked with 0.1% Triton X-100 in 4% normal goat serum for 30 min, followed by overnight incubation with primary antibodies. After incubation, the samples were washed with PBS and incubated with secondary antibodies for 1 h. Finally, the samples were washed with PBS and imaged using a confocal microscope (Zeiss, LSM 880).

[0091] 16. Quantification

[0092] After staining with cell markers for immune cell quantification, at least 10 retinal section images (20x objective) were taken from each mouse. Ibal, CD68, CD45, or CD4 positive cells were counted in all retinal layers, then normalized by dividing the cell counts by the measured area to determine the cell density per mm2. At least 163 Ibal+, 93 CD68+, 106 CD45+, and 10 CD4+ cells were counted per mouse, with 3-4 mice used for quantification per condition, as indicated in the figure legends. 2

[0093] ​For quantification of p-STAT3 positive RGCs, RGCs showing bright p-STAT3 signal accumulated in the nucleus were identified as positive cells. The positive rate was calculated by dividing the number of p-STAT3 positive RGCs by the total number of Tuj 1 positive RGCs. A minimum of 10 retinal section images (63x objective) were taken, with an average of 73 RGCs analyzed per mouse, to calculate the percentage of p-STAT3 positive RGCs.

[0094] Whole-mount stained retinas were imaged using a confocal microscope to quantify surviving RGCs. Twelve images were taken from different areas of each retina. Tuj 1 positive RGCs were then counted randomly per mm 2 to avoid bias, with a minimum of 212 RGCs counted per mouse. The RGC survival rate (%) after injury for different groups was calculated using the following formula:

[0095] RGC survival rate (%) = (number of surviving RGCs per group / average number of RGCs in the uninjured control group) x 100%

[0096] The average number of RGCs in 6 uninjured mice was (2952 ± 239) / mm 2 .

[0097] To quantify the number of regenerated axons, optic nerves were longitudinally cryosectioned at 8 μm thickness. CTB-FITC signal was amplified by immunostaining with FITC antibody and secondary antibody (goat anti-rabbit, Alexa Fluor TM 555; Invitrogen, #A-21429). Regenerated axons were quantified by capturing five images from each optic nerve. Axon count was determined using the following formula:

[0098] Axon number = πr 2 x (n / d) / 8 μm,

[0099] where r represents the radius of the optic nerve, n / d is the average axon count per average nerve width at the designated counting site, and the section thickness is 8 μm.

[0100] 17. Animal behavior

[0101] Five C57BL / 6 mice received vehicle intravitreally in both eyes, while another 5 mice received S-A hydrogel intravitreally in both eyes. Animal behavior was monitored using overhead video cameras for 10 minutes at 1, 5, and 14 days post-injection. After recording, a blinded observer analyzed the videos to determine if the mice exhibited eye-rubbing behavior or any signs of distress.

[0102] 18. Statistical analysis

[0103] Data are presented as mean ± standard deviation (SD) unless otherwise specified. Sample size (n) for each experiment is detailed in the corresponding figure legends and in the Methods section. Comparisons between two groups were performed using unpaired two-tailed t-test. For comparing results across multiple experimental groups, one-way or two-way ANOVA was performed followed by post-hoc multiple analysis. Statistical significance for all experiments was defined as *P < 0.05 and **P < 0.01. All data analysis was performed using GraphPad Prism 8.0 software.

[0104] Naturally occurring spider silk proteins are known for their robust phase transition from a liquid condensate to a solid fiber upon physical stimuli. The most obvious stimulus from a biological system when it comes to injectable materials for in vivo therapeutic agent delivery is the sudden change in temperature, i.e., from ambient temperature (approximately 23 °C or less than approximately 23 °C) to body temperature (approximately 37 °C). The spidroin-SpyTag protein (4%) after brief sonication undergoes a rapid sol-gel transition upon temperature increase from room temperature to 37 °C. This heat-triggered gelation confers injectability to the system.

[0105] By taking advantage of the SpyTag / SpyCatcher chemistry (see literature: M. A. Calhoun, S. A. Bentil, E. Elliott, J. J. Otero, J. O. Winter, R. B. Dupaix, Beyond Linear Elastic Modulus: Viscoelastic Models for Brain and Brain Mimetic Hydrogels. ACS Biomater. Sci. Eng. 5, 3964-3973 (2019)), spidroin-SpyTag can be covalently modified with various bioactive proteins, thus forming a versatile platform capable of sustained release of therapeutic agents in vivo. To demonstrate the effectiveness of this approach, we performed a proof-of-principle experiment in which spidroin-SpyTag modified with SpyCatcher-fused ciliary neurotrophic factor (SpyCatcher-CNTF) was injected into the vitreous of the mouse eye. This intervention enabled long-term neuroprotection and promoted axon regeneration after optic nerve injury.

[0106] The results of this study highlight the ability of recombinant spider silk proteins to serve as a universal platform for injectable protein therapeutics. The unique properties of this protein, including its heat-induced phase transition behavior and genetically encoded click chemistry features, have tremendous potential in addressing a wide range of biomedical challenges beyond CNS axon regeneration.

[0107] All patents, patent applications, provisional applications, and publications (including all charts) referred to or cited herein are hereby incorporated by reference in their entirety, as if each were incorporated individually. In the event of inconsistencies between the disclosure of the present specification and the disclosures of the herein incorporated reference documents, the present specification shall control.

[0108] The following examples illustrate the process of carrying out the present application. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise indicated.

[0109] Example 1. Preparation and functionalization of new injectable spidroin hydrogels

[0110] We designed and fabricated a recombinant spidroin, which we named spidroin-SpyTag (S-A). This protein solution rapidly transforms from a sol to a gel state at body temperature (37 °C) upon brief sonication, rendering it injectable Figure 1 and Figures 2A-2H ). In addition, the stiffness of the resulting hydrogels can be tailored by adjusting the protein concentration. Hydrogels formed at 40 mg / mL (G’ ~ 1.2 kPa) and 30 mg / mL (G’ ~ 0.5 kPa) exhibit stiffness levels comparable to those of neural tissues (G’ ~ 0.15-1.5 kPa) (see literature: M. A. Calhoun, S. A. Bentil, E. Elliott, J. J. Otero, J. O. Winter, R. B. Dupaix, Beyond Linear Elastic Modulus: Viscoelastic Models for Brain and Brain Mimetic Hydrogels. ACS Biomater. Sci. Eng. 5, 3964-3973 (2019); M. Iwashita, N. Kataoka, K. Toida, Y. Kosodo, Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain. Dev. 141, 3793-3798 (2014)). Furthermore, the resulting S-A hydrogels exhibit an erosion rate of less than 30% Figures 2A-2H ) when exposed to various salt solutions for up to 30 days. We conducted preliminary studies to understand the mechanism of hydrogel formation. Circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM) analysis revealed that hydrogel formation is associated with an increase in the content of β-sheets and the growth and entanglement of protein fibrils Figures 2A-2H .

[0111] Another novel aspect of the present invention is the incorporation of Spy chemistry into the system. The integration of Spy chemistry enables the hydrogels to be functionalized, loaded with proteins with a wide range of biological activities, including CNTF, laminin, insulin-like growth factor 1, and osteopontin Figures 3A-3C ) The covalent immobilization of protein therapeutics on the hydrogel network offers the potential for slow release kinetics, thus having a more sustained effect in vivo compared to traditional physical encapsulation methods. This injectable material system, combined with its ability to be multifunctionally decorated with proteins, establishes a versatile platform for the delivery of protein therapeutics. Due to its wide applicability, the present technology can address a variety of chronic diseases and injuries, not limited to neuronal injuries and neurodegenerative diseases.

[0112] Example 2 Culturing neurons with spidroin-SpyTag hydrogels

[0113] N2A cells were highly viable after 1 day on blank S-A hydrogels. To improve the adhesion and viability of N2A cells, we prepared two new functional hydrogels, B-CNTF-B hydrogels and B-LM-B hydrogels, by coupling the gel precursors S-A with B-CNTF-B and B-LM-B, respectively. After 3 days, the cell viability on blank S-A hydrogels decreased slightly [(92 ± 3) %], while the cell viability on B-CNTF-B hydrogels and B-LM-B hydrogels remained high (about 97 and 98 %, respectively). In addition, the cell density observed on B-CNTF-B hydrogels and B-LM-B hydrogels was up to four and two times higher, respectively, compared to S-A hydrogels, indicating that the immobilization of CNTF and laminin successfully promoted cell growth and proliferation Figures 4A-4C

[0114] We also examined the suitability of these hydrogels for culturing dorsal root ganglion (DRG) neurons, a commonly used cell system for studying axon regeneration (see literature: K. Liu, A. Tedeschi, K. K. Park, Z. He, Neuronal intrinsic mechanisms of axon regeneration. Annu. Rev. Neurosci. 34, 131-152 (2011)). When the coating layer comprised of gel loaded with laminin (i.e., B-LM-B hydrogels), the cell density on this coating layer was comparable to that on PDL / laminin Figures 5A-5D ​), indicating that S-A hydrogels modified with laminin ligands via Spy chemistry are ideal matrices to support the attachment of primary DRG neurons. Moreover, while cells only exhibited limited neurite outgrowth on blank S-A hydrogels, enhanced neurite outgrowth (about 1.3-fold, Figures 5A-5D ), CNTF is a neurotrophic factor that activates JAK-STAT3, thus promoting neurite outgrowth (see literature: V. Pernet, S. Joly, D. Dalkara, N. Jordi, O. Schwarz, F. Christ, D. V. Schaffer, J. G. Flannery, M. E. Schwab, Long-distance axonal regeneration induced by CNTF gene transfer is impaired by axonal misguidance in the injured adult optic nerve. Neurobiol. Dis. 51, 202-213 (2013)). These results together demonstrate that spidroin-SpyTag is a cell-compatible and versatile material system that can be used to encapsulate bioactive proteins, thus enabling the modulation of neuronal behavior in vitro.

[0115] Example 3 In vivo degradability and immunogenicity of spidroin-SpyTag hydrogels

[0116] After intravitreal injection into the eye, S-A hydrogels (3 wt%) exhibited a moderate rate of degradation in vivo; two weeks after injection, histological analysis revealed that there was remaining material in the vitreous that was distributed along the surface of the retina, while after four weeks the gel was completely degraded, while avoiding any significant deformation of the retina Figure 6 ).

[0117] To assess their immunogenicity in the retina, we injected either vehicle or S-A hydrogels directly into the vitreous after optic nerve injury in C57BL / 6 mice. The retina was collected at 1, 5, 14, or 28 days after injection and stained for common immune cell markers to identify specific immune cell populations. At all time points, the number of microglia (Ibal positive) and helper T cells (CD4 positive) was comparable in the presence of S-A hydrogels to that in the case of treatment with vehicle Figures 7A-7D). In addition, we observed that 1 day after injection, there was a greater amount of activated macrophages and microglia (CD68 positive) and hematopoietic cells (CD45 positive) in samples treated with S-A hydrogels. However, no significant differences were found between carrier-treated and S-A hydrogel-treated groups in these two biomarkers of immune cells at 5, 14 and 28 days after injection Figures 7A-7D ).

[0118] We monitored the behavior of the animals after intravitreal injection of S-A hydrogels into the eyes of mice to determine whether these protein hydrogels induced any abnormal behavior, such as eye rubbing. Observations 1, 5 and 14 days after injection showed that mice injected with hydrogels rubbed their eyes very little, comparable to carrier-injected mice. Overall, after injection of S-A hydrogels, animals showed normal behavior without any signs of distress.

[0119] Example 4B - CNTF-B spider silk protein hydrogels for prolonged STAT3 activation

[0120] To assess the ability of S-A hydrogels to prolong CNTF signaling in vivo, we performed optic nerve injury in C57BL / 6 mice and immediately applied carrier (Tris buffer), blank S-A hydrogel, B-CNTF-B solution (15 mM) or B-CNTF-B hydrogel (S-A + 15 mM B-CNTF-B) into the vitreous. Then, we collected retinas at different time points after injection (1, 5, 14 or 28 days) and subjected them to phospho-STAT3 (p-STAT3) immunostaining. Quantitative analysis of p-STAT3 positive RGCs showed that one day after treatment, the carrier induced the least amount of STAT3 phosphorylation in RGCs (<10%), while both B-CNTF-B solution and B-CNTF-B hydrogel led to a significant increase in p-STAT3 levels Figures 8A-8B ). Differences between CNTF delivery methods became prominent over time. Five days after injection, mice treated with B-CNTF-B solution showed a significant decrease in the amount of STAT3 phosphorylation Figures 8A-8B . In contrast, mice that received B-CNTF-B hydrogel treatment maintained a consistently high level of STAT3 phosphorylation, with approximately 60% of RGCs showing p-STAT3 positivity over two weeks (5 and 14 days) Figures 8A-8B . Notably, even four weeks after injection, when hydrogels are considered to have completely degraded, approximately 35% of RGCs in B-CNTF-B hydrogel-treated mice remained p-STAT3 positive, surpassing the other groups Figures 8A-8B), suggesting that spidroin-SpyTag gels can significantly extend the functional duration of CNTF in vivo. Surprisingly, the administration of blank S-A hydrogels slightly increased the level of phosphorylated STAT3 (p-STAT3) in RGCs after one day, which can be attributed to the inherent ability of spider silk proteins to activate JAK-STAT3 Figures 8A-8B ) in general, our research results establish the feasibility of using thermoresponsive protein materials for sustained protein delivery and signaling in vivo. This approach avoids the need for multiple injections when using protein solutions and reduces the risk of over-activation of certain signaling pathways, which is a potential complication associated with viral delivery.

[0121] Example 5B-CNTF-B hydrogels enhance neuroprotection and axon regeneration

[0122] We employed a model of optic nerve injury to evaluate the efficacy of spidroin-SpyTag hydrogels in promoting neuroprotection in vivo. The retina and optic nerve were collected at 2 and 4 weeks after injury, and CTB-FITC was injected intravitreally 2 days before sacrifice to label RGC axons. RGC survival was quantified using whole-mount Tujl staining. After two weeks, neither treatment with B-CNTF-B solution nor treatment with S-A hydrogels significantly protected RGCs. However, B-CNTF-B hydrogels significantly improved RGC survival Figures 9A-9D ) after two weeks. After four weeks, when the gels were completely degraded, RGC survival decreased but was still significantly higher than that under other conditions. These findings demonstrate the ability of B-CNTF-B hydrogels to elicit sustained neuroprotection in vivo.

[0123] Adult CNS injury often leads to permanent motor and sensory dysfunction. Among numerous relevant factors, CNS axon regeneration is considered crucial for functional recovery [see references: K. Liu, A. Tedeschi, K. K. Park, Z. He, Neuronal intrinsic mechanisms of axon regeneration. Annu. Rev. Neurosci. 34, 131-152 (2011); Z. He, Y. Jin, Intrinsic Control of Axon Regeneration. Neuron 90, 437-451 (2016)]. We investigated the effect of B-CNTF-B hydrogels on axon regeneration within the injured optic nerve. In the control group treated with Tris buffer, there was almost no axon regeneration at the injury site two weeks after injury Figures 10A-10D). Treatment with B-CNTF-B solution or treatment with blank S-A hydrogel modestly improved axon regeneration, with about 530 and about 650 axons extending beyond 0.2 mm, respectively. Without being bound by any theory, this can be due to short-term activation of STAT3 by B-CNTF-B solution or weak activation of STAT3 by blank hydrogel Figures 8A-8B ). Notably, B-CNTF-B hydrogel significantly enhanced axon regeneration, with over 1500 axons regenerated at the lesion site and with lengths beyond 0.2 mm, and some even beyond 1.5 mm Figures 10A-10D ) At the 2-week time point, B-CNTF-B hydrogel was comparable to adenovirus-mediated CNTF gene delivery in promoting axon regeneration [see C. Yang, X. Wang, J. Wang, X. Wang, W. Chen, N. Lu, S. Siniossoglou, Z. Yao, K. Liu, Rewiring Neuronal Glycerolipid Metabolism Determines the Extent of Axon Regeneration. Neuron 105, 276-292.e5 (2020); X. Wang, C. Yang, X. Wang, J. Miao, W. Chen, Y. Zhou, Y. Xu, Y. An, A. Cheng, W. Ye, M. Chen, D. Song, X. Yuan, J. Wang, P. Qian, A. R. Wu, Z. Y. Zhang, K. Liu, Driving axon regeneration by orchestrating neuronal and non-neuronal innate immune responses via the IFNy-cGAS-STING axis. Neuron 111, 236-255.e7 (2023)]. At 4 weeks post-lesion, the effect of CNTF solution was diminished Figures 10A-10D ) In contrast, axon regeneration was still robust in mice treated with B-CNTF-B hydrogel, with nearly 1400 axons regenerated beyond 0.2 mm at the lesion site, and some axons regenerated up to 2 mm distal to the lesion Figures 10A-10D ) Interestingly, consistent axon regeneration was observed in the optic nerve treated with blank hydrogel at both 2 and 4 weeks, albeit with modest magnitude, suggesting that the recombinant spider silk protein has intrinsic ability to promote axon regeneration. These results together demonstrate the feasibility of using spidroin-SpyTag as a simple, sustained protein delivery system to promote optic nerve axon regeneration.

[0124] Covalent immobilization is critical for maintaining CNTF activity in vivo

[0125] To further elucidate the role of covalent immobilization in CNTF delivery, we used a mouse model of optic nerve injury and compared S-A hydrogels physically mixed with 15 mM CNTF (a commercially available CNTF lacking the SpyCatcher (B) domain) to covalent B-CNTF-B hydrogels. While both systems elicited high levels of p-STAT3 at 1 and 5 days post-injection, there was a significant difference in their STAT3 activation over time ( Figures 11A-11F ). At 14 days post-injection, over 60% of RGCs were p-STAT3 positive in animals treated with covalently bound B-CNTF-B hydrogels, compared to only 31% in animals receiving physically mixed CNTF hydrogels. This difference was amplified over time; at 28 days post-injection, 41% of RGCs remained p-STAT3 positive in mice treated with B-CNTF-B hydrogels, three times the level observed in mice treated with CNTF hydrogels (approximately 13%). Moreover, administration of B-CNTF-B hydrogels significantly improved RGC survival and axon regeneration at 14 and 28 days post-injection compared to CNTF hydrogels ( Figures 11A-11F ). These results demonstrate the clear advantage of covalent immobilization in maintaining neurotrophic factor activity.

[0126] It is to be understood that the embodiments and implementations described herein are for illustrative purposes only and that various modifications or changes in light thereof will be obvious to persons skilled in the art and are intended to be included within the spirit and purview of this application and scope of the appended claims. Furthermore, any element or limitations of any application or implementation disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) of any other application or implementation disclosed herein, and all such combinations are within the scope of the present application and are intended to be embraced by the claims.

[0127] Exemplary implementations

[0128] Implementation 1. An injectable protein delivery system comprising a recombinant spidroin-SpyTag, wherein the spidroin-SpyTag is in the form of an injectable hydrogel, and wherein the spidroin-SpyTag is covalently coupled to one or more biologically active agents.

[0129] Implementation 2. The system of implementation 1, wherein the one or more biologically active agents comprises one or more protein therapeutics.

[0130] Embodiment 3. The system of embodiment 2, wherein the one or more protein therapeutic agents comprise one or more of ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), or osteopontin (OPN).

[0131] Embodiment 4. A method for delivering a therapeutic agent to a central nervous system (CNS) targeted tissue, the method comprising administering an effective amount of the injectable protein delivery system of embodiment 1 to a subject’s targeted CNS tissue.

[0132] Embodiment 5. The method of embodiment 4, wherein the subject is a mammal.

[0133] Embodiment 6. The method of embodiment 5, wherein the mammal is a mouse or a human.

[0134] Embodiment 7. The method of any one of embodiments 4-6, wherein the spidroin-SpyTag transitions from a sol state to a gel state with injectability upon brief sonication and incubation at a temperature of about 37 °C.

[0135] Embodiment 8. The method of any one of embodiments 4-7, wherein the spidroin-SpyTag is covalently coupled to one or more protein therapeutic agents.

[0136] Embodiment 9. The method of embodiment 8, wherein the one or more protein therapeutic agents comprise one or more of ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN).

[0137] Embodiment 10. The method of any one of embodiments 4-9, wherein an effective amount of the hydrogel comprising spidroin-SpyTag covalently coupled to one or more bioactive agents is administered to a subject by intravitreal, intrathecal, intramuscular, intradermal, intracranial, intraspinal, or epidural injection.

[0138] Embodiment 11. The method of embodiment 10, wherein the subject is affected by a CNS condition or injury.

[0139] Embodiment 12. The method of embodiment 11, wherein the CNS condition or injury comprises a spinal cord injury, traumatic brain injury, stroke, glaucoma, muscle dystrophy, muscle hypertrophy, metabolic myopathy, or muscle paralysis.

[0140] Embodiment 13. The method of embodiment 11 or 12, wherein the site of injury includes the optic nerve and retinal tissue.

[0141] Embodiment 14. The method of embodiment 12, wherein the hydrogel is injected into the site of CNS injury to promote neuroprotection and axonal regeneration.

[0142] Embodiment 15. The method of embodiment 13, wherein the hydrogel is injected into the injury site to promote neuroprotection and axonal regeneration.

[0143] Embodiment 16. The method of any one of embodiments 4 to 15, wherein the hydrogel releases CNTF in the injury site to promote axonal regeneration by prolonging STAT3 signaling.

Claims

1. An injectable protein delivery system comprising a recombinant spider silk protein spidroin-SpyTag, wherein the spidroin-SpyTag is in an injectable hydrogel form, and wherein the spidroin-SpyTag is covalently coupled to one or more biologically active agents.

2. The system of claim 1, wherein the one or more biologically active agents comprise one or more protein therapeutics.

3. The system of claim 2, wherein the one or more protein therapeutics comprise one or more of ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN).

4. A method for delivering a therapeutic agent to a central nervous system (CNS) targeted tissue, the method comprising administering to a target CNS tissue of a subject an effective amount of the injectable protein delivery system of claim 1.

5. The method of claim 4, wherein the subject is a mammal.

6. The method of claim 5, wherein the mammal is a mouse or a human.

7. The method of claim 4, wherein the spidroin-SpyTag is converted from a sol state to a gel state upon brief sonication and incubation at a temperature of about 37 °C, with injectability.

8. The method of claim 7, wherein the spidroin-SpyTag is covalently coupled to one or more protein therapeutics.

9. The method of claim 8, wherein the one or more protein therapeutics comprise one or more of ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF1), laminin, or osteopontin (OPN).

10. The method of claim 4, wherein an effective amount of the hydrogel comprising spidroin-SpyTag covalently coupled to one or more protein therapeutics is administered to the subject by intravitreal, intrathecal, intramuscular, intradermal, intracranial, intraspinal, or epidural injection.

11. The method of claim 10, wherein the subject is affected by a CNS disorder or injury.

12. The method of claim 11, wherein the CNS disorder or injury comprises a spinal cord injury, traumatic brain injury, stroke, glaucoma, muscle dystrophy, muscle hypertrophy, metabolic myopathy, or muscle paralysis.

13. The method of claim 12, wherein the site of injury comprises optic nerve and retinal tissue.

14. The method of claim 12, wherein the hydrogel is injected in the site of CNS injury to promote neuroprotection and axonal regeneration.

15. The method of claim 13, wherein the hydrogel is injected in the site of injury to promote neuroprotection and axonal regeneration.

16. The method of claim 12, wherein the hydrogel releases CNTF in the site of injury to promote axonal regeneration by prolonging STAT3 signaling.

17. The method of claim 13, wherein the hydrogel releases CNTF in the lesion site to promote axonal regeneration by prolonging STAT3 signaling.

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