WO2024155961A1 - Viral vector functionalized core-shell scaffold - Google Patents

Abstract

Scaffolds, including Nerve Guidance Scaffolds (NGSs), and methods of making and using scaffolds are provided. In some embodiments, an NGS is used for the connecting damaged nerves together through surgical repair, optionally aided by expression of proteins from adeno-associated viral (AAV) vectors. In some embodiments, NGSs bridge two or more nerve endings to fix damaged nerves using a singular scaffold; methods of making the NGSs are also provided.

Description

Docket No.5200.2392-001 (INV-23052) Viral Vector Functionalized Core-Shell Scaffold RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 63/439,946, filed on January 19, 2023. The entire teachings of the above application are incorporated herein by reference. GOVERNMENT SUPPORT [0002] This invention was made with government support under Grant No. 80NSSC18K0305 and 80NSSC21K0039 awarded by the National Aeronautics and Space Administration (NASA). The government has certain rights in the invention. BACKGROUND [0003] An estimated 20 million Americans suffer from peripheral nerve deficits. Discontinuities of peripheral nerves lead to motor and sensory deficits that severely impact patient quality of life. Current treatments often fail to restore meaningful function, and healthcare costs associated with treatment and treatment failures are enormous. Surgical repair of peripheral nerve gaps often requires interposition nerve grafting to guide and support regenerating axons. Limitations of nerve autografting include the need for a second operative site, limited availability in cases of polytrauma, and donor site morbidity. [0004] Further, success of nerve grafting procedures can be length-dependent: axon regeneration over distances exceeding several centimeters is hampered by gradual downregulation of pro-regenerative transcription factors within axotomized neurons and Schwann cells. Accordingly, functional recovery following long nerve grafting procedures is poor, yielding long-term patient disability and financial burden to society. [0005] There is a need for therapies that address the current limitations of grafting, including nerve grafting (e.g., autografting). SUMMARY [0006] One embodiment provides a scaffold, e.g., a scaffold for insertion into a tissue gap, the scaffold comprising: one or more porous materials, one or more polymers, and one or more gene delivery vehicles. In some aspects of an embodiment, the gene delivery vehicle - 1 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) is a vector, for example, a viral vector, e.g., an adeno-associated viral (AAV) vector. In some aspects of an embodiment, the scaffold is a nerve guidance scaffold (NGS). In another aspect of an embodiment, the scaffold comprises a solid cross section. The solid cross section may have one or more tubular holes throughout its length to enable passthrough of tissue in a tissue gap to reconnect two or more ends of damaged tissue in some embodiments. In one aspect, the scaffold defines one or more tubular holes within a boundary of the scaffold throughout its length. [0007] In another aspect of an embodiment, the scaffold is freeze-cast. In some aspects of embodiments, at least one porous material comprises one or more cores, one or more shells, or any combination thereof. In an aspect of some embodiments, at least one shell or at least one core is longitudinally porous. In other aspects of some embodiments, at least one shell is radially porous. [0008] In some embodiments, at least one polymer is a biopolymer selected from the group of chitosan, chitin, collagen, gelatin, cellulose, alginate, agar, agarose, soy protein, hyaluronic acid, elastin, silk, fibrin, or any combination thereof. In some other embodiments, at least one polymer comprises at least one polymer selected from the group of polylactic acid (PLA), polyglutamic acid (PGA), poly lactic-co-glycolic acid (PLGA), polycaprolactone, polydioxanone, solubilized basement membrane matrix, or any combination thereof. [0009] In other embodiments, one or more freeze-cast chitosan-based shells are adventitia-like, one or more collagen-based porous cores are endoneurium-like, or one or more freeze-cast chitosan-based shells are endoneurium-like and one or more of the collagen- based porous cores are adventitia-like. In some aspects of embodiments, one or more porous cores comprise collagen. In other aspects of some embodiments, one or more porous cores comprise a collagen-nanocellulose composite (CNC). In other aspects of some embodiments, one or more porous cores comprise chitosan. In some aspects of embodiments, one or more porous cores comprise a chitosan-nanocellulose composite. In aspects of some embodiments, one or more cores comprise about 1-10% w/v collagen. In other aspects of some embodiments, one or more shells comprise about 1-10% w/v chitosan. In other aspects of some embodiments, one or more cores comprise about 1-10% w/v collagen-nanocellulose. Other aspects of some embodiments include one or more cores comprise about 1-10% w/v chitosan-nanocellulose. - 2 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) [0010] In another embodiment, one or more cores are coated with laminin. In some aspects of embodiments, one or more cores are coated with about 1-100 μg/mL of laminin. [0011] In some other aspects of embodiments, one or more cores or one or more shells has non-directional porosity. In other aspects of embodiments, the scaffold comprises radially oriented porosity, longitudinally oriented porosity, non-directional porosity, or a combination thereof. In some aspects of embodiments, the scaffold, or an element therein, is radially porous, longitudinally porous, non-directionally porous, or a combination thereof. [0012] In another embodiment, the scaffold comprises a vector, e.g., an AAV vector, which comprises a concentration gradient in the scaffold. [0013] In another aspect, a method of regenerating damaged nerves is provided, said method comprising attaching each nerve end to a scaffold of an embodiment, thereby connecting them and promoting nerve regeneration. In aspects of embodiments, a method of inhibiting nerve regeneration in a peripheral nervous system in a subject in need thereof is provided, said method comprising administering the scaffold of some embodiments to the subject, thereby inhibiting nerve regeneration. In other aspects of embodiments, in the damaged nerves are peripheral nerves. A method of treating a nervous system deficit in a subject in need thereof is provided, said method comprising attaching the scaffold of some aspects of embodiments to damaged nerve ends of the subject to connect damaged nerve ends. [0014] In some aspects of embodiments, the nervous system deficit is at least one of: a neuroma, amyotrophic lateral sclerosis (ALS), carpal tunnel syndrome, Guillain-Barre syndrome, peripheral neuropathy, a peripheral nerve injury, congenital nerve absence, peripheral nerve pain, or spinal cord repair. [0015] Another embodiment describes a method of making a nerve guidance scaffold (NGS), the method comprising: forming a core by either freeze casting a collagen, collagen- cellulose, chitosan, or chitosan-nanocellulose solution/slurry in a mold, or freeze extruding a collagen, collagen-cellulose, chitosan, or chitosan-nanocellulose solution, freeze drying the core, freeze casting a chitosan solution in a mold, thereby forming a shell, freeze drying the shell, and sliding the shell over the core, thereby assembling the NGS. In aspects of some embodiments, the NGS is sterilized prior to use in a subject. In other aspects of some embodiments, the core of the NGS is coated with laminin prior to sliding the shell over the - 3 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) core. In some aspects of embodiments, the NGS is compatible with a vector, e.g., a viral vector, e.g., an adeno-associate viral (AAV) vector, wherein the NGS is dipped in an AAV vector solution, injected directly into the core of the NGS, or freeze cast together in either the core solution, shell solution, or both solutions. In other aspects of some embodiments, multiple cores are crosslinked together, thereby forming a longer core scaffold. [0016] In some aspects, a method of inhibiting disorganized growth of a nerve, said method comprising contacting the nerve with the scaffold of other embodiments, is provided. In some aspects of embodiments, a method of mediating nerve function is provided, said method comprising contacting the nerve with the scaffold of other embodiments. In some embodiments, contacting the nerve with the scaffold promotes ingrowth of axonal and Schwann cells through the scaffold core and shell. [0017] In some embodiments, a ureteral stent is provided. In some embodiments, the stent comprises a shell of some embodiments, or a scaffold comprising a shell, wherein the shell comprises chitosan and further wherein the shell is leakproof. In other embodiments, a method for regenerating blood vessels is provided, the method comprising implantation of a scaffold of some embodiments connecting two or more blood vessels in a subject in need thereof. [0018] In some embodiments, the vector, e.g., the AAV vector, encodes exogenous proteins. [0019] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, e.g., the method comprising a) forming a core by either freeze casting a collagen, b) freeze drying the core, c) optionally cross-linking the core; if crosslinking in solvent, cross-linking, washing, d) optionally core-coating with laminin, e) optionally flash-freezing the core for biopsy punching, f) optionally biopsy punching the core to remove a layer formed during freeze casting, g) optionally flash freezing and freeze drying the core, and h) optionally sterilizing the core of the scaffold. [0020] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) freeze casting a collagen, collagen- cellulose, chitosan, or chitosan-nanocellulose solution in a mold, thereby forming a shell, b) freeze-drying the shell, c) neutralizing the shell, d) optionally flash freezing and freeze-drying the shell, and e) optionally sterilizing the shell. - 4 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) [0021] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) freeze extruding a collagen, collagen- cellulose, chitosan, or chitosan-nanocellulose solution, thereby forming a core, b) freeze- drying the core, and c) optionally sterilizing the core. [0022] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) freeze extruding a collagen, collagen- cellulose, chitosan, or chitosan-nanocellulose solution, thereby forming a shell, b) freeze- drying the shell, and c) optionally sterilizing the shell. [0023] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) a core of some other embodiments, b) a shell of some other embodiments, c) wetting the core and shell, sliding the shell over the core, thereby assembling the scaffold, d) optionally flash freezing and freeze drying, and e) optionally sterilizing the scaffold. [0024] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) spray coating a dry core of some embodiments to form a shell, b) air drying the spray-coated core, c) optionally neutralizing the spray-coated core in a case of chitosan or chitosan-nanocellulose, d) optionally crosslinking the spray-coated core in a case of collagen or collagen-nanocellulose, and e) optionally sterilizing the spray coated core. [0025] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) brush coating a dry core of some embodiments to form a shell, b) freezing the brush-coated core, c) freeze drying the brush- coated core, d) optionally neutralizing the brush-coated core in a case of chitosan or chitosan- nanocellulose, e) optionally crosslinking the brush-coated core in a case of collagen or collagen-nanocellulose, and f) optionally sterilizing the brush coated core. [0026] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) spray freezing a shell of some embodiments onto a core of some embodiments, b) optionally acclimatizing the core coated by spray freezing to about -80 ºC, c) freeze drying the core coated by spray freezing, d) optionally neutralizing the core coated by spray freezing in a case of chitosan or chitosan- nanocellulose, e) optionally crosslinking the core coated by spray freezing in a case of - 5 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) collagen or collagen-nanocellulose, and f) optionally sterilizing the core coated by spray freezing. [0027] In another embodiment, a method of making a scaffold, e.g., a nerve guidance scaffold (NGS), is provided, the method comprising a) coaxially freeze extruding a collagen, collagen-cellulose, chitosan, or chitosan-nanocellulose solution, thereby forming a core-shell assembly b) freeze drying the core-shell assembly, c) optionally neutralizing the core-shell assembly in a case of chitosan or chitosan-nanocellulose, d) optionally crosslinking the core- shell assembly in a case of collagen or collagen-nanocellulose, and e) optionally sterilizing the core-shell assembly. [0028] In another embodiment, a scaffold of some embodiments with or without one or more gene delivery vehicles is provided, e.g., for in vitro or in vivo follicle culture. In other aspects of embodiments, a scaffold with or without one or more gene delivery vehicles is provided for in vitro or in vivo ovarian tissue culture. In aspects of an embodiment, a scaffold with or without one or more gene delivery vehicles is provided, e.g., to serve as in vitro or in vivo seminiferous tubule construct. [0029] In some aspects, the gene delivery vehicle comprises a viral or non-viral vector. [0030] In an embodiment, a scaffold comprises a shell and a core, e.g., a freeze-cast chitosan-based shell and a freeze-cast collagen-based core, wherein the shell, the core, or both the shell and the core are porous. [0031] Another embodiment provides a scaffold for insertion into a tissue gap, the scaffold comprising: one or more porous materials and one or more polymers. [0032] In some embodiments, the scaffold does not comprise a gene delivery vehicle. BRIEF DESCRIPTION OF THE DRAWINGS [0033] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. [0034] FIG.1 is a schematic summarizing a research platform for study of viral vector functionalization of bioengineered nerve guidance scaffolds and their applications. [0035] FIG.2 is a schematic for a rapid spatial protein profiling of implantable biomaterials and host tissue response. - 6 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) [0036] FIG.3A is a scanning electron microscopy image of an example embodiment of a nerve guidance scaffold (NGS) core-shell structure. [0037] FIG.3B is a schematic representation of an example embodiment of a rodent sciatic nerve model repaired with an adeno-associated virus (AAV) vector impregnated with NGS. [0038] FIG.3C is an intraoperative photograph of an example embodiment of interposition gap repair of mouse sciatic nerve with a core-shell NGS. [0039] FIG.4 is an example embodiment of a NGS performance assessment in a Sox10- Venus x Thy1-CFP mouse model. Scaffold explants harvested four weeks following interposition repair of a sciatic nerve defect were imaged transversely whole-mounted and axially following vibratome-sectioning using multiphoton microscopy. Varying degrees of Schwann cell and axonal ingrowth into collagen-based scaffold cores were observed. [0040] FIG.5A is a confocal microscopy image of an example embodiment of chitosan shells and collagen cores soaked in a vehicle control for AAV vector functionalization of core-shell NGS. [0041] FIG.5B is a confocal microscopy image of an example embodiment of chitosan shells and collagen cores soaked in AAV8-CAG-tdTomato vector solution. DETAILED DESCRIPTION [0042] A description of example embodiments follows. [0043] Congenital absence or injury to peripheral nerves yields devastating consequences. As peripheral neurons retain their ability to regenerate axons and neurotize distant targets, surgery may be employed to re-establish critical sensory and motor functions. Where gaps exist between nerve trunks and vital tissue, the gold standard for repair entails nerve autografting, wherein segments of noncritical peripheral nerve trunks are harvested for use in defect bridging. [0044] Success of nerve grafting procedures is length-dependent: axon regeneration over distances exceeding several centimeters is hampered by gradual downregulation of pro- regenerative transcription factors within axotomized neurons and Schwann cells. Accordingly, functional recovery following long nerve grafting procedures is poor, yielding long-term patient disability and financial burden to society. There is a need for methods and - 7 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) devices, e.g., bioengineered devices, that address the current limitations of nerve autografting. [0045] In some embodiments, the invention provides targeted viral vector gene therapy and bioengineering techniques to bring to market an off-the-shelf, acellular, functionalized nerve guidance scaffold, whose clinical performance rivals that of nerve autografts over short and long-distances. In some embodiments, nerve guidance scaffolds are impregnated with adeno-associated viral (AAV) vectors encoding growth factors, e.g., for neural regeneration. Such scaffolds can yield performance similar to or superior to nerve grafts, over long regeneration distances. [0046] Example aspects of some embodiments include nerve guidance scaffolds comprising a mesoneurium/epineurium-like chitosan shell that bridges the defect and provides protection from fibrous tissue infiltration while permitting nutrients, oxygen, and waste transfer, and a laminin-coated endoneurium-like collagen/cellulose nanofiber or chitosan-based core with aligned pore morphology to support and direct axonal extension across a nerve gap. [0047] Peripheral nerve injuries frequently yield loss of vital motor and sensory functions. Microsurgical repair of nerve injuries using interposition grafts is often indicated to re-establish neural input to critical distal targets. Nerve autografts are the gold standard for use in bridging peripheral nerve gaps, but their harvest yields donor site functional loss, and their performance is suboptimal over long regeneration distances. There is a critical need for high-performing off-the-shelf nerve autograft alternatives. Herein, in some embodiments, viral vector gene therapy is used to functionalize novel bioengineered nerve guidance scaffolds, e.g., scaffolds comprising freeze-cast chitosan-based cylindrical shells and discrete collagen-based porous cores. Sciatic nerves of Sox10-Venus / Thy1-CFPr (expressing specific fluorescent reporter proteins in Schwann cells and axons) were transected and repaired with nerve guidance scaffolds impregnated with adeno-associated virus (AAV) serotype 8 (AAV.8 vectors) encoding a distinct reporter transgene. Repair sites were harvested after four weeks and imaged by multiphoton and confocal microscopy to assess axonal and Schwann cell ingrowth and transgene expression. Cores comprising laminin- coated collagen cellulose nanofiber demonstrated superior axonal and Schwann cell ingrowth compared to cores comprising bovine and rat collagen. Transgene expression within scaffolds - 8 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) was confirmed, demonstrating proof-of-concept of AAV vector-functionalization of novel core-shell nerve guidance scaffolds for peripheral nerve repair as shown in FIG.4. [0048] An estimated 20 million Americans suffer from peripheral nerve deficits (1-5). Discontinuities of peripheral nerves lead to motor and sensory deficits that severely impact patient quality of life (6-8). Current treatments often fail to restore meaningful function (9- 11), and healthcare costs associated with treatment and treatment failures are enormous (5, 6, 12). Surgical repair of peripheral nerve gaps often requires interposition nerve grafting to guide and support regenerating axons. Limitations of nerve autografting include the need for a second operative site, limited availability in cases of polytrauma, and donor site morbidity. To circumvent these limitations, several biocompatible nerve autograft alternatives have been developed and brought to market. U.S. Food and Drug Administration approved nerve graft alternatives include acellular nerve allografts (Axogen Avance) or biocompatible bioengineered constructs (e.g., NeuroFlex™, NeuroGen®, Neurolac®, NeuroMatrix™, Neurotube®, and SaluBridge™ (13) made from collagen, chitosan, polyglycolic acid (PGA), or PCL substrates (14). Nerve graft alternatives may demonstrate regenerative performance comparable to autografts over short distances but are generally contraindicated for repair of critical nerves or nerve gaps exceeding 3-4 centimeters (cm). [0049] Though superior to current alternatives, interposition nerve autograft repair of long nerve gaps often yields suboptimal functional outcomes. Peripheral nerve regeneration over long distances is hampered by progressive downregulation of pro-regenerative factors within Schwann cells (SC) lacking axonal contact for prolonged periods (15-21). Schwann cell activity regulates axonal regeneration (22). Upon denervation, SCs undergo phenotype switching with downregulation of genes supporting myelination and upregulation of genes encoding trophic factors that support axonal extension, including nerve growth (NGF), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) (22- 26). The regeneration-supportive SC phenotype is time-dependent. In murine models, denervation periods exceeding two months yield drastic reductions in nerve graft support for regeneration (21, 22, 27). Owing to the slow rate of axonal regeneration of approximately 1 mm per day in humans (28), SCs in the distal portions of long nerve grafts lack axonal contact for long periods, and gradually become less supportive of regeneration (26, 29-34). Suboptimal functional recovery following long-gap nerve autograft repair occurs secondary to axonal growth arrest, yielding inadequate neurotization of distal targets. Therapies targeted - 9 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) at maintaining SC repair phenotype during prolonged denervation carry potential to enhance clinical outcomes in peripheral nerve repair. [0050] Over the past three decades, advances in microfabrication have enabled design and production of nerve guidance scaffolds (NGSs) mirroring the internal architecture of nerve grafts. Advancements in NGS production have also enabled local delivery of nerve growth factors and supporting cells to promote an appropriate environment for nerve regeneration (14). Though incorporation of growth factors implicated in Schwann cell migration and proliferation into NGSs has demonstrated potential to enhance their performance (35), challenges associated with factor encapsulation and timed release of factors over a time frame relevant to nerve regeneration over long distances has prevented successful commercialization of such approaches. Though SC seeding of nerve scaffolds has proven effective at enhancing axonal extension over longer distances (36-43), cell seeding of bioengineered constructs remains commercially infeasible due to the costs and complexities associated with autogenous cell isolation and expansion, and the risks of immunosuppression therapy where allogenic cells are used. [0051] Gene therapy functionalization of NGSs represents an unexplored avenue for realizing the potential of bioengineered constructs in surgery. Progress in viral vector gene therapy has yielded means for targeted biologic functionalization of bioengineered constructs. Direction injection of peripheral nerves with viral vectors targeting SCs carrying genes known to promote neurite outgrowth has potential to enhance neural regeneration (44-46). AAV vectors are an ideal vehicle for drug delivery to peripheral nerves. Current AAV vectors have a transgene packaging capacity of ~4.7 kb (47, 48), sufficient for carrying genes encoding fluorescent reporters and growth factors critical to neural regeneration. In contrast to lentiviruses, AAV vectors remain primarily episomal, posing minimal risk of insertional mutagenesis (49, 50). In contrast to adenovirus vectors, AAV vectors pose little risk to humans, requiring only biosafety level 1 precautions for injection, and elicit a much smaller immune response (51). Prior work has demonstrated negligible serum cytokine responses to systemic infection of a murine model with the AAV vector serotypes proposed for use in embodiments herein (52, 53). [0052] In some example embodiments, the NGS would require no immunosuppression, carry no risk of infectious disease transmission, and be cost-effective. It would completely biodegrade over an appropriate length of time and carry negligible risk of mutagenesis. - 10 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) Biological functionalization of NGSs is a feature to enhance their performance. Though early approaches to NGS functionalization largely focused on incorporation of exogenous growth factors impacting neurite outgrowth (Rich KM, Alexander TD, Pryor JC, Hollowell JP. Nerve growth factor enhances regeneration through silicone chambers. Experimental neurology. 1989;105(2):162-70. Epub 1989/08/01. PubMed PMID: 2753116.; Pfister LA, Papaloizos M, Merkle HP, Gander B. Nerve conduits and growth factor delivery in peripheral nerve repair. J Peripher Nerv Syst.2007;12(2):65-82. Epub 2007/06/15. doi: 10.1111/j.1529- 8027.2007.00125.x. PubMed PMID: 17565531.; Lee AC, Yu VM, Lowe JB, 3rd, Brenner MJ, Hunter DA, Mackinnon SE, Sakiyama-Elbert SE. Controlled release of nerve growth factor enhances sciatic nerve regeneration. Experimental neurology.2003;184(1):295-303. Epub 2003/11/26. PubMed PMID: 14637100.), evidence suggests that the improved performance of nerve autografts, as compared to commercialized products for nerve repair, is chiefly explained by the presence of regeneration-supportive Schwann cells (Chen YY, McDonald D, Cheng C, Magnowski B, Durand J, Zochodne DW. Axon and Schwann cell partnership during nerve regrowth. Journal of neuropathology and experimental neurology. 2005;64(7):613-22. Epub 2005/07/27. PubMed PMID: 16042313.; Colen KL, Choi M, Chiu DT. Nerve grafts and conduits. Plastic & Reconstructive Surgery.2009;124(6 Suppl):e386- 94. doi: PubMed PMID: 19952706.). Accordingly, senescence of ingrowing Schwann cells within NGSs is thought responsible for axonal growth arrest observed over distances longer than a few centimeters (30, 17, 36, Chen YY, McDonald D, Cheng C, Magnowski B, Durand J, Zochodne DW. Axon and Schwann cell partnership during nerve regrowth. Journal of neuropathology and experimental neurology.2005;64(7):613-22. Epub 2005/07/27. PubMed PMID: 16042313.; Iijima Y, Ajiki T, Murayama A, Takeshita K. Effect of Artificial Nerve Conduit Vascularization on Peripheral Nerve in a Necrotic Bed. Plastic and Reconstructive Surgery Global Open.2016;4(3):e665. doi: 10.1097/GOX.0000000000000652. PubMed PMID: PMC4874309.; Cattin A-L, Burden Jemima J, Van Emmenis L, Mackenzie Francesca E, Hoving Julian J, Garcia Calavia N, Guo Y, McLaughlin M, Rosenberg Laura H, Quereda V, Jamecna D, Napoli I, Parrinello S, Enver T, Ruhrberg C, Lloyd Alison C. Macrophage- Induced Blood Vessels 2015;162(5):1127-39. doi: 10.1016/j.cell.2015.07.021. PubMed PMID: PMC4553238. Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves. Cell.). Though Schwann cell seeding has proven effective at enhancing axonal extension along bioengineered NGSs (36-43), such approaches remain commercially infeasible due to - 11 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) the costs and complexity associated with autogenous Schwann cell isolation and expansion, and the risks of immunosuppression therapy necessary, where allogenic Schwann cells are used. [0053] Though incorporation of growth factors implicated in Schwann cell migration and proliferation into NGSs has proven effective (35), this approach has yet to be commercialized owing to challenges associated with factor encapsulation and timed release. [0054] Some embodiments of the invention employ adeno-associated virus (AAV) vector gene delivery, e.g., to functionalize NGSs to achieve low-cost, long-term, tunable NGSs with potential for timed release of growth factors within the NGS environment, circumventing the costs and complexities associated with incorporation of live cells and exogenous growth factors. Gene therapy functionalization of NGSs is an avenue for realizing the potential of bioengineered constructs in surgery. [0055] Direct injection of peripheral nerves with viral vectors targeting Schwann cells carrying genes known to promote neurite outgrowth has already shown promise of enhancing neural regeneration (44-46). Herein, some embodiments involve optimal AAV vector subtypes, transgenes, and dosing for biological functionalization of bioengineered NGSs using a novel fluorescent reporter mouse platform for high-throughput study of neural regeneration. In some embodiments, examples of subtypes include AAV8 and AAV1. Examples of transgenes include hormones, such as insulin growth hormone 1 (IGF1), and growth factors, such as nerve growth factor (NGF), with examples of dosing including about 5-10 μL of 10¹² - 10¹³ gc/mL. [0056] In some embodiments, AAV vectors are a vehicle for drug delivery to peripheral nerves. Current AAV vectors have a transgene packaging capacity ~4.7 kb (47, 48), sufficient for packaging genes encoding fluorescent reporters and growth factors critical to neural regeneration. In contrast to lentiviruses, AAV vectors remain primarily episomal, posing minimal risk of insertional mutagenesis (49, 50). In contrast to adenovirus (AV) vectors, AAV vectors pose little risk to humans, requiring only biosafety level 1 precautions for injection, and elicit a much smaller immune response (51). Prior work has demonstrated negligible serum cytokine responses to systemic infection of a murine model with the AAV vector serotypes in some embodiments herein (52, 53). [0057] AAV vectors demonstrate serotype-dependent tropism for a wide variety of cell types, including dividing and non-dividing cells, with onset of transgene expression in vivo - 12 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) occurring within four days of administration (50, Zinn E, Vandenberghe LH. Adeno- associated virus: fit to serve. Current opinion in virology.2014;8:90-7. Epub 2014/08/17. doi: 10.1016/j.coviro.2014.07.008. PubMed PMID: 25128609; PMCID: PMC4195847.; Reimsnider S, Manfredsson FP, Muzyczka N, Mandel RJ. Time Course of Transgene Expression After Intrastriatal Pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 Transduction in the Rat. Molecular Therapy.2007;15(8):1504-11.). AAV vectors yield transgene expression over several months, a timescale perfectly suited to peripheral nerve regeneration over long distances (48, Chen HH, Mack LM, Choi SY, Ontell M, Kochanek S, Clemens PR. DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle. Human gene therapy.1999;10(3):365-73. Epub 1999/02/27. doi: 10.1089/10430349950018814. PubMed PMID: 10048389.; Fan X, Brun A, Segren S, Jacobsen SE, Karlsson S. Efficient adenoviral vector transduction of human hematopoietic SCID-repopulating and long-term culture-initiating cells. Human gene therapy. 2000;11(9):1313-27. Epub 2000/07/13. doi: 10.1089/10430340050032410. PubMed PMID: 10890741.; Vassalli G, Bueler H, Dudler J, von Segesser LK, Kappenberger L. Adeno- associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors. International journal of cardiology.2003;90(2-3):229-38. Epub 2003/09/06. PubMed PMID: 12957756.; Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther.2008;16(6):1073-80. Epub 2008/04/17. doi: 10.1038/mt.2008.76. PubMed PMID: 18414476.). Undoubtedly, AAV vector-based gene therapy will revolutionize the management of sensorimotor peripheral nerve disorders (Miller AD. Human gene therapy comes of age. Nature.1992;357(6378):455- 60. Epub 1992/06/11. doi: 10.1038/357455a0. PubMed PMID: 1608446.; Kuzmin DA, Shutova MV, Johnston NR, Smith OP, Fedorin VV, Kukushkin YS, van der Loo JCM, Johnstone EC. The clinical landscape for AAV gene therapies. Nat Rev Drug Discov. 2021;20(3):173-4. Epub 2021/01/27. doi: 10.1038/d41573-021-00017-7. PubMed PMID: 33495615.). The FDA approved an AAV vector gene therapy for an inherited blinding retinal disease, with the first procedure in the U.S. performed in 2018 (Day S. Mass. Eye and Ear performs first FDA-approved gene therapy procedure for inherited disease. In: Day S, editor. Boston, MA: Mass. Eye and Ear Media Relations; 2018.; Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L, Conlon TJ, Boye SL, Flotte TR, Byrne BJ, - 13 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) Jacobson SG. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Human gene therapy.2008;19(10):979-90. Epub 2008/09/09. doi: 10.1089/hum.2008.107. PubMed PMID: 18774912; PMCID: PMC2940541.). A second AAV vector-based gene therapy for treatment of spinal muscular atrophy targeting lower motor neurons also gained FDA approval in 2019 (Hoy SM. Onasemnogene Abeparvovec: First Global Approval. Drugs.2019;79(11):1255-62. Epub 2019/07/05. doi: 10.1007/s40265-019-01162-5. PubMed PMID: 31270752.). [0058] In some embodiments, AAV vectors are employed to functionalize NGSs up to about 50 mm in length to express distinct growth factors to enhance Schwann cell ingrowth and subsequent axonal regeneration. Multiphoton microscopy and machine-learning based segmentation can be employed for rigorous quantification of neural regeneration relative to controls using a laboratory’s high-throughput platform (FIG.1). Prior work has demonstrated that increased neurotrophic factor expression may result in improved axonal regeneration over long distances (Wang Y, Li WY, Jia H, Zhai FG, Qu WR, Cheng YX, Liu YC, Deng LX, Guo SF, Jin ZS. KLF7- transfected Schwann cell graft transplantation promotes sciatic nerve regeneration. Neuroscience.2017;340:319-32. Epub 2016/11/09. doi: 10.1016/j.neuroscience.2016.10.069.; Sanna MD, Ghelardini C, Galeotti N. HuD-mediated distinct BDNF regulatory pathways promote regeneration after nerve injury. Brain research. 2017;1659:55-63. Epub 2017/01/24. doi: 10.1016/j.brainres.2017.01.019. PubMed PMID: 28111162; Bareyre FM, Garzorz N, Lang C, Misgeld T, Buning H, Kerschensteiner M. In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration. Proc Natl Acad Sci U S A.2011;108(15):6282-7. Epub 2011/03/31. doi: 10.1073/pnas.1015239108. PubMed PMID: 21447717; PMCID: PMC3076857.). In some aspects of embodiments, targeted AAV vector transduction of cells within the NGS environment will serve as an endogenous source of secreted growth factors over the course of axonal regeneration along the scaffolds. Though exogenous delivery of growth factors targeted regenerating axons or denervated Schwann cells has proven effective in enhancing nerve regeneration (35, Rich KM, Alexander TD, Pryor JC, Hollowell JP. Nerve growth factor enhances regeneration through silicone chambers. Experimental neurology. 1989;105(2):162-70. Epub 1989/08/01. PubMed PMID: 2753116.; Pfister LA, Papaloizos M, Merkle HP, Gander B. Nerve conduits and growth factor delivery in peripheral nerve repair. J - 14 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) Peripher Nerv Syst.2007;12(2):65-82. Epub 2007/06/15. doi: 10.1111/j.1529- 8027.2007.00125.x. PubMed PMID: 17565531.; Lee AC, Yu VM, Lowe JB, 3rd, Brenner MJ, Hunter DA, Mackinnon SE, Sakiyama-Elbert SE. Controlled release of nerve growth factor enhances sciatic nerve regeneration. Experimental neurology.2003;184(1):295-303. Epub 2003/11/26. PubMed PMID: 14637100.; Chen YY, McDonald D, Cheng C, Magnowski B, Durand J, Zochodne DW. Axon and Schwann cell partnership during nerve regrowth. Journal of neuropathology and experimental neurology.2005;64(7):613-22. Epub 2005/07/27. PubMed PMID: 16042313.; Colen KL, Choi M, Chiu DT. Nerve grafts and conduits. Plastic & Reconstructive Surgery.2009;124(6 Suppl):e386-94. doi: PubMed PMID: 19952706.; Moore AM, Wood MD, Chenard K, Hunter DA, Mackinnon SE, Sakiyama- Elbert SE, Borschel GH. Controlled delivery of glial cell line-derived neurotrophic factor enhances motor nerve regeneration. The Journal of hand surgery.2010;35(12):2008-17. Epub 2010/11/03. doi: 10.1016/j.jhsa.2010.08.016. PubMed PMID: 21035963.), challenges associated with growth factor encapsulation and timed release have hampered commercialization. One prior study employed gene therapy to establish an endogenous source of secreted growth factors to enhance long gap nerve regeneration (Marquardt LM, Ee X, Iyer N, Hunter D, Mackinnon SE, Wood MD, Sakiyama-Elbert SE. Finely Tuned Temporal and Spatial Delivery of GDNF Promotes Enhanced Nerve Regeneration in a Long Nerve Defect Model. Tissue engineering Part A.2015;21(23-24):2852-64. doi: 10.1089/ten.TEA.2015.0311. PubMed PMID: 26466815.); therein ex-vivo lentiviral vector transduction of cultured Schwann cells to overexpress glial cell-derived neurotrophic factor (GDNF) was employed, with subsequent seeding of transduced cells within a nerve allograft. [0059] Instead, in some embodiments provided herein, scaffolds, for example, NGSs, comprise AAV vectors. The vectors will be used for efficient transduction of NGSs, e.g., to express growth factors, e.g., pro-regenerative growth factors. Examples of growth factors that can be encoded by an AAV vector include, but are not limited to, IGF1, NGF, glial cell line- derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). In some embodiments, the NGSs are used to bridge nerve defects, e.g., defects of about 70 mm in length or longer in nerves, e.g., sensory, motor, or mixed peripheral nerves. AAV vector- based gene therapy is already FDA-approved for two indications, one of which targets lower motor neurons. - 15 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) [0060] As described herein, freeze-cast NGSs for impregnation with AAV vectors to encode growth factors for neural regeneration were developed in an iterative process supported by in vivo testing. The great strength of freeze-cast NGSs is that the scaffold properties can be adjusted through materials choice, processing parameters, and degree of cross-linking. Scaffolds with chitosan shells and cores of two different compositions, a collagen-nanocellulose composite and chitosan were used. [0061] To characterize Schwann cell transcriptional states and immune responses within and along the scaffolds in response to variation of pore sizes and stiffness high-plex profiling were employed, and results were compared against results obtained with fresh and long-term denervated isografts (FIG.2). Histology, the traditional tool for the study of cell types and tissue morphology, provides correlation between structure and function. It does not, however, reveal more detailed quantitative molecular information such as protein types, location, and abundance, required for an objective and quantitative measure of implant performance. Recently developed molecular techniques address this knowledge gap and are increasingly being implemented to quantify gene expression and to assist with better informed decision- making, when evaluating and selecting biomaterials (Vegas AJ, Veiseh O, Doloff JC, Ma M, Tam HH, Bratlie K, Li J, Bader AR, Langan E, Olejnik K. Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nature biotechnology.2016;34(3):345-52.; Doloff JC, Veiseh O, Vegas AJ, Tam HH, Farah S, Ma M, Li J, Bader A, Chiu A, Sadraei A. Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates. Nature materials.2017;16(6):671-80.; Sadtler K, Wolf MT, Ganguly S, Moad CA, Chung L, Majumdar S, Housseau F, Pardoll DM, Elisseeff JH. Divergent immune responses to synthetic and biological scaffolds. Biomaterials.2019;192:405-15.; Veiseh O, Doloff JC, Ma M, Vegas AJ, Tam HH, Bader AR, Li J, Langan E, Wyckoff J, Loo WS. Size- and shape- dependent foreign body immune response to materials implanted in rodents and non-human primates. Nature materials.2015;14(6):643-51.; Jiang K, Weaver JD, Li Y, Chen X, Liang J, Stabler CL. Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages. Biomaterials. 2017;114:71-81.; Divakar P, Reeves J, Gong J, Kolling FWt, Jack Hoopes P, Wegst UGK. High-plex expression profiling reveals that implants drive spatiotemporal protein production and innate immune activation for tissue repair. Acta Biomater.2022;138:342-50. Epub - 16 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) 20211019. doi: 10.1016/j.actbio.2021.10.018. PubMed PMID: 34673228.). Initially limited to a more global analysis of RNA, it was possible to obtain through a technique called digital spatial profiling (DSP) site-specific information on both protein presence and quantity within a well-defined tissue region (Bentley-Hewitt KL, Hedderley DI, Monro J, Martell S, Smith H, Mishra S. Comparison of quantitative real-time polymerase chain reaction with NanoString® methodology using adipose and liver tissues from rats fed seaweed. New biotechnology. 2016;33(3):380-6.; Merritt CR, Ong GT, Church S, Barker K, Geiss G, Hoang M, Jung J, Liang Y, McKay-Fleisch J, Nguyen K. High multiplex, digital spatial profiling of proteins and RNA in fixed tissue using genomic detection methods. BioRxiv.2019:559021.). Collecting first bulk RNA information to define the most appropriate panel for DSP, then spatial and bulk information in parallel, determining the effect of implant-driven protein profiles on the spatiotemporal performance of NGSs to evaluate and compare the regenerative outcomes achieved with virgin and AAV-impregnated cores in comparison with those achieved with fresh and long-term denervated isografts. Uniquely offered by DSP is the ability to co-register gene expression, protein expression, and histomorphological changes in a highly sensitive and accurate spatiotemporal manner. When combined, these techniques provide insight to the growth factors and signaling promoting axonal growth. [0062] For DSP, regions of interest (ROI) of 100–200 µm diameter were selected on formalin-fixed, paraffin- embedded tissue slides (Kulkarni MM. Digital multiplexed gene expression analysis using the NanoString nCounter system. Current protocols in molecular biology.2011;94(1):25B.10.1-25B.10.7.; Malkov VA, Serikawa KA, Balantac N, Watters J, Geiss G, Mashadi-Hossein A, Fare T. Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System. BMC research notes. 2009;2(1):1-9.) and sampled for the identification and quantification of individual proteins present, using a unique protein-specific, oligo-conjugated antibody-barcoding and digital counting technique (Kulkarni MM. Digital multiplexed gene expression analysis using the NanoString nCounter system. Current protocols in molecular biology.2011;94(1):25B.10.1- 25B.10.7.; Malkov VA, Serikawa KA, Balantac N, Watters J, Geiss G, Mashadi-Hossein A, Fare T. Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System. BMC research notes.2009;2(1):1-9.). This evaluation enabled a paradigm shift in implant development and assessment by facilitating rapid quantitation of proteins and their distribution within implants and surrounding tissue (FIG.2). - 17 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) [0063] The scaffolds and scaffold materials that were tested or were planned to be tested are described in Tables 1 and 2, respectively. Table 1. Live mice experiments with NGSs. *proximal longitudinal (qualitative), distal axial (quantitative axon counts), **Whisking, EMg, muscle weight. (I.D.: inner diameter, O.D.: outer diameter)

Table 2. Live rat experiments with NGSs. *proximal longitudinal (qualitative), distal axial (quantitative axon counts), **Whisking, EMg, muscle weight. (I.D.: inner diameter, O.D.: outer diameter)

[0064] Herein, the potential of multifunctional core-shell design NGSs comprising biochemical cues to enhance regeneration of peripheral nerves across a gap was assessed. In some embodiments provided herein, the NGSs comprise (e.g., consist of): i) an adventitia- like shell that bridges the defect and provides protection from fibrous tissue infiltration while permitting nutrient, oxygen, and waste transfer and/or ii) an endoneurium-like core, e.g., with aligned pore morphology to support and direct axonal extension across the gap; and, optionally, iii) biochemical cues (e.g., laminin, fibronection, type IV collagen) to enhance regeneration. The adventitia-like shell can bridges the defect and provides protection from fibrous tissue infiltration while permitting nutrient, oxygen, and waste transfer, and the endoneurium-like core with aligned pore morphology can support and direct axonal extension across the gap. Further, laminin can enhance regeneration. In some embodiments, the NGSs - 18 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) are freeze-casted. In some embodiments, advantages of freeze-casting over prior approaches to NGS manufacturing include the ability to tailor and optimize structural, mechanical, and/or biochemical cues for a given application (54-56). In some embodiments, the scaffold architecture emulates that of peripheral nerve trunks through its composition and processing parameters. [0065] Herein, in some embodiments, neural tissue cultures and a high-throughput rodent model of sciatic nerve injury are utilized to inform optimal designs of NGSs. As described herein, application of freeze casting as a processing technique enabled manufacture of NGSs with specific structural and mechanical properties and use of a novel collagen-nanocellulose (CNC) composite. In some embodiments the immersion of NGS cores in AAV vector solution, e.g., at the time of implantation, yielded targeted transduction of cells within the scaffold environment and enabled controlled release of endogenous growth factors to enhance nerve regeneration across long gaps. DEFINITIONS [0066] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. [0067] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, exemplary materials and methods are described herein. [0068] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” [0069] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, wherein two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third - 19 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0070] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include”, as well as variations thereof, such as “comprises” and “comprising”, are to be construed in an open, inclusive sense, e.g., “including, but not limited to.” The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open- ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element or step not specified in the claim; and (iii) “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.” [0071] “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 or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the disclosure, claims, result or embodiment, “about” means within one standard deviation per the practice in the art, or can mean a range of ± 20%, ± 10%, ± 5%, ±4, ±3, ±2 or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Examples. For example, an “about” 10% w/v of collagen is a range of 8% to 12% at a range of ± 20%, a plausible standard deviation to weight percent. [0072] In some example embodiments, nerve guidance scaffolds (NGS) are designed to mirror the internal structure of nerve grafts, containing a cylindrical inner core with a continuous, highly-aligned honeycomb-like porosity that parallels the cylinder axis, enabling the proximal nerve end to extend and reconnect with the distal nerve end(s). The inner core is - 20 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) surrounded by a cylindrical outer shell with a radially-oriented porosity made of the same or different material than the inner core. The stiffness of the shell may be similar to or higher than the stiffness of the core. [0073] In some embodiments, the treated nerves are peripheral nerves, for example, nerves of the brachial plexus such as musculocutaneous, radial, median, and ulnar nerves, nerves of the thoracoabdominal, such as intercostals, subcoastal, iliohypogastric, ilioinguinal, lateral cutaneous of thigh, and genitofemoral nerves, nerves of the lumbar plexus, such as obturator, femoral, muscular branches of femoral, and saphenous nerves, nerves of the sacral plexus, such as sciatic, tibial, common peroneal, deep peroneal, superficial peroneal, and sural nerves, pudendal nerves, and cranial nerves such as vagus nerves. [0074] In some embodiments, the treated nerves are central nerves part of the central nervous system including cerebrum, cerebellum, brainstem, and spinal cord nerves. [0075] Freeze-casting, also referred to as ice-templating, is a manufacturing process based on directional solidification of a solution or slurry (suspension, colloid). Water is a frequently used solvent by many others can be used. Applying well-defined processing conditions (e.g. mold type and shape, thermal gradient, applied cooling rate), solvent crystal growth and templating can be controlled to form a porous solid with a well-defined pore architecture. Undercooling a solution or slurry, solvent crystals nucleate and grow directionally along a temperature gradient, when one is applied. During solidification as phase separation into a solvent rich or a pure solvent (typical for aqueous systems) and a second phase occurs with the second phase being increasingly upconcentrated between the growing solvent crystals. The growing solvent crystals template the second phase, thereby defining the architecture of the scaffold which emerges once the sample is fully solidified and the solvent phase has been removed by, for example, lyophilization (freeze drying) or solvent exchange. [0076] tdTomato is a basic orange fluorescent protein derived from Discosoma sp. It is a slowly-maturing, very bright tandem dimer with low acid sensitivity. Herein its sequence was encoded into AAVs to confirm transgene expression within an NGS environment. [0077] Cellulose nanofiber (CNF) is a material composed of nanosized cellulose fibrils with a high aspect ratio, possessing pseudo-plastic characteristics and exhibiting thixotropy, which is the property of certain gels or fluids that are thick (viscous) under normal - 21 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) conditions, but become less viscous when shaken or agitated. When the shearing forces are removed, the gel regains much of its original state. [0078] “Collagen” described herein encompasses the definition of “collagen” as known to those skilled in the art, and, in some embodiments, may also be defined as but not limited to components such as rat tail tendon, porcine, bovine collagen, collagen sourced from marine sources, such as jellyfish, fish, and/or sponges. Types of collagen include, but are not limited to, telocollagen, atelocollagen, more or less pre-fibrillated collagen. [0079] “Cellulose” described herein encompasses the definition of “cellulose” as known to those skilled in the art and, in some embodiments, may also be defined as but not limited to components such as fibrillated and/or nanocrystalline nanocellulose, micro-cellulose, and bacterial cellulose. [0080] “Chitosan” described herein encompasses the definition of “chitosan” as known to those skilled in the art and, in some embodiments, may also be defined as but not limited to components such as a large group of polymers which differ in degree of deacetylation and molecular withs, both of which define their properties. [0081] “Gene delivery” and “gene delivery vehicle” described herein includes a process and means for introducing foreign genetic material, such as but not limited to nucleic acid such as DNA or RNA, into a host, wherein the gene delivery or gene delivery vehicle must reach a genome of a host to induce gene expression by delivering a foreign gene into host for integration into a genome or replication to occur independently of the genome. The foreign genetic material can be part of a vector, wherein the vector may be viral or non-viral. In some embodiments, the gene delivery vehicle is a viral vector, e.g. AAV. In other embodiments, the gene delivery vehicle may be a non-viral vector. Examples of non-viral vectors are well known in the art. [0082] The term “encodes exogenous proteins” refers to gene delivery, e.g., viral gene delivery utilizing a virus to inject genetic material (e.g. DNA, RNA, etc.) inside of a host cell and leverages the virus’ ability to replicate and implement their own genetic material. [0083] Transduction is the process that describes virus-mediated insertion of DNA into the host cell. Viruses prevent degradation via lysosomes of the genetic material being delivered. Viruses for gene delivery include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus. Virus gene delivery may contain genetic material of up to about 5 kilobases (kb) that may be delivered to a host cell. In some - 22 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) embodiments, the gene delivery may induce expression of growth factors including but not limited to: adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic proteins, ciliary neurotrophic factor family (ciliary neurotrophic factor, leukemia inhibitory factor, interleukin-6), colony-stimulating factors, epidermal growth factor, ephrins, erythropoietin, fibroblast growth factor, fetal bovine somatotrophin, glial cell line-derived neurotrophic factor family of ligands, interleukins, keratinocyte growth factor, migration- stimulating factor, macrophage-stimulating protein, myostatin, neuregulins, neurotrophins, placental growth factor, platelet-derived growth factor, renalase, T-cell growth factor, thrombopoietin, transforming growth factors, tumor necrosis factor-alpha, vascular endothelial growth factor, wnt singaling pathway, and nerve growth factor. [0084] The term “subject” or “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. In some examples, the subject is pediatric or geriatric. In some example, the subject is an infant, child, adolescent or adult. In [0085] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disease or condition, e.g., central nervous system (CNS) or peripheral nervous system (PNS) injury, and/or symptoms associated therewith. Moreover, treatment includes the partial or complete regeneration of nerve fibers in a subject. It will be appreciated that, although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. [0086] As used herein, the term “central nervous system disease, disorder, or condition” refers to any disease, disorder, or trauma that disrupts the normal function or communication of the brain or spinal cord. The CNS and PNS injuries which can be treated according to the present invention are diverse and will be easily understood by the skilled person. Without limitation, there may be mentioned brain and spinal cord injuries due, to neurosurgery, trauma, ischemia, hypoxia, neurodegenerative disease, metabolic disorder, infectious disease, compression of the intervertebral disc, tumors, and autoimmune disease. [0087] As used herein, the term “therapeutically active molecule” or “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes, This term includes - 23 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) pharmaceuticals, e.g., small molecules, treatments, remedies, biologicals, devices, and diagnostics, including preparations useful in clinical screening, prevention, prophylaxis, healing, imaging, therapy, surgery, monitoring, and the like. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixtures or combinations thereof, including, for example, DNA nanoplexes, ribozyme, shRNA, RNAi. The term also includes carbohydrates and polypeptides such as an antibody, antibody fragment, scFV, and enzymes. The term further includes radiotherapeutic agents; extracellular matrix components; free radical scavengers; chelators; antioxidants; anti- polymerases; photodynamic therapy agents gene therapy agents; and the like, Pharmaceutically active agents include but are not limited to any of the specific examples disclosed herein. Those of ordinary skill in the art will recognize also numerous other compounds that fall within this category and are useful according to the invention, Examples include a growth factor, e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), or glial cell-line derived neurotrophic factor (GNDF), a steroid, an anti-inflammatory agent, an analgesic agent, a sedative, a peptide agent, a biopolymeric agent, an antimicrobial agent, an enzyme (e.g., chondroitinase ABC (chABC) or sialidase), a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, precinisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesolide, naproxene, acetominophen, ibuprofen, ketoprofen and piroxicam, celocoxib, refocoxib, and N-[2- (cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; analgesic agents such as salicylates; sedatives such as benzodiazapines and barbiturates: antimicrobial agents such as penicillins, cephalosporins, and macrolides, including tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline, chloratriphenicol, rifampicin, ciprofloxacin, tobramycin, gentamycin, erythromycin, penicillin, sulfonamides, sulfadiazine, sulfacetamide, sulfatmethizole, solfisoxazole, nitrofurazone, sodium propionate, minocycline, doxycycline, vaticomycin, kanamycin, cephalosporins such as cephalothin, cephapirin, cetazolin, cephalexin, cephardine, cefadroxil, cefamandole, cefoxitin, cefaclor, cefuroxime, - 24 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) cefonicid, ceforanide, cefitaxime, moxalactam, cetizoxime, ceftriaxone, cefoperazone; acids such as DNA sequences encoding for biological proteins and antisense oligonucleotides; and other pharmacological agents that have been shown to promote axonal regeneration such as paclitaxel (TAXOL®). The term also refers to combinations of any of the therapeutic agents disclosed herein. The scaffolds, e.g., NGSs, described herein may be combined with other therapy treatments, e.g., those stated as therapeutic agents herein, biological agents, biological molecules, biological therapeutics, scrambler therapy, spinal cord stimulation, plasma exchange, physical therapy, surgery, or any combination thereof. [0088] As used herein, the term “biological agent,” “biological molecule,” or “biological therapeutic” is intended to mean a subset of therapeutic agents that are a polypeptide or nucleic acid molecule, in specific embodiments, the biological therapeutic is an agent that induces or enhances nerve growth, e.g., a neurotrophic agent. Examples of useful neurotrophic agents are αFGF (acidic fibroblast growth factor), FGF (basic FGF), NGF (nerve growth factor), BDNF (brain derived neurotrophic factor), CNTF (ciliary neurotrophic factor), MNGF (motor nerve growth factor), NT-3 (neurotrophin-3), GDNF (glial cell line- derived neurotrophic factor), NT4/5 (neurotrophin4/5), CM101, (heat shock protein-27), IGF- I (insulin-like growth factory, IGF-II (insulin-like growth factor 2), PDGF (platelet derived growth factor) including PDGF-BB and PDGF-AB, ARIA (acetylcholine receptor inducing activity), LIF (leukemia inhibitory factor), VIP (vasoactive intestinal peptide), GGF (glial growth factor), and IL-1 (interleukin-1). In a preferred embodiment, the biological therapeutic is NGF or GNDF. In embodiments, the biological therapeutic is an antibody, antibody fragment, or scFV that induces or enhances nerve growth, e.g., an antibody specific for any of the neurotrophic agents described herein, in other embodiments, the biological therapeutic is a ribozyme, shRNA, or RNAi that induces or enhances nerve growth, e.g., an RNA molecule specific for any of the neurotrophic agents described herein. [0089] As used herein, the term “scaffold” refers to a structure comprising a biocompatible material that provides a surface suitable for adherence and proliferation of cells. A scaffold may further provide mechanical stability and support. A scaffold be in a particular shape or form so as to influence or delimit three-dimensional shape or form assumed by a population of proliferating cells. Such Shapes or forms include, but are not limited to, films (e.g., a form with two-dimensions substantially greater than the third - 25 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) dimension), ribbons, cords, sheets, flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc. [0090] As used herein, “biocompatible” means the ability of an object to be accepted by and to function in a recipient without eliciting a significant foreign body response (such as, for example, an immune, inflammatory, thrombogenic, or the like response). For example, when used with reference to one or more of the polymeric materials of the invention, biocompatible refers to the ability of the polymeric material for polymeric materials) to be accepted by and to function in its intended manner in a recipient. [0091] As used herein, “therapeutically effective amount” refers to that amount of a therapeutic agent alone that produces the desired effect (such as treatment of a medical condition such as a disease or the like, or alleviation of a symptom such as pain) in a patient. In some aspects, the phrase refers to an amount of therapeutic agent that, when incorporated into a composition of the invention, provides a preventative effect sufficient to prevent or protect an individual from future medical risk associated with a particular disease or disorder. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the bioactive agent required to treat and/or prevent the progress of the condition. [0092] Demonstrated herein is the feasibility of freeze casting biomaterials in the construction of neuronal scaffolds. In various embodiments, the scaffolds of the present invention can be constructed from a variety of polymer compositions, including, but not limited to, chitosan, chitin, cellulose, alginate, agar, gelatin, soy protein, hyaluronic acid collagen, elastin, and silk alone or in combination with any other polymer composition, in any concentration and in any ratio. In one embodiment, the scaffolds of the present invention comprise chitosan, either separately or in combination with one or more other materials. Chitosan is a polysaccharide and is a partially deacetylated derivative of chitin. Chitosan is cationic in nature, and allows for modifications with other molecules, Such as glycosaminoglycans. Chitosan provides many options for ionic and covalent modifications and cross linking (e.g. with genipin), allowing mechanical properties and swelling to be adjusted and tailored for a particular application. Chitosan is also preferable for its relatively easy processing requirements. In another embodiment, chitosan may be used in combination with other materials, such as with gelatin or alginate. [0093] Freeze-cast polymer structures were created across a range of processing conditions and sample sizes, and the structure property correlations were elucidated, with - 26 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) particular emphasis on the local conditions required for ridge formation to occur in the Solidifying structure and how these ridges serve to strengthen the elastically-dominated deformation in both dry and wet conditions. [0094] In one aspect, polymer solutions having varying amounts of polymer dissolved in an acidic solution is used. The concentration of the acid can be adjusted depending on the amount of polymer dissolved. In one aspect, the acidic solution is about 1% (v/v) acetic acid. In one embodiment, the amount of polymer in solution is between about 0.5-5% (w/v) and any whole or partial increments therebetween. For example, the amount of polymer in solution (w/v) can be about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5% or about 5%. In a preferred embodiment, the amount of polymer in solution is about 2.4% (w/v). In other various embodiments, the polymer is dissolved in at least one of water, acid, acetic acid, camphene, or camphene-naphthalene. [0095] In another aspect, polymer solutions having varying amounts of chitosan dissolved in an acidic solution is used. The concentration of the acid can be adjusted depending on the amount of chitosan dissolved. In one aspect, the acidic solution is 1% (v/v) acetic acid. In one embodiment, the amount of chitosan in solution is between about 0.5-5% (w/v) and any whole or partial increments therebetween. For example, the amount of chitosan in solution (w/v) can be about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5% or about 5%. In a preferred embodiment, the amount of chitosan in solution is about 2.4% (w/v). In other various embodiments, the chitosan is dissolved in at least one of water, acid, acetic acid, camphene, or camphene-naphthalene. [0096] In one aspect, polymer solutions can include varying amounts of gelatin in combination with varying amounts of chitosan, each dissolved in an acidic solution. The concentration of the acid can be adjusted depending on the amount of gelatin in combination with chitosan that is dissolved. In one aspect, the acidic solution is 1% (v/v) acetic acid. In one embodiment, the amount of gelatin in solution is between about 1-10% (w/v) and any whole or partial increments there between. For example, the amount of chitosan in solution (w/v) can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%. In a one embodiment, the amount of chitosan in solution is about 5.5% (w/v). In one embodiment, the polymer solution includes a combination of about a 2.4% (w/v) chitosan solution and about a 5.5% (w/v) gelatin solution. In other various - 27 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) embodiments, the gelatin in combination with varying amounts of chitosan is dissolved in at least one of water, acid, acetic acid, camphene, or camphene-naphthalene. [0097] In an alternative embodiment, alginate is used as a scaffold material, either separately or in combination with one or more other materials. Alginate is easily processed, water soluble, and non-immunogenic. Alginate is a biodegradable anionic polysaccharide with free hydroxyl groups that offer easy gelling. Alginate is a derivative of brown seaweed that has been used for a various medical applications from impression casting in dentistry to medical bandages. The ability to be cast easily and proof of biocompatibility make alginate a desirable material for use in the present invention. Alginate absorbs and holds water well, making it ideal for injury repair where a moist environment is ideal for healing. Previous studies have shown promising results on alginate’s process ability and properties for use in nerve repair Suzuki, et al., 1999, NeuroReports 10:2891-2894: Nunamaker, et al., 2011, J. Mech Behav Biomed 4:16-33; Pattani, et al., 2009, Mol. Pharmaceutics.6 (2):345-352: Sobani, et al., 2010, Surg Neural Int 1,93. [0098] As contemplated herein, other polymer materials may be used, either separately or in any combination, and in any concentration, in the creation of the scaffolds of the present invention. Such additional or alternative materials may include, without limitation, collagen, elastin, agar, hydroxyapatite, PVA, agarose, PHBHHx (poly(3-hydroxybutyrate-co-3- hydroxy-hexanoate)), BGAL (1,2,3,4,6-pentaacetyl a-Dgalactose), PCL, Alginate/CPC, and Soy Protein Isolate, for example. In one alternative embodiment, the polymer may be a polyelectrolyte complex mixture (PEC) formed from a 1:1 solution of chitosan and alginate. In yet another embodiment, the scaffold may formed from analginate/calcium carbonate/glucono-delta-lactone mixture, Such as 0.5-5% alginate, 0.5-15 g/L calcium carbonate, and 1-50 g/L gluconon-delta-lactone in a ratio of 2:1:1 (alginate:CaCO:GDL). [0099] Depending on the materials and material ratios in mixture, the scaffolds may optionally be crosslinked. For example, after freezing and drying, a PEC mixture formed from a 1:1 solution of chitosan and alginate may be crosslinked in calcium chloride. In another example, analginate based scaffold can be at least partially pre-gelled (by addition of CaCO:GDL), or crosslinked, to maintain the scaffold for freeze casting, drying and subsequent crosslinking with CaCl. [00100] Additionally, the scaffolds as described herein can be coated fully or in-part with a variety of compounds, to alter the surface charge of the scaffold material, and in certain - 28 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) embodiments, to promote cell growth. For example, the scaffold can beat least partially coated with a polypeptide, Such as polylysine or polyornitine, or glycoproteins. In another example laminin, either separately or in combination with a polypeptide, can be used to at least partially coat the scaffold. It should be appreciated that any polypeptide, glycoprotein or combination thereof, may be used to coat the scaffolds of the present invention. [00101] The aforementioned polymer solutions can be freeze cast in various sized mold systems as would be understood by those skilled in the art, and further described in the examples presented herein. When pipetting the polymer solutions into small molds, air bubble formation is avoided by placing a micropipette on the open end of one of the mold grooves and repeatedly flushing the entire canal system until the residual air was flushed out. [00102] In one aspect of the present invention, the rate of cooling is highly controlled, as the size and alignment of pores, as well as the formation of ridges, is affected by the cooling rate. In one embodiment, controlling thermal transfer from the cold finger to the mold can be accomplished with a tightly fitting secondary mold (copper) placed on the bottom of the primary mold so as to provide a controlled thermal transfer from the cold finger upon which the primary mold is placed. In some embodiments, the secondary mold at least partially covers the primary mold, and can leave the top of the primary mold open to ambient air conditions. In one embodiment, the rate of cooling is controlled by controlling the temperature at one end of the mold. In another embodiment, the rate of cooling is controlled by controlling the temperature at more the one end of the mold. In a further embodiment, the rate of cooling is controlled by transitioning the mold through a temperature gradient. [00103] In another aspect of the present invention, the cold finger can be cooled down at various rates. In one embodiment, the cooling rate can range between about 0.1-100 degrees Celsius per minute C/min) and any whole or partial increments there between. In a preferred embodiment, the cooling rate can range between about 1-10° C/min, and any whole or partial increments therebetween. For example, the cooling rate (C/min) can be about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. The cooling rate can be achieved by, for example, the use of a PID controller, a band heater wrapped around the cold finger, a thermocouple imbedded under the surface of the cold finger and submerging the opposite end of the cold finger in a bath of liquid nitrogen. In this way, the band heater counteracts the thermal diffusion of the liquid nitrogen and power is slowly reduced by the PID controller to - 29 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) ensure an even cooling rate. The cold finger can be held steady at about 5°C to begin, and slowly lowered to about -150° C. Upon reaching -150° C, the cold finger can be held steady until the entire sample is frozen solid. [00104] In one aspect of the invention, the overall morphology of the freeze-cast polymer scaffolds is characterized by regions of aligned pores, or lamellae. In another aspect, ridges are formed along the lamellae and can protrude substantially perpendicular and uniformly in only one direction of the lamellae wall, creating a series of substantially parallel grooves between ridges. In one embodiment, the ridges along the lamellae are spaced about 1-50 μm apart, and any whole or partial increments therebetween. In preferred embodiments, the spacing between ridges can be about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, or about 50 μm. Formation of these ridges is controlled by manipulation of the local freezing front velocity and cooling rate experienced by the sample during freeze casting. In one embodiment, ridges are generated along the lamellae when the cooling rate is equal to or greater than about 6°C/min. The formation of ridges, as well as the spacing between ridges, can also vary depending on the type and combination of polymer materials used. [00105] In another aspect, the scaffolds include excellent pore alignment, with pore diameters of between about 10-200 μm, and any whole or partial increments therebetween. In certain embodiments, pore diameters can range between about 10-50 μm, and any whole or partial increments therebetween. In preferred embodiments, pore sizes can be about 10 μM, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, or about 50 μm. The selection of pore size within the scaffold can be controlled by manipulation of the local freezing front velocity and cooling rate experienced by the sample during freeze casting. The selection of pore size can also be controlled by selection of the type and combination of polymer materials used. [00106] In another aspect, the scaffolds have a Young's modulus of about 1-15 kPa, and any whole or partial increments there between. In preferred embodiments, the modulus can be about 1 kPa, about 2 kPa, about 3 kPa, about 4 kPa, about 5 kPa, about 6 kPa, about 7 kPa, about 8 kPa, about 9 kPa, about 10 kPa, about 11 kPa, about 12 kPa, about 13 kPa, about 14 kPa, or about 15 kPa. In a preferred embodiment, the modulus can be 3-5 kPa. The resulting modulus of various portions of the scaffold can be controlled by manipulation of the local freezing front velocity and cooling rate experienced by the sample during freeze casting. The - 30 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) modulus can also be controlled by selection of the type and combination of polymer materials used. [00107] In yet another aspect, the scaffolds have a plateau strength of about 0.1-1 kPa. In preferred embodiments, the plateau strength can be about 0.1 kPa, about 0.2 kPa, about 0.3 kPa, about 0.4 kPa, about 0.5 kPa, about 0.6 kPa, about 0.7 kPa, about 0.8 kPa, about 0.9 kPa, or about 1 kPa. The resulting plateau strength of various portions of the scaffold can be controlled by manipulation of the local freezing front velocity and cooling rate experienced by the sample during freeze casting. The plateau strength can also be controlled by selection of the type and combination of polymer materials used. The plateau strength can also be controlled by selection of the type and combination of crosslinkers used. [00108] For example, in one embodiment, the properties of the scaffold are controlled through directional freezing, scaffold composition and the degree of cross-linking. The resulting scaffold structure preferably has a substantially aligned porosity, where porosity is approximately 97%, and the Young's modulus is preferably in the range of 3-5 kPa. In other embodiments where the scaffold has approximately 97% porosity, the Young's modulus of gelatin can be in the range of 11.6-17.6 kPa in the dry state and in the range of 25.6-30.4 kPa in the wet state. As may be the case with chitosan, the Young's modulus can be in the range of 12-2.7 kPa in the wet state. [00109] In another aspect of the present invention, the scaffolds described herein can be used to guide nerve growth, as well as tissue growth, while best matching the material properties of the native tissue. Thus, the present invention includes a method of guiding the nerve growth through the core of the scaffold that is improved with the AAV vector containing growth proteins. [00110] Pharmaceutical Formulations [00111] In some embodiments, the scaffolds, e.g., NGSs are formulated into pharmaceutical compositions suitable for administration to a subject such as a mammal, e.g., a human patient. The compositions typically comprise one or more scaffolds, e.g., NGSs, of the present invention and a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient” includes suitable solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. The - 31 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) compositions also can contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. [00112] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known in the art. The administration may be, for example, local and direct. [00113] In some embodiments, pharmaceutical compositions contain, in addition to an NGS of the invention, an immune stimulating agent or immune modulating agent. The pharmaceutical composition optionally can be employed with other therapeutic modalities, such as surgery. [00114] Toxicity and therapeutic efficacy of the composition of the invention can be determined by conventional procedures. Generally, a therapeutically effective amount of an NGS or any composition described herein is in the range of about 0.1 mg/kg to 100 mg/kg, preferably about 0.1 mg/kg to 50 mg/kg. The amount administered will depend on variables such as the type and extent of disorder or indication to be treated, the overall health of the subject, the pharmaceutical formulation, and the route of administration. [00115] Administration frequency can vary, depending on factors such as route of administration, dosage amount, and the disease being treated. [00116] Methods of Treatment [00117] In some embodiments described herein are method of treating a subject (e.g., a patient) afflicted with a nerve deficit or disorder, comprising administering to the subject a pharmaceutical composition comprising a pharmacologically effective amount of a scaffold, e.g., an NGS. [00118] The administered pharmaceutical composition may further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents, and preservatives, as appropriate. [00119] While any suitable carrier known to those of ordinary skill in the art may be employed in the compositions, the type of carrier will typically vary depending on the mode - 32 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) of administration. The therapeutic compositions may be formulated for any appropriate manner of administration. [00120] The amount administered to the host will vary depending upon what is being administered, the purpose of the administration, the state of the host, the manner of administration, the number of administrations, interval between administrations, and the like. These can be determined empirically by those skilled in the art and may be adjusted for the extent of the therapeutic response. Factors to consider in determining an appropriate dose include, but is not limited to, size and weight of the patient, the age and sex of the patient, the severity of the symptom, the stage of the disease, method of delivery of the agent, half-life of the agents, and efficacy of the agents. [00121] Determining the dosages and times of administration for a therapeutically effective amount are well within the skill of the ordinary person in the art. For example, an initial effective dose can be estimated from cell culture or other in vitro assays. A dose can then be formulated in animal models to generate a circulating concentration or tissue concentration, including that of the IC50 as determined by the cell culture assays. [00122] In addition, toxicity and therapeutic efficacy are generally determined by using experimental animals. Guidance is found in standard reference works, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th Ed. (Hardman, J. G. et al., eds.) McGraw-Hill, New York, N.Y. (2001). [00123] For the purposes of this invention, the methods of administration are chosen depending on the condition being treated and the pharmaceutical composition. Administration of the pharmaceutical compositions may be through a single route or concurrently by several routes. [00124] In some embodiments, the scaffolds and compositions may be administered just once, or once per month, a bi-week, a day, a few or several times per day, or even multiple times per day, depending upon, among other things, the indication being treated and the judgment of the prescribing physician. [00125] The amount of a scaffold, e.g., an NGS, needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering the cells for therapeutic purposes, the cells are given at a pharmacologically effective dose. "Pharmacologically effective amount" or "pharmacologically effective dose" refers to an amount sufficient to produce the desired - 33 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms or manifestations of the disorder or disease. [00126] As an illustration, administration of an NGS to a patient suffering from a nerve disorder provides a therapeutic benefit not only when the underlying condition is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the condition. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized. Pharmacologically effective dose, as defined above, will also apply to therapeutic compounds used in combination with the cells, as further described below. [00127] Preferably, the effect will result in a quantifiable change of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized. [00128] An effective amount that will treat the disorder will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%. EXEMPLIFICATION Example 1: Nerve Guidance Scaffold Nerve Regeneration in Mice [00129] Nerve guidance scaffolds (NGSs) were prepared by sliding a freeze-cast shell comprising chitosan over a core comprising various types of collagen and collagen mixtures as shown in the scanning electron microscopy images of the NGS in FIG.3A. The sciatic nerve of double fluorescent mice (Thy1-CFP and Sox10-Venus) was transected as shown in FIG.3B and then an NGS was sewn to connect the proximal and distal nerve stumps as shown in FIG.3B and pictured in FIG.3C. Each of the NGS core compositions were assessed four weeks following implantation across a sciatic nerve gap. Those made of laminin coated collagen cellulose nanofiber (CNF) demonstrated the most robust ingrowth of Schwann cells and axons (FIG.4) in double fluorescent mice (Thy1-CFP and Sox10-Venus). Scaffolds comprising bovine and rat-based collagen cores showed minimal axonal and Schwann cell ingrowth, with growth predominantly confined to the potential space between - 34 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) the core and shell lumen. Cores coated in laminin demonstrated enhanced ingrowth compared to non-coated replicas. [00130] Scaffold cores were impregnated in vector solutions of AAV.8 (2.2x10¹³ viral genomes/mL, 10 μL per scaffold) encoding tdTomato or vehicle controls following immersion of transected proximal sciatic nerve stumps using a reservoir technique for 10 minutes. Immunofluorescence staining was performed to amplify tdTomato reporter signal. Transduction was observed only within scaffolds immersed in AAV.8 vector solutions. [00131] These results suggest the potential of bioengineered core-shell design NGSs functionalized with exogenous proteins and AAV vectors to achieve endogenous production of growth factors within scaffold environment to enhance their neural regenerative performance. [00132] Axonal and Schwann cell ingrowth into novel core-shell design nerve guidance scaffolds functionalized with biochemical cues has been demonstrated as shown (compare FIG.5A and B). The vehicle control in FIG.5A shows minimal Schwann cells and axons from the immunofluorescence staining compared to the AAV.8 vector shown in FIG.5B, having about the entire core of the NGS filled with Schwann cells and axons, indicating regeneration of the sciatic nerve in mice. Proof-of-principal of targeted transduction of cells within scaffold environments to express specific gene products using AAV vectors has been demonstrated. In some example embodiments, constructs are capable of bridging sensory, motor, and mixed nerve defects exceeding 70 mm. [00133] METHODS Nerve Guidance Scaffold Preparation [00134] Scaffolds of 12 mm shell length and 10 mm core length were manufactured. [00135] The type I bovine collagen solution was prepared by soaking 2% w/v collagen (Type I bovein collagen, lyophilized fibrous powder, Advanced BioMatrix, CA, USA) in 1% v/v acetic acid (glacial, EMD Millipore, MA, USA) for 12 hours before homogenization in an ice bath with a homogenizer (152, Fisher scientific, MA, USA) at its 2/3 power for 4 hours. The collagen-CNF solution was prepared by blending the 2% wv type I bovine collagen mixture and the 2% w/v CNF mixture (diluted from the as received cellulose nanofibrils solution, BioPlus, American Process Inc., Atlanta, GA) in 1:1 ratio. The type I rat tail collagen solution (8 mg/mL solution, Advanced BioMatrix, CA, USA) was used as received. - 35 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) All three solutions were then mixed in a planetary orbital mixer (SpeedMixer DAC 150 FVZ- K, FlackTek, Landrum, SC, USA) for 2 mins at 2000 rpm, then stored at 4°C. They were homogenized again in the planetary orbital mixer for 1 min at 2000 rpm before freeze casting. [00136] The slurries and solution were freeze cast into nerve scaffold cores through previously described method [Caruso, I., Yin, K., Divakar, P., Wegst, U.G.K., 2023. Tensile properties of freeze-cast collagen scaffolds: How processing conditions affect structure and performance in the dry and fully hydrated states. Journal of the Mechanical Behavior of Biomedical Materials 105897. https://doi.org/10.1016/j.jmbbm.2023.105897; Divakar, P., Yin, K., Wegst, U.G.K., 2019. Anisotropic freeze-cast collagen scaffolds for tissue regeneration: How processing conditions affect structure and properties in the dry and fully hydrated states. J Mech Behav Biomed Mater 90, 350–364. https://doi.org/10.1016/j.jmbbm.2018.09.012; Mohan, S., Hernandez, I.C., Wang, W., Yin, K., Sundback, C.A., Wegst, U.G.K., Jowett, N., 2018. Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model. Laryngoscope 128, E386–E392. https://doi.org/10.1002/lary.27439; Riblett, B.W., Francis, N.L., Wheatley, M.A., Wegst, U.G.K., 2012. Ice-Templated Scaffolds with Microridged Pores Direct DRG Neurite Growth. Advanced Functional Materials 22, 4920–4923. https://doi.org/10.1002/adfm.201201323; Yin, K., Divakar, P., Hong, J., Moodie, K.L., Rosen, J.M., Sundback, C.A., Matthew, M.K., Wegst, U.G.K., 2018. Freeze-cast Porous Chitosan Conduit for Peripheral Nerve Repair. MRS Adv 3, 1677–1683. https://doi.org/10.1557/adv.2018.194]. Briefly, the solution was injected into cylindrical polytetrafluoroethylene mold which 7 bores of 1.8 mm diameter and 4 mm length sealed with a copper bottom covered with a paraffin film. The mold was placed with the copper bottom on freeze caster [Wegst, U.G.K., Schecter, M., Donius, A.E., Hunger, P.M., 2010. Biomaterials by freeze casting. Philos Trans A Math Phys Eng Sci 368, 2099–121. https://doi.org/10.1098/rsta.2010.0014] and frozen with 1 °C/min applied cooling rate until - 150°C was reached. Once fully frozen, the mold was placed in a freezer (Model 5705, VWR, PA, USA) for 20 mins to equilibrate it to -20 °C. The samples were demolded with an arbor press, lyophilized at 0.008 mbar and -85 °C coil temperature in a lyophilizer (FreeZone 6 Plus, Labconco, MO, USA) for 24 hours. The freeze dried core scaffolds were crosslinked in 6 millimolar (mM) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 98+%, Alfa Aesar, MA, USA) and 33 mM N-Hydroxysuccinimide (NHS, 98+%, Alfa Aesar, - 36 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) MA, USA) in ethanol (200 proof, Koptec, PA, USA) for 6 hours on an orbital shaker (VWR, PA, USA), followed by three washes of 1 hour, 12 hours, and 1 hour in distilled water with gentle palpitating after each wash. [00137] Half of the core scaffolds were coated with laminin by submerging in a 25 µg/mL laminin (Sigma-Aldrich, MO, USA) in phosphate buffered saline (PBS, Biotechnology grade, VWR, PA, USA) solution at 37 °C for 12 hours, and then washed in distill water for 3 ^ 20minutes. The core scaffolds were flash frozen in liquid nitrogen, punched with a 2 mm inner diameter biopsy punch to remove the skin layer, cut to 5 mm length with a razor blade (Astar, St. Petersberg, Russia), and stored in distilled water before assembling. [00138] To freeze cast the scaffold shell, a 3.5 w/v% chitosan in 1.5 v/v% acetic acid in distilled water solution was prepared on a roller mixer (W348923-A, Wheaton, NJ, USA) at 10 rpm for 24 hours. The chitosan solution was homogenized in the planetary orbital mixer (1 minute, 2000 rpm) and injected into the void between a coaxially fixed brass rod (1.8mm diameter) in an aluminum tube (3mm inner diameter), and frozen for 20 min in a -80°C freezer (Model 5705, VWR, PA, USA). Once fully frozen, the chitosan tube was demolded and lyophilized at 0.008 mbar and -85°C (FreeZone 6 Plus, Labconco, MO, USA) for 24 hours, and neutralized by 6 hours immersion in 0.4 v/v% sodium hydroxide solution in 95% ethanol, and washed for 1 hour-12 hours-1 hour in distilled water. The shells were cut into 7 mm length with the razor blade. [00139] Scaffolds were assembled by manual sliding of cores into center of shells with microforceps. The core-shell assemblies were then flash frozen and lyophilized. Scaffolds were placed in sealed sterilization pouches and sterilized using ethylene oxide gas (Nelson Laboratories, Sterigenics, Salt Lake City, UT). Structure Analysis by Scanning Electron Microscopy [00140] Transverse and longitudinal sections on the core-shell assembly were platinum coated for 1 minutes (Hummer 6.2, Anatech, CA, USA), and imaged by scanning electron microscopy (Scios 2, Thermo Fisher Scientific, OR, USA). Adeno-associated viral vector [00141] Two AAV backbones driving the expression of Tdtomato were used in these experiments. The AAV expression plasmid pAAV-CAG-tdTomato (codon diversified) was - 37 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) selected, which was a gift from Edward Boyden (Addgene plasmid # 59462 ; http://n2t.net/addgene:59462). This AAV transgene expression cassette was packaged in AAV8 (Addgene) and vector stock diluted to 2.2 x 10¹³ viral genomes per mL of sterile phosphate buffered saline (PBS) with 0.001% Pluronic F-68. The chicken β-actin (CAG) promoter is a ubiquitous active promoter. Vector solutions were titered using quantitative TaqMan PCR assay as previously described (57), and dropet digital PCR with a probe and primers targeting the ITRs, respectively. The ddPCR assay and corresponding titer determination was validated using a reference virus of a known titer. The vectors were stored at −80 °C prior to use. Development of Double Transgenic Mice Model [00142] Transgenic murine models were used to visualize regeneration of the peripheral nervous system. B6.Cg-Tg(Thy1-CFP)23Jrs/J (common name Thy1-CFP 003710, Jackson Laboratory) express cyan fluorescent protein (CFP) in axons of the motor and sensory peripheral nervous system and some neurons of the central nervous system. Tg(Sox10- Venus)1Okn (common name Sox10-Venus), provided via cryo-recovery (kind gift of S Shibata), highly express mVenus variant of YFP, a green-yellow fluorescent protein, in oligodendrocytes (Schwann cells) (58). These two lines were back crossed 10 generations with wildtype C57Bl/6J and tested via SNP genome scanning analysis to ensure they were >99% congenic (The Jackson Laboratory, Bar Harbor, ME). The two lines were cross bred to produce a double transgenic model expressing CFP and mVenus respectively in peripheral nerve axons and Schwann cells. Animal Surgery [00143] All animal surgeries were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with study approval by the Massachusetts Eye and Ear Animal Care Committee. [00144] Eighteen double transgenic Sox10-Venus/Thy1-CFP mice (8 female), ages 12-35 weeks were placed under 2% isoflurane anesthesia in 1L O₂, buprenorphine (0.05 mg/kg subcutaneously) and meloxicam (5.0 mg/kg subcutaneously) were administered for intraoperative analgesia. Sciatic nerves were transected at the midthigh through a 1 cm skin incision. Proximal sciatic nerve stumps were immersed in viral vector or control solution - 38 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) (n=1 mice per core design, 5 microliters (μL) of solution) for 10 minutes using a reservoir technique. Scaffold cores were then impregnated with 5 μL of pluronic control or AAV.8 vector solution immediately prior to interposition of the scaffolds across the defect (n=3 mice/scaffold group), and proximal and distal nerve stumps secured within the shells abutting the cores using 11-0 nylon epineural sutures under high stereoscopic magnification by a skilled microsurgeon. Wounds were closed using absorbable sutures, and mice recovered from anesthesia. Mice provided analgesia daily for three days and monitored daily for signs of distress. NGS harvest and processing [00145] At four weeks, animals underwent CO₂ euthanasia and cardiac perfusion using 2% phosphate-buffered paraformaldehyde solution. Repair sites were harvested and drop-fixed in 2% paraformaldehyde overnight. Scaffolds were divided in half into proximal and distal portions using a scalpel. Proximal nerve-scaffold segments where then longitudinally sectioned with a scalpel to unroof the shells, and whole-mounted for longitudinal imaging via multiphoton microscopy. Optical clearing of proximal segments was achieved by 36 hour immersion and subsequent imaging in refractive index matching solution (EasyIndex, LifeCanvas Technologies). Distal scaffold-nerve segments were vibratome sectioned at 250 μm for cross-sectional imaging. [00146] Free-floating immunofluorescence protocol (59) was performed on additional distal sections and imaged cross-sectionally. Floating sections were blocked and permeabilized for 30 minutes with buffer (0.3% Triton X-100, 3% donkey serum in 1XTBS). Primary antibody (rabbit anti-RFP Min X Hu Ms and Rt Serum Proteins; Rockland 600-401- 379) was incubated for two nights at 1:200 in antibody buffer (0.3% Triton X-100, 1% donkey serum in 1XTBS) at 37°C. After washing sections in 1XTBS, fluorophore-conjugated secondary antibody (donkey anti-rabbit conjugated AlexaFluor 594, Invitrogen cat. A- 215207) was incubated in antibody buffer at 1:500 dilution for 2 hours at RT. All steps occurred under light-tight conditions and on an orbital shaker (Thermo Scientific MaxQ 4450). Prior to imaging, sample was washed in 1XTBS and then immersed in refractive index matching solution (EasyIndex, LifeCanvas Technologies, Cambridge, MA) for 12 hours at 4°C. - 39 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) NGS multiphoton imaging [00147] Multiphoton imaging was achieved using a commercial multiphoton microscope (TrimScope II, LaVision Biotec) powered by a dual-output femtosecond laser (Insight X3, SpectraPhysics). The laser system provides one tunable output (680-1300 nm, < 120 fs) and one fixed output (1045 nm, < 200 fs). The fixed output was employed for 2-photon excitation (2PE) of Venus while tuning wavelength was set at 820 nm for sequentially imaging CFP, Alexa 594 and SHG labelled structures. Beam intensity was software-controlled independently via high-speed electro-optical modulators for volumetric stack images. Images were acquired with a set of galvanometer mirrors. A motorized XYZ-stage was used to acquire wide field-of-view volumetric imaging. A glycerol-immersion objective lens (CLr Plan-Neofluar 20x, WD 5.6 mm, Carl Zeiss) with correction collar was used for imaging thick sections. A dichroic beamsplitter was employed to separate excitation and emission light (T680lpxxr, Chroma Technology Corp, Bellows Falls, VT, USA). Three secondary dichroic mirrors (Semrock FF435-Di01, Chroma T495lpxr-UF1, and Semrock Di02-R635- 25x36) were used to split the fluorescence and SHG signal. Emission light was further filtered (Semrock FF01-458/64, Semrock ET 525/50 nm, FF01-650/60, Semrock) and collected in a non-descanned detection path using four high-quantum efficiency GaAsP photomultiplier tubes (H 7422-40 and H 7422-50, Hamamatsu). Images were averaged three times to improve signal-to-noise ratio. Microscope control was achieved via open-source software (ImSpector Pro, LaVision BioTec).3D-rendering and image stitching were performed using commercial image-analysis software (Bitplane Imaris 9.2; Oxford Instruments, Zurich, Switzerland). NGS confocal imaging [00148] 250 μm sections were imaged on a confocal microscope (TCS SP8, Leica microsystems, Germany) with a high numerical aperture glycerol immersion lens (HC PL APO CS263X 1.30 GLYC, Leica Microsystems, Germany). Samples were imaged using an Argon laser (488, 514 nm) and a 561 nm diode-pumped solid-state laser. Fluorescence emission was separated and filtered into four detection channels (465-450 nm, 520–550 nm, and 650-750 nm) using an acousto-optical beam splitter. The fluorescence signal was sequentially collected using two photomultiplier tubes and two hybrid detectors. Confocal images were line averaged 3 times with unidirectional laser scan (700 Hz). A motorized stage - 40 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) was used for tile scan imaging of longitudinal scaffold. The images were stitched using the same software used for imaging acquisition (LAS X, Leica Microsystems, Germany). Rat-Cross-Facial Nerve Gap Model [00149] Male and female Lewis rats, 16-22 wk old (150 – 350 g) will be placed under general anesthesia using 1-3% isoflurane in 1L O₂. Buprenorphine (0.05 mg/kg SQ) and meloxicam (2.0 mg/kg SQ) will be administered. A custom titanium head-restraint device will be implanted as previously described (155). Briefly, a 1 cm sagittal incision will be made through the scalp atop the occiput, periosteum over the occipital and parietal bones removed, and titanium devices secured using six titanium microscrews. Two additional small longitudinal incisions will be made over right and left cheeks. The right buccal branch of the facial nerve will then be sharply transected distally and reflected superiorly for end-to-end coaptation of the proximal stump to a 50 millimeter (mm) long NGS (4 types, N= 8 per group) or sciatic nerve isograft (fresh N= 8, or denervated N=8) using two 11-0 nylon sutures under high stereoscopic magnification using our laboratory’s operating microscope. Scaffolds (or grafts) will be tunneled subcutaneously across the face for coaptation of the distal end to the distal stump of the contralateral buccal branch using two 11-0 nylon sutures. The left marginal branch of the facial nerve will be resected to remove its contribution to whisking function on the recipient side. Skin incisions will be closed with interrupted 5-0 nylon sutures. Antibiotic and bite deterrent ointment will be applied. Animals will recover from anesthesia on a warming pad before returning to the animal housing facility. Meloxicam (2.0 mg/kg) will be administered daily for two days postoperatively, and animals monitored for signs of distress. Fresh and Denervated Rat Isograft Harvest [00150] Male and female Lewis rats, ages 10-12 weeks, weighing 150-300 g, will be placed under 1-3% isoflurane anesthesia in 1L O₂. For fresh isograft harvest, animals will be euthanized by 5% isoflurane and open thoracotomy, and both sciatic nerve harvested along their entire length to obtain 5 cm long lengths for immediate implantation into recipient animals. Donor animals providing deneverated isografts will be administered buprenorphine (0.05 mg/kg SQ) and meloxicam (2.0 mg/kg SQ) for intraoperative analgesia. Sciatic nerves will be transected proximally and distally, and placed within a subcutaneous pocked in the - 41 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) dorsum. Wounds will be closed and mice recovered from anesthesia, and provided meloxicam daily for two days and monitored for signs of distress. After 10 weeks, animals will be euthanized by CO₂ inhalation, and isografts harvested for immediate use in recipient animals. Rat Functional Testing [00151] Beginning two weeks after surgery, rats will undergo twice weekly two-minute long functional assessment of whisking function via high-speed videography (acA800-510um Basler ac, while in head-restraint on a miniature conveyor belt as previously described (Jowett N. Design of a neural prosthesis for facial ranimation and assessment in a rat model. Montreal, QC, Canada: McGill University; 2021.; Jowett N, Kearney RE, Knox CJ, Hadlock TA. Toward the bionic face: A Novel Neuroprosthetic Device Paradigm for Facial Reanimation Consisting of Neural Blockade and Functional Electrical Stimulation. Plastic and Reconstructive Surgery.2019;143(1):63e-76e. doi: 10.1097/prs.0000000000005164.). Right and left C-1 whiskers are defined by linear regression lines through five landmark points automatically tracked from captured video using custom Python script developed in our laboratory powered by a deep neural network (DeepLabCut) (Mathis A, Mamidanna P, Cury KM, Abe T, Murthy VN, Mathis MW, Bethge M. DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nat Neurosci.2018;21(9):1281-9. doi: 10.1038/s41593-018-0209-y.; Nath T, Mathis A, Chen AC, Patel A, Bethge M, Mathis MW. Using DeepLabCut for 3D markerless pose estimation across species and behaviors. bioRxiv.2018:476531. doi: 10.1101/476531.). Whisker displacements are defined as the angle formed by the right and left C-1 whisker with the coronal plane of the face, defined as an axis orthogonal to the sagittal midline. An open-source toolkit for the identification of biomedical systems in MATLAB (R2018a, The MathWorks Inc, Natick, MA) is employed for whisker signal processing (Kearney RE. Tools for the identification of biomedical systems. December 2020 ed. Online: GitHub; 2003.). Linear interpolation is employed for missing data points. First derivatives are taken and signals detrended. A nonequispaced fast fourier transform of 5000 bin length is employed for generation of power spectra for comparison of whisking activity between right and left sides and between injured and uninjured animals. Mean differences in root-mean-square power and areas under power - 42 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) spectra curves between right and left sides between groups will be compared using one-way ANOVA with Dunnett post hoc with alpha set at 0.05. Rat cross-facial nerve conduction tests [00152] At eight or 16 weeks following end-to-side motor nerve transfer, animals will be placed back under isoflurane general anesthesia with buprenorphine and meloxicam analgesia. Right and left cheek incisions will be meticulously re-opened. A silicone-sheathed nerve cuff with bipolar platinum-iridium electrodes (MicroProbes for Life Sciences) will be positioned about the proximal right donor buccal nerve, and a two-channel highly flexible conductive polymer electrode array (Ripple LLC) positioned atop the left whisker pad musculature. A grounding electrode will be placed into the hind leg, and differential EMG signals from left whisker musculature in response to increasing current-controlled charge- balanced square wave amplitudes at a repetition rate of 1Hz will be recorded using a commercial signal conditioner and signal acquisition system and bundled software (CyberAmp 380, Digidata 1322A, Clampex 10, Molecular Devices, Sunnyvale, CA). Signals will be captured using a sampling rate of 10 kHz at 16-bit resolution, with an input high-pass filter at 10 Hz, an input differential gain of 10-100, a low pass filter at 1000 Hz and a total gain of 1000. Mean maximum compound muscle action potential will be compared between experimental groups using one-way analysis of variance (ANOVA) with post hoc Dunnetts. Animals will then be euthanized and scaffolds harvested. Two harvested scaffolds/isografts from each group of eight animals will be employed for RNA bulk analysis. Rat cross-facial scaffold/isograft histomorphometry [00153] Scaffolds (N=6 per group) will be fixed in 4% PFA, paraffin embedded and cross-sectioned at 2 µm or 7 µm. Thin sections through the proximal, mid, and distal scaffolds / isografts will be mounted on silane-coated glass slides, stained with a myelin dye (FluoroMyelin® Green, 1:300 dilution, Molecular Probes), and cover-slipped according to previously described methods (Mohan S, Hernandez IC, Wang W, Yin K, Sundback CA, Wegst UGK, Jowett N. Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model. Laryngoscope.2018;128(11):E386-E92. Epub 20180810. doi: 10.1002/lary.27439. PubMed PMID: 30098047.; Wang W, Kang S, Coto Hernandez I, Jowett N. A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated - 43 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) Axon Count and Its Application to Cross-Facial Nerve Grafting. Plast Reconstr Surg. 2019;143(3):771-8. Epub 2019/01/03. doi: 10.1097/PRS.0000000000005338. PubMed PMID: 30601328; PMCID: PMC7147971.). Temporal stacks of 120 images will be acquired with a widefield microscope (Axio Imager A.2; Carl Zeiss, Oberkochen, Germany), with a 40×/1.3 objective lens to generate individual SRRF images using a post-processing algorithm (NanoJ-SRRF [Jowett N, Pineda II R. Corneal neurotisation by great auricular nerve transfer and scleral-corneal tunnel incisions for neurotrophic keratopathy. Br J Ophthalmol.2018. Epub 2018/11/25. doi: 10.1136/bjophthalmol- 2018-312563. PubMed PMID: 30470713.]) in open-source image analysis software (ImageJ/Fiji [Vacanti CA. The history of tissue engineering. Journal of cellular and molecular medicine.2006;10(3):569-76. Epub 2006/09/23. PubMed PMID: 16989721; PMCID: PMC3933143.]). Myelinated axons will be segmented, quantified, and compared between groups as above. Shell Manufacture [00154] Freeze-cast porous chitosan tubular scaffolds: The mold for freeze casting was a coaxially fixed tube (3.0 mm inner diameter, 80 mm in length), rod (2 mm in diameter, 100 mm in length) and space holder combination of different materials. The different materials included aluminum, 316 stainless steel, or copper for the tube, 316 stainless steel or copper for the rod, aluminum or epoxy (plastic) for the holder and were used in any combination thereof. The chitosan solution was injected between tube and rod. The mold was then frozen in the freezer (HF–5017W-PA, VWR, PA, USA) at -80 C for 20 min. The frozen chitosan tubes were demolded and lyophilized at 0.008 mbar and -85 C coil temperature in a lyophilizer (Freezone 6 Plus, Labconco, MO, USA) for 24 h. The freeze-dried chitosan tubes were neutralized by 15 min immersion in 0.4% w/v sodium hydroxide (reagent grade, anhydrous, Sigma-Aldrich, MO, USA) in 95% ethanol (200 proof, Koptec, PA, USA), followed by 6 h of washing in deionized water. The tubes were either i) flash frozen and lyophilized for spray coating and mechanical testing, or ii) stored in the frozen state for spray freezing and brush freezing. Core-Shell Manufacture [00155] Spray coating: The dry tubes were attached coaxially and horizontally to a spinning motor on the bed of a 3-axis stage. An atomizer nozzle (NS60K, Sonaer Inc., NY, - 44 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) USA) was attached to the moving head of the stage. The chitosan solution (1.2% w/v) was fed into the nozzle by a syringe pump (NE-300, New Era Pump Systems Inc., NY, USA) at a pumping rate of 10mL/h, and atomized at 60 kHz, 95% power. Spray coating of the 8 mm long tube was conducted by spinning the tube at 60 rpm, and nozzle motion rate of 1 mm/s along the axial direction of the tube for two roundtrips at a vertical distance of 10 mm upon the tube. Filtered air was blown onto the tube afterwards to facilitate drying the coated layer. [00156] Spray freezing: For spray freezing, a stainless-steel dwell pin (1.8 mm diameter) was gently drilled into the lumen of the frozen CSP tube replacing the ice core. The pin was attached to the same spraying system with a 15 mm thick layer of dry ice at 15 mm below the spinning axis. Chitosan solution (1.2% w/v) was fed at a rate of 40 mL/h into the nozzle. Spray freezing was conducted at a spinning rate of 120 rpm, and nozzle motion rate of 1 mm/s for one round trip. The tubes were first acclimatized in the -80 C freezer, then lyophilized. [00157] Brush coating followed by freezing: A nylon artistic brush (size #0, Bomega) was attached to the stage instead of the atomizer nozzle. The brush was wet by the 3.5% w/v chitosan solution, and 0.25 mL additional chitosan solution was added onto both sides of the brush. Brush coating was conducted by spinning the tube at 120 rpm, and a brush motion rate of 1 mm/s for two roundtrips. The tip of the brush was just in contact with the tube. Finally, the brush-coated tubes were transferred into a -80 C freezer and not removed until fully frozen, then lyophilized. - 45 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) REFERENCES 1. Lundborg G. Richard P. Bunge memorial lecture. Nerve injury and repair--a challenge to the plastic brain. Journal of the peripheral nervous system : JPNS.2003;8(4):209-26. Epub 2003/12/04. doi: 10.1111/j.1085-9489.2003.03027.x. PubMed PMID: 14641646. 2. Ootes D, Lambers KT, Ring DC. 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An ice-templated, linearly aligned chitosan-alginate scaffold for neural tissue engineering. Journal of Biomedical Materials Research Part A.2013;101(12):3493-503. doi: https://doi.org/10.1002/jbm.a.34668. 57. Maguire CA, Balaj L, Sivaraman S, Crommentuijn MH, Ericsson M, Mincheva- Nilsson L, Baranov V, Gianni D, Tannous BA, Sena-Esteves M. Microvesicle-associated AAV vector as a novel gene delivery system. Molecular Therapy.2012;20(5):960-71. 58. Shibata S, Yasuda A, Renault-Mihara F, Suyama S, Katoh H, Inoue T, Inoue YU, Nagoshi N, Sato M, Nakamura M. Sox10-Venus mice: a new tool for real-time labeling of neural crest lineage cells and oligodendrocytes. Molecular brain.2010;3(1):1-14. 59. Potts EM, Coppotelli G, Ross JM. Histological-based stainings using free-floating tissue sections. JoVE (Journal of Visualized Experiments).2020(162):e61622. [00158] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. [00159] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. - 50 - 3878724.v1

Claims

Docket No.5200.2392-001 (INV-23052) CLAIMS What is claimed is: 1. A scaffold for insertion into a tissue gap, the scaffold comprising: one or more porous materials; one or more polymers; and one or more gene delivery vehicles. 2. The scaffold of claim 1, wherein at least one gene delivery vehicle is an adeno- associated viral (AAV) vector. 3. The scaffold of claim 1, wherein the scaffold is a nerve guidance scaffold (NGS). 4. The scaffold of claim 1, wherein the scaffold comprises a solid core. 5. The scaffold of claim 4, wherein the solid core has one or more tubular holes throughout its length to enable passthrough of tissue in a tissue gap to reconnect two or more ends of damaged tissue. 6. The scaffold of claims 1-5, wherein the scaffold is freeze-cast. 7. The scaffold of claims 1-6, wherein at least one porous material comprises one or more cores, one or more shells, or any combination thereof. 8. The scaffold of claim 7, wherein at least one shell or at least one core is longitudinally porous. 9. The scaffold of claim 7, wherein at least one shell is radially porous. 10. The scaffold of claim 1, wherein at least one polymer is a biopolymer selected from chitosan, chitin, collagen, gelatin, cellulose, alginate, agar, agarose, soy protein, hyaluronic acid, elastin, silk, fibrin, or any combination thereof. 11. The scaffold of claim 1, wherein at least one polymer comprises at least one polymer selected from polylactic acid (PLA), polyglutamic acid (PGA), poly lactic-co-glycolic acid (PLGA), polycaprolactone, polydioxanone, solubilized basement membrane matrix, or any combination thereof. - 51 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) 12. The scaffold of claim 7, wherein: a) one or more shells are adventitia-like, freeze-cast chitosan-based shells, b) one or more cores are endoneurium-like, collagen-based porous cores, or c) one or more shells are adventitia-like, freeze-cast chitosan-based shells and one or more cores are endoneurium-like, collagen-based porous cores. 13. The scaffold of claim 7, wherein one or more pores are porous cores comprising collagen. 14. The scaffold of claim 14, wherein one or more cores are porous cores comprising a collagen-nanocellulose composite (CNC). 15. The scaffold of claim 7, wherein one or more cores are coated with laminin. 16. The scaffold of claim 15, wherein one or more cores are coated with about 1-100 μg/mL of laminin. 17. The scaffold of claim 7, wherein one or more cores comprise about 1-10% w/v collagen. 18. The scaffold of claim 7, wherein one or more shells comprise about 1-10% w/v chitosan. 19. The scaffold of claim 7, wherein one or more cores or one or more shells has non- directional porosity. 20. The scaffold of claim 1, wherein the scaffold comprises radially-oriented porosity, longitudinally-oriented porosity, non-directional porosity, or a combination thereof. 21. The scaffold of claim 2, wherein the AAV vector comprises a concentration gradient in the scaffold. 22. A method of regenerating damaged nerves, said method comprising attaching each nerve end to the scaffold of any one of claims 1-21, thereby connecting them and promoting nerve regeneration. - 52 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) 23. A method of inhibiting nerve regeneration in a peripheral nervous system in a subject in need thereof, said method comprising administering the scaffold of any one of claims 1-21 to the subject, thereby inhibiting nerve regeneration. 24. The method of claim 22, wherein the damaged nerves are damaged peripheral nerves. 25. A method of treating a nervous system deficit in a subject in need thereof, said method comprising attaching the scaffold of any one of claims 1-21 to damaged nerve ends of the subject to connect the damaged nerve ends. 26. The method of claim 25, wherein the nervous system deficit is at least one of: a neuroma, amyotrophic lateral sclerosis (ALS), carpal tunnel syndrome, Guillain-Barre syndrome, peripheral neuropathy, a peripheral nerve injury, congenital nerve absence, peripheral nerve pain, or spinal cord repair. 27. A method of making a nerve guidance scaffold (NGS), the method comprising: a) forming a core by either: i) freeze casting a collagen, collagen-cellulose, chitosan, or chitosan- nanocellulose solution/slurry in a mold, or ii) freeze extruding a collagen, collagen-cellulose, chitosan, or chitosan- nanocellulose solution; b) freeze drying the core; c) freeze casting a chitosan solution in a mold, thereby forming a shell; d) freeze drying the shell; and e) sliding the shell over the core, thereby assembling the NGS. 28. The method of claim 27, wherein the NGS is sterilized prior to use in a subject. 29. The method of 27 or 28, wherein the core of the NGS is coated with laminin prior to step e) of method 27. 30. The method of any one of claims 27-29, wherein the NGS is compatible with an adeno-associate viral (AAV) vector, and wherein: a) the NGS is dipped in an AAV vector solution, b) the AAV is injected directly into the core of the NGS, or - 53 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) c) the NGS and the AAV are freeze cast together in either the core solution, shell solution, or both solutions. 31. The method of any one of claims 27-30, comprising crosslinking multiple cores together, thereby forming a longer core scaffold. 32. A method of inhibiting disorganized growth of a nerve, said method comprising contacting the nerve with the scaffold of any one of claims 1-21. 33. A method of mediating a function of a nerve, said method comprising contacting the nerve with the scaffold of any one of claims 1-21. 34. The method of claim 33, wherein contacting the nerve with the scaffold promotes ingrowth of axonal and Schwann cells through the scaffold core and shell. 35. A ureteral stent comprising the scaffold of claim 7, wherein the shell comprises chitosan and wherein the shell allows directional flow of liquid. 36. A method for regenerating blood vessels, the method comprising implantation of the scaffold of claim 7 connecting two or more blood vessels in a subject in need thereof. 37. The scaffold of claim 2, wherein the AAV vector is functionalized with one or more exogenous proteins. 38. The scaffold of claim 7, wherein the scaffold comprises one or more porous cores which comprise chitosan. 39. The scaffold of claim 38, wherein the scaffold comprises one or more porous cores which comprise a chitosan-nanocellulose composite. 40. The scaffold of claim 7, wherein the scaffold comprises one or more porous cores which comprise about 1-10% w/v collagen-nanocellulose. 41. The scaffold of claim 7, wherein the scaffold comprises one or more porous cores which comprise about 1-10% w/v chitosan-nanocellulose. 42. A scaffold, comprising: - 54 - 3878724.v1 Docket No.5200.2392-001 (INV-23052) a freeze-cast chitosan-based shell and a freeze-cast collagen-based core, wherein the shell, the core, or both the shell and the core are porous. - 55 - 3878724.v1