WO2025255571A1 - Ask neurogenic and neurotrophic reprogramming of the skin - Google Patents

Abstract

Methods and compositions for preventing and/or treating nerve damage, e.g., nerve damage associated with diabetic polyneuropathy, are provided herein. The methods comprise administering ASK, a cocktail of transcription factors, to the subject, which maintains or increases sensation. The presently disclosed subject matter further comprises methods and compositions for transplanting skin tissue administered with ASK.

Description

ASK NEUROGENIC AND NEUROTROPHIC REPROGRAMMING OF THE SKIN

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/657,536 filed June 7, 2024, and U.S. Provisional Application No. 63/727,915 filed December 4, 2024, priority to each of which is claimed, and the contents of each of which are incorporated by reference in their entireties.

SEQUENCE LISTING

A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on May 30, 2025, is entitled “072396.1085_ST26. xml”, and is 26,657 bytes in size

FIELD OF THE INVENTION

The disclosed subject matter relates to methods for reprogramming skin fibroblasts. The disclosed subject matter further relates to methods for treating or preventing diabetic neuropathy of the skin.

BACKGROUND

Diabetic polyneuropathy (DPN) is the degradation of nerves causing pain and numbness that affects a large number of diabetic patients. There is currently no known cure for DPN. Previous research evaluated methods of treating DPN through neurogenic reprogramming of skin in vivo [16, 182], These methods employed tissue nanotransfection (TNT) technology for delivering transcription factors to the skin. However, these methods failed to rescue the loss of sensation in the skin. As such, there remains a need for new therapies for treating DPN that are capable of restoring skin sensation to patients.

SUMMARY

The presently disclosed subject matter provides methods of preventing and/or treating nerve damage in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOX10; and c. a nucleic acid encoding KROX20. In certain embodiments, ASK comprises ASO-miR200b. In certain embodiments, ASK comprises a nucleic acid encoding ASO-miR200b. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK is administered using tissue nanotransfection. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO- miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1 : 10: 1 to about 10:1:10; from about 1 : 10: 1 to about 1:1:1, from about 1:1:1 to about 10:1:10, from about 1 : 1 : 10 to about 10:10:1, from about 1 : 1 : 10 to about 1 : 1 : 1, or from about 1:1:1 to about 10:10:1. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2: 1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10:1 to about 1:1, or from about 1 : 1 to 1 : 10. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10: 1, about 5: 1, about 2: 1, about 1 : 1, about 1 :2, about 1 :5, or about 1 : 10. In certain embodiments, the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 1 : 1. In certain embodiments, the nerve damage is associated with diabetic polyneuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or pre-diabetic type II diabetes. In certain embodiments, ASK is administered to skin tissue of the subject. In certain embodiments, the skin tissue comprises skin fibroblasts. In certain embodiments, the skin fibroblasts are reprogrammed to a cell population comprising Schwann cells. In certain embodiments, the method further comprises transplanting skin tissue administered with ASK to another location of the subject.

The presently disclosed subject matter further provides pharmaceutical compositions comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO- miR200b; b. a nucleic acid encoding SOXIO; and c. a nucleic acid encoding KROX20. In certain embodiments, ASK comprises ASO-miR200b. In certain embodiments, ASK comprises a nucleic acid encoding ASO-miR200b. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOXIO to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1:10:1 to about 10:1:10; from about 1 : 10: 1 to about 1:1:1, from about 1 : 1 : 1 to about 10:1:10, from about 1:1:10 to about 10:10:1, from about 1:1:10 to about 1:1:1, or from about 1:1:1 to about 10:10:1. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO- miR200b to nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2:1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1. In certain embodiments, the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10:1 to about 1:1, or from about 1:1 to 1:10. In certain embodiments, the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10. In certain embodiments, the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 1:1. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the skin tissue comprises skin fibroblasts that are reprogrammed to a cell population comprising Schwann cells. In certain embodiments, the skin tissue comprises non-reprogrammed skin fibroblasts.

The presently disclosed subject matter further provides an in vivo method of enhancing nerve innervation in a skin graft in a subject in need thereof, comprising administering to the skin graft a therapeutically effective amount of a composition comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOXIO; and c. a nucleic acid encoding KROX20. In certain embodiments, ASK comprises ASO-miR200b. In certain embodiments, ASK comprises a nucleic acid encoding ASO-miR200b. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO- miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO- miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng. In certain embodiments, ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK is administered using tissue nanotransfection. In certain embodiments, the molar ratio of ASO- miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10: 1 : 1 to about 1:10:10, from about 10: 1 : 1 to about 1:1:1, from about 1 : 1 : 1 to about 1:10:10, from about 1 : 10: 1 to about 10:1:10; from about 1:10:1 to about 1:1:1, from about 1:1:1 to about 10:1:10, from about 1:1:10 to about 10:10:1, from about 1 : 1 : 10 to about 1 : 1 : 1, or from about 1 : 1 : 1 to about 10: 10: 1. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2:1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10:1 to about 1 : 1, or from about 1 : 1 to 1 : 10. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 1:1. In certain embodiments, the skin graft comprises skin fibroblasts.

The presently disclosed subject matter provides methods of preventing and/or treating nerve damage in a subject in need thereof, comprising administering a therapeutically effective amount of: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOX10; and c. a nucleic acid encoding KROX20. In certain embodiments, the ASO- miR200b or a nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOX10, and the nucleic acid encoding KROX20 are administered sequentially. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO- miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered in an amount ranging from between about 1 ng and about 500 ng. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered in an amount ranging from between about 150 ng and about 200 ng. In certain embodiments, the nucleic acid encoding SOX10 is administered in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, the nucleic acid encoding SOX10 is administered in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, the nucleic acid encoding KROX20 is administered in an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, the nucleic acid encoding KROX20 is administered in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered using tissue nanotransfection. In certain embodiments, the nucleic acid encoding SOX10 is administered using tissue nanotransfection. In certain embodiments, the nucleic acid encoding KROX20 is administered using tissue nanotransfection. In certain embodiments, the nerve damage is associated with diabetic polyneuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or prediabetic type II diabetes. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOX10, and the nucleic acid encoding KROX20 are administered to skin tissue of the subject. In certain embodiments, the skin tissue comprises skin fibroblasts. In certain embodiments, the skin fibroblasts are reprogrammed to a cell population comprising Schwann cells.

DESCRIPTION OF DRAWINGS

The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, without departing from the scope of this disclosure.

Figures 1A-1C illustrate an approach for rescuing Diabetic Peripheral Neuropathy (DPN) in db/db mice using tissue nanotransfection of ASK (TNTASK). Figure 1A shows a schematic diagram showing experimental procedure in db/+ and db/db mice. Mice were treated with either control plasmid and control LNA, or the combination of Sox 10, Krox20, and LNA anti-miR-200b by TNT. Figures 1B-1C show confirmation of the TNT -based delivery of reprograming factors to db/db mouse skin 72 hours post TNT evident by abundance of SoxlO, Krox20 (Figure IB) and reduced level of miR200b (Figure 1C). db/db: BKS.Cg- Dock7m+/+Leprdb/J strain; db/+: heterozygous littermate controls.

Figures 2A-2C show neurogenic changes associated with fibroblasts from dermal papillary origin (MEF2C). TNTASK reprograming of the skin resulted in neurogenic fate change of dermal fibroblasts as indicated by the expression of NF200 in dermal papillary fibroblasts. Skin samples were collected two weeks post TNT. Figure 2A shows representative images of immunohistochemistry (IHC) with merged channels of MEF2C and NF200 and their colocalization patterns. Scale bar indicates 20 pm. Figure 2B shows colocalization of MEF2C and NF200 determined by Pearson's correlation (n=6; one-way ANOVA). Figure 2C shows the MEF2C and NF200 colocalized elements quantified across the groups and plotted graphically. Data are expressed as mean ± SEM (n= 5-6 in db/+ mock and db/db ASK; n=6 in d/db mock, analyzed by one-way ANOVA).

Figures 3A-3B show that TNTASK increased NGF production in fibroblasts from dermal sheath origin. TNTASK reprograming of the skin results in increased NGF production in fibroblasts from dermal sheath origin as indicated by the expression of NGF in dermal sheath fibroblasts (MYL9). Skin samples were collected two weeks post TNT. Figure 3A shows representative images of IHC with merged channels of MYL9 and NGF and their colocalization patterns (scale bar indicates 20 pm). Figure 3B shows colocalization of MYL9 and NGF determined by Pearson's correlation (n=6; one-way ANOVA). Data are expressed as mean ± SEM (n= 5-6 in db/+ MOCK and db/db ASK; n=6 in d/db MOCK, analyzed by one-way ANOVA). The presented data corresponds to samples collected two weeks post TNT.

Figures 4A-4D show that TNTASK increased NGF production in skin of db/db mice. TNTASK reprograming of the skin results in increased NGF production as indicated by the expression of NGF in Skin samples were collected two weeks post TNT. Figures 4A-4B show immunostaining of NGF in epidermis after two weeks (Figure 4A; scale bar indicates 20 pm) and quantification of the IHC images (Figure 4B). Figures 4C-4D show immunostaining of NGF in epidermis after 21 weeks (Figure 4C) and quantification of the IHC images (Figure 4D). Scale bar indicates 50 pm. Data in Figures 4B was analyzed by one-way ANOVA and Figure 4C was analyzed by Students T-test.

Figure 5 shows that TNTASK increased neurotrophins (NGF & NP2) production in the footpad skin of db/db mice. NGF, NP2 and BDNF were assessed in footpad skin using custom conjugated antibodies with nucleotide barcodes for multiplexed detection through PhenoCycler-Fusion 2.0. Footpads were harvested 21 weeks post TNTASK. The analysis was performed on 5 pm thick plantar skin. Scale bar indicates 10 pM.

Figures 6A-6D show that TNTASK improved the mechanical allodynia and thermal hyperalgesia in the diabetic mice. Figure 6A shows schematic illustration of the Dynamic plantar apparatus with vonfrey filament exerting mechanical pressure on the plantar surface of the mice’s hind paws. Figure 6B shows schematic illustration of the Thermal plantar apparatus. Figure 6C shows hind paw withdrawal latency in response to the vonfrey mechanical pressure. Figure 6D shows hind paw withdrawal latency in response to the heat generated by the infrared light was measured at 2,6,18 weeks post TNT resulting in a decreased hyperalgesia in the TNTASK treated group (db/db ASK) compared to the diabetic group (db/db MOCK), almost equal to the nondiabetic control animals (db/+ MOCK). All the data are represented as mean ± SEM; one-way ANOVA, n=6-8 (db/+MOCK; db/db MOCK; db/db ASK).

Figure 7 shows nerve conductivity measurements of diabetic mice and littermate controls after 1 Iw post TNTASK. The data are represented as mean ± SEM; one-way ANOVA n=9 (db/+MOCK); 8 (db/db MOCK) and 8 (db/db ASK).

Figure 8 shows that reprogramming with TNTASK protected PGP9.5+ nerve fiber density in footpad skin of db/db mice. PGP9.5 -immunoreactive fibers in 5 pm thick plantar footpads of diabetic mice are shown. Innervation in the footpad was quantified by the area of the epidermis occupied by PGP 9.5 -immunoreactive fibers. Footpads were collected 21 weeks post TNTASK.

Figure 9 show that reprogramming with TNTASK protected PGP9.5+ and Tuj l nerve fiber density in footpad skin of db/db mice. PGP9.5 and Tuj l -immunoreactive fibers (intraepidermal nerve fibers (lENFs) in the 5 pm thick footpad skin were concurrently assessed using custom conjugated antibodies with nucleotide barcodes for multiplexed detection through PhenoCycler-Fusion 2.0. Footpads were collected 21 weeks post TNTASK.

Figures 10A-10B show that TNTASK associated cytokine leads from the protein array and their validation by ELISA. Figure 10A shows cytokines having more than 1.5-fold change across the groups pertaining to the TNTASK only (n=6 pooled). Figure 10B show the ELISA based validation of cytokines PTX2 (NP2) using the individual samples (n=4-5 in db/+ MOCK; 6 in both db/db MOCK and db/db ASK). The presented data corresponds to samples collected two weeks post-TNT.

Figure 11A-11D show NP2 expression at various timepoints post TNT. Figure 11A shows representative figures of NP2 IHC at 2w post TNT. Figure 1 IB shows quantification of the intensity profile of NP2 2w post TNT (n=5 in db/+ MOCK; 6 in db/db MOCK and db/db ASK). Figure 11C shows representative figures of NP2 IHC at 21w post TNT. Figure 11D shows quantification of the intensity profile of NP2, 21 w post TNT (n=5 in db/+ MOCK; 6 in db/db MOCK and db/db ASK). Data in B and E were analyzed by one-way ANOVA.

Figures 12A-12D show scRNA-seq data (GSE142471; 2 unwounded skin and 3 wounded at d4 post-wound; 5 mice) of 26,723 cells were analyzed. Briefly, scRNA-seq samples were filtered to exclude low quality cells, normalized using log transformation with 10,000 as a scaling factor, and clustered using Seurat in R. Figure 12A shows UMAP plot with identified cell types. Figure 12B shows a second round of clustering using the Louvain algorithm in Seurat for extracted fibroblasts 8,920 cells). Figure 12C shows fibroblast subsets annotated to specific fibroblasts subtypes [34], Figure 12D shows dotplot representing marker genes for fibroblast subtype identification.

Figures 13A-13E show post-TNT (72h) in situ hybridization (ISH). Figures 13A-13C shows gene expression for SOX10 (Figure 13 A), KROX20 (Figure 13B), and miR200b (Figure 13C). EP: Empty Plasmid, n=3-4, *p<0.05. Figure 13D shows ISH counterstained with DAPI Scale bar indicates 0.01 mm. Figure 13E shows that ASO inhibits epidermal (epi) miR200b expression (brightfield).

Figure 14 shows Spatial protemics: Akoya PhenoCycler Fusion in situ workflow permits detection of multiple cellular proteins of diverse lineages in the same tissue.

Figures 15A-15B show TNT ASK rescued PGP9.5+ nerve fiber density in footpad skin of 27wk old db/db mice. Figure 15 A shows relative density (RD) of intra-epidermal nerve fibers in the footpad as detected by IHC. Sample nerve fiber staining shown. Footpads were collected 21 w post-ASK treatment. Mean SEM; one-way ANOVA, *p<0.05, n=6-7. Figure 15B shows spatial proteomics (Akoya) imaging of merged GAP43+ (regenerative neuron) and PGP9.5+ in footpads. Skin epidermis with white arrowheads showing higher abundance of colocalized GAP43/PGP9.5 fibers following TNT ASK. Footpads were collected 21 wk post-TNT treatment. Scale bar indicates 40 pm.

Figures 16A-16C show that TNT ASK rescued MPZ in sciatic nerve (21 weeks post- TNT) and S100P in dorsal skin (2 wk post-TNT) of db/db diabetic mice. Figures 16A-16C show quantification of IHC & Akoya Phenocycler spatial proteomics data were quantified for MPZ (Figure 16A), Substance P (Figure 16B), and SlOOp (Figure 16C). Substance P rescue was not significant. All data represented as mean SEM; one-way ANOVA, **p<0.001, *p<0.05, ns=non-significant.

Figures 17A-17C show spatial proteomic (Akoya Phenocycler Fusion) analyses of 27wk old db/db footpad. TNT was performed at 6wk of age and outcomes measured 21 weeks thereafter. Figure 17A shows visualization of the nerve bundles [sensory neuron (NF200); regenerative neurons (GAP43); differentiating neurons (TUJ1)] with Schwann cells [SlOOb (regenerative); myelinating (MBP); differentiation (SOXIO)] and sensory transducing receptors [Navi.6 (Nociceptor) and TRPV4 (mechanoceptor)] in post-TNT skin along with an endothelial marker (CD31) and merged nerve markers (N). Scale bar indicates 0.4mm. Figure 17B shows a heatmap which reveals decreased expression of these markers in the db/db mice with DPN. Figure 17C shows that small and large nerve fiber density was less in db/db, which was rescued by TNT ASK.

Figures 18A-18B show that TNT ASK treatment of the hind limb skin improved mechanical allodynia and thermal sensitivity in diabetic mice. Figures 18 A- 18B show hind paw withdrawal latency in response to Von Frey mechanical pressure (Figure 18 A) or the thermal plantar heat (Figure 18B) generated by the infrared light (50W intensity). All measurements were taken at 2- and 18-week post-TNT. Results show improved thermoception and mechanical allodynia in TNTASK treated group compared to the TNTEP group. Control db/+ EP treated animals are represented as a dashed line (mean data). Mean SEM; unpaired T-test, **p<0.001, *p<0.05, ns=not significant. TNTABM was not effective.

Figures 19A-19F show that TNTASK restored A-fiber myelination and Remak bundle formation while also reducing myelin infoldings. 21 -wk post-TNT data. Morphometric analysis (TEM) was performed on a transection of the sciatic nerve trunk. Figures 19A-19C show myelin fiber density (Figure 19 A), myelinated area (Figure 19B), and myelin infoldings (Figure 19C) from TEM imaging. Figure 19D shows the number of Remak bundles in the cross-sectional area of the sciatic nerve. Figure 19E shows axons per Remak bundle, mean SEM. one-way ANOVA, n=3-4, **p<0.001, *p<0.05. Figure 19F shows TEM images of sciatic nerves in 27-week-old mice. Scale bar indicates 10 pm. Red arrow indicates myelin infoldings in A-fibers. White outlines highlight Remak bundles.

Figures 20A-20F show that TNTASK improved nerve conductivity in db/db mice. Figures 20A-20B show that nerve conductivity in non-diabetic m+/db and db/db mice was quantified utilizing synaptic transistors (Figure 20A) in response to stimulation (Figure 20B) as reported [107], Figure 20C shows that the synaptic transistor was placed proximate to the sciatic nerve to form an ad hoc synaptic junction [107] which is excitable during neuronal communication. Figure 20D shows sciatic nerve cross-sections of db/+ and db/db. Scale bar indicates 500 nm. Figure 20E shows depolarization curves for db/+ and db/db. Figure 20F shows mean ionic nerve conductance in diabetic mice after 11 weeks post-TNTASK treatment. Mean SEM; unpaired t-Test. n=6-9, *p<0.05.

Figures 21A-21C show that neurogenic TUJ1 co-localized with dermal papilla B and reticular fibroblasts following TNTASK of db/db diabetic skin. Figures 21A-21B show that TNTASK enhanced TUJ1 co-localization with: dermal papilla (MEF2C) (Figure 21A) and reticular (MGP) (Figure 2 IB) fibroblasts 2 weeks post-TNT. Figure 21C shows that TNTASK rescued TUJ1 expression in dorsal skin of db/db as detected by Akoya spatial proteomics analysis, mean SEM; one-way ANOVA, *p<0.05,ns=not significant.

Figures 22A-22D show that TNTASK enriched neurofilament (NF200) in db/db skin which colocalized with dermal papilla fibroblasts. Figures 22A-22B show that NF200+ neurofilament were rescued (Figure 22A) and colocalized (Figure 22B) with MEF2C+ dermal papilla fibroblasts following TNTASK treatment of db/db mouse skin. Figure 22C shows NF200 expression which was quantified in Figure 22A. Figure 22D shows colocalization which was quantified in Figure 22B. Mean SEM; one-way ANOVA, n=5-6, 8-week old mice,*p<0.05; **p<0.001; ns = not significant. Scale bar indicates 20 pm.

Figures 23A-23H show spatial biology of the skin. Figure 23A shows Xenium work flow. Figure 23B shows cell segmentation image of skin showing cells with like transcriptome in same color; overall tissue architecture display epidermis (diamond) and dermis (triangle) - demarcations apply to C & D as well. Figure 23 C shows magenta dots depicting nerve specific transcripts (coding PGP9.5 & NF200). Dots in epidermis mark nerve terminals. Dots in the dermis mark dermal nerve fibers. Green dots in dermis represent COL1A2 genes pan marking fibroblasts. Figure 23D shows blue dots in dermis marking MEF2C+ dermal papillary fibroblasts. Thus visualization of specific fibroblast subpopulation is enabled. Figure 23E shows zoomed in image of a single Vasculogenic Fibroblast expressing COL1 A2 (green dots) and vascular genes vWF/CDH5 (red dots). Figures 23F shows peripheral nerve fiber (PGP9.5) in green surrounded by NGF (red) expressing fibroblasts in dermis. Figure 23G shows that fibroblasts within 10 micron of the nerve fiber express more NGF than ones away (>35 micron) from the nerve fiber. Figure 23H shows peripheral nerve fascicle in non-diabetic and diabetic skin.

Figure 24A-24D show TNTASK induced neurotrophic enrichment of the skin with induced NGF as one marker. Figure 24A shows study design. Figures 24B-24C show ELISA data at 6 days post- TNT (Figure 24B) and 21 days post- TNT (Figure 24C). mean SEM; unpaired t-Test, n=4-10, *p<0.05, **p<0.001. Figure 24D shows that NGF co-localized with MEF2C (DP marker) and MGP (reticular fibroblast marker) 2wk post- TNT ASK. Pearson’s coefficient data show that NGF colocalized with MGP+ and MEF2C+ fibroblasts independent of treatment or diabetic condition.

Figures 25A-25B show isolation of fibroblast-specific exomes plasmid design and validation. Figure 25 A shows Coll Al promoter-driven plasmids encoding CD9, CD63 or CD81 with “in frame” mNeonGFP (mN-GFP) reporter [76], Figure 25B shows fibroblastspecific expression of mN-GFP. Scale bar indicates 20pm.

Figures 26A-26C show super-resolution dSTORM imaging of single exosome. Figure 26A shows dSTORM image showing single particle localization. Scale bar indicates 10 pm. Figure 26B shows single exosome CD63+ and ALIX+ (exosome markers). Scale bar indicates 50 pm. Figure 26C Single exosome membrane staining.

Figures 27A-27H show isolation, characterization, and nanoscale imaging of Exonb. Figure 27A shows Exonb isolation procedure. SEM images show mNeonGFP-tagged immunomagnetic beads, beads with exosomes trapped and post-elution. Figure 27B shows nanotracking analysis of particle size peak at 103 nm. Figure 27C shows TEM image of Exonb. Figure 27D shows dSTORM image of Exonb showing the presence of tetraspannins markers CD9, CD63 and CD81. Inset. Zoomed image of an exosome with 3 color localization. Figure 27E shows antibody array of Exonb showing specific markers of exosomes. Figure 27F shows quantitative analysis using CODI software showing distribution of tetraspannins markers on Exonb. Figure 27G shows endosomal pathway showing early, intermediate and late endosomal markers. Figure 27H shows flow cytometry of murine Exonb on mN-GFP-trap magnetic beads showing binding of RAB5APE and RAB7AFITC antibody. The presence of RAB5APE and RAB7AFITC validates endosomal origin.

Figures 28A-28D show that fibroblast targeted dynasore encapsulates fluorescent PLGA nanoparticles (Npdynasore). Figure 28A shows fibroblast targeted Npdynasore preparation. EDC = l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Figure 28B shows fluorescence conjugation. Figure 28C shows Fourier Transform Infrared Spectroscopy showing antibody conjugation. Figure 28D shows NTA showing no effect on size. Before (black) and after conjugation (red).

Figures 29A-29E shows “Eat Me Not” exosomes to block cellular update. CD47-tagged Exo will be produced by wound-tissue but not taken up by fibroblasts. Figure 29A shows plasmid design. Figure 29B shows experimental design. Figure 29C shows super-resolution confocal microscopic images showing GFP and RFP expression (black arrowhead) in multivesicular bodies (MVB) in cells 24h post-transfection. Scale bar indicates 10 pm. Figure 29D shows flow cytometry of the exosomes isolated from conditioned media post transfection showing the presence of both RFP and GFP reporter. Figure 29E shows live-cell confocal images showing blocked uptake (black arrowhead) of “eat me not” Exo by cells using LSM 880. Scale bar indicates 10pm.

Figures 30A-30B show TNT ASK rescue of peripheral limb perfusion in db/db diabetic mice. Blood flow studied using PeriScan™ PSI laser speckle flowmetry. Figure 30A shows that TNTASK improved perfusion in feet of db/db mice (n = 6). Figure 30B shows immunohistochemical analysis showing that vascularization marker CD31 was lower in db/db than db/+. This was rescued by TNTASK. These data are represented as mean SEM; one-way ANOVA, 8 week old mice, ***p<0.001, *p<0.05.

Figures 31A-31B show that TNTASK induced NP2 in db/db mouse skin. Figure 31A shows ELISA of NP2 21wk post- TNT. Figure 3 IB shows post-TNTASK NP2 and NGF expression were co-localized. Scale bar indicates 50 pm. Mean SEM; one-way ANOVA or unpaired t-Test, n=4-6, 27wk old mice (ELISA) ***p=0.05.

Figures 32A-32F show that TNTASK suppressed AMP ARI and mGLURl expression in dorsal skin of db/db diabetic mice. Figures 32A-32B show AMP ARI (Figure 32A) and mGLURl (Figure 32B) expression at 2 weeks post-TNT. Figures 32C-32D show AMP ARI (Figure 32C) and mGLURl (Figure 32D) expression at 21 weeks post-TNT. Mean ± SEM; one-way ANOVA, *p<0.05. Figures 32E-32F show H4C images for 2 weeks post-TNT (Figure 32E) and 21 -wk post-TNT (Figure 32F). Scale bar indicates 50 pm.

Figure 33 illustrates an approach for rescuing metabolically acquired Peripheral Neuropathy (PN) induced by a high-fat diet (HFD) in C57BL6/J mice (Diet-Induced Obesity (DIO)) using TNTASK. Schematic diagram showing experimental procedure in C57BL6/J and DIO mice. C57BL6/J and DIO mice were treated with either control plasmid, and control LNA or SoxlO, Krox20 and LNA anti-miR-200b by TNT. C57BL6/J mice: fed with standard diet (SD) (10% k.cal) and DIO mice: diet-induced obesity mice are the C57BL6/J mice fed with HFD (60% k.cal).

Figures 34A-34D show that TNTASK improved the mechanical allodynia and thermal hyperalgesia associated with metabolically acquired peripheral neuropathy in DIO mice. Figure 34A shows schematic illustration of the Dynamic plantar apparatus with vonfrey filament exerting mechanical pressure on the plantar surface of the mice’s hind paws. Figure 34B shows schematic illustration of the Thermal plantar apparatus with infrared light stimuli at 50-mW per square cm-intensity used to assess the thermal hyperalgesia of both the hind paws. Figure 34C shows hind paw withdrawal latency in response to the mechanical pressure generated by the monofilament was measured at baseline, 2, and 11 weeks post TNT resulting in an improvement of neuropathy associated mechanical allodynia in the TNTASK treated group (HFD ASK) compared to the diabetic group (HFD mock), almost equal to the nondiabetic control animals (SD mock). Figure 34D shows hind paw withdrawal latency in response to the heat stimuli generated by the IR light was measured at baseline, 2, and 11 weeks post TNT resulting in a decreased hyperalgesia in the TNTASK treated group (HFD ASK) compared to the diabetic group (HFD mock), almost equal to the nondiabetic control animals (SD mock). All the data are represented as mean ± SEM; one-way ANOVA, n=9-18 (SD mock; HFD mock; HFD ASK).

Figures 35A-35D show that TNTASK improved the hypoesthesia associated with metabolically acquired peripheral neuropathy in DIO (HFD) mice. Figure 35 A shows schematic illustration of the Hot plate test apparatus with heat stimuli generated by heating and maintaining the plate at 50°C exerting heat sensitivity along with nociception on the plantar surface of the mice’s hind paws. Figure 35B shows latency in lick the hind paw in response to the aesthetic stimuli generated by heat was measured at baseline, 2, and 11 weeks post TNT resulting mitigation of hypoesthetia in the TNTASK treated group (HFD ASK) compared to the diabetic group (HFD mock), almost equal to the nondiabetic control animals (SD mock). Figures 35C-35D show latency to wall rearing on hind paws (Figure 35C) and jumping (Figure 35D) as an avoidance response to the aesthetic stimuli of hot plate was measured at baseline, 2, and 11 weeks post TNT resulting early rearing response in the TNTASK treated group (HFD ASK) compared to the diabetic group (HFD mock), almost equal to the nondiabetic control animals (SD mock). However similar jumping response was seen at 11 weeks post TNT. All the data are represented as mean ± SEM; one-way ANOVA, n=9-18 (SD mock; HFD mock; HFD ASK.

Figures 36A-36C show that TNTASK rescue the peripheral limb perfusion in metabolically acquired peripheral neuropathy in DIO (HFD) mice. Blood flow studied using PeriScan™ PSI laser speckle flowmetry. Figure 36A shows baseline perfusion changes between the SD and HFD fed C57BL6/J mice. n=10-30. Figures 36B-36C provide images (Figure 36B) and quantification (Figure 36C) supporting that improved perfusion in the hind limbs of HFD fed mice (DIO) by TNTASK treatment (HFD ASK, 2 weeks post treatment) was observed compared to the mock treatment (HFD mock) almost to the level of the SD fed control mice (SD mock). All the data are represented as mean ± SEM; one-way ANOVA, n=9-18 (SD mock; HFD mock; HFD ASK).

Figure 37 shows that TNT ASK improved nerve conductivity in mice with metabolically acquired peripheral neuropathy. Progression of PN in SD and HFD fed C57BL/6 mice was quantified using IXTA high-voltage stimulator and software, LabScribe. Stimulator with a custom amplitude stimulation controller (StimD) was placed in the Achilles tendon of the limb with two recording electrodes; proximal (Rec P) and distal (Rec D) placed 1.5cm apart at the sciatic nerve location to measure the time taken for the stimuli to travel from distal to proximal location, which gives us the conduction velocity of sciatic nerve. TNT ASK treatment improved the conduction velocity in the sciatic nerve location of the HFD fed mice compared to the mock treatment; p=0.0004, n=9-18 animals per group. All the data are represented as mean ± SEM; one-way ANOVA.

Figures 38A-38B show that reprogramming with TNT ASK protected PGP9.5+ nerve fiber density in footpad skin of metabolically acquired peripheral neuropathy in DIO (HFD) mice. Figure 38A shows PGP9.5 -immunoreactive fibres in 5mm thick plantar footpads of SD and HFD fed mice. Innervation in the footpad was quantified by the area of the epidermis occupied by PGP 9.5 -immunoreactive fibres. Footpads were collected 11 weeks post TNTASK. Figure 38B shows that TNTASK treatment ameliorated the loss of PGP9.5 fiber density in the footpads of the HFD fed mice compared to the mock treatment TNTASK group p=0.004, n=9- 18 animals per group. All the data are represented as mean ± SEM; one-way ANOVA.

DETAILED DESCRIPTION

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

1. Definitions;

2. ASK Therapy; and

3. Methods of Treatment.

1. Definitions

To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them.

As used in this specification and the following claims, the terms “comprise” (as well as forms, derivatives, or variations thereof, such as “comprising” and “comprises”) and “include” (as well as forms, derivatives, or variations thereof, such as “including” and “includes”) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms “comprise” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Other than where noted, all numbers expressing quantities of ingredients, reaction conditions, geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

Accordingly, these terms are intended to not only cover the recited element(s) or step(s), but can also include other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms “a”, “an”, and “the” when used in conjunction with an element can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Therefore, an element preceded by “a” or “an” does not, without more constraints, preclude the existence of additional identical elements.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. A range can be construed to include the start and the end of the range. For example, a range of 10% to 20% (i.e., range of 10%-20%) can includes 10% and also includes 20%, and includes percentages in between 10% and 20%, unless explicitly stated otherwise herein.

It is understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), that while specific reference of each of the various individual and collective combinations and permutations of these elements may not be explicitly disclosed, each is specifically contemplated and described herein.

As used herein, the term “optionally,” is meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “optionally includes an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

As used herein, “administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including topical, transcutaneous, transdermal, intra-joint, intradermal, intralesional, via an implanted reservoir, subcutaneous, , and the like. “Concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

As used herein, the terms “decrease”, “decreasing”, “reduce”, “reducing”, “reduction”, “inhibit”, “inhibiting”, or “inhibition” refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease. In certain embodiments, the decrease is statistically significant.

As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.

As used herein, the term “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

As used herein, a “therapeutically effective amount” or “effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term “therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

As used herein, the term “pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

As used herein, the term “control” refers to an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.” A positive control demonstrates that a particular result can be achieved under experimental conditions. A negative control demonstrates that a particular result is not achieved under baseline conditions.

As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or a veterinary patient.

As used herein, the term “patient” refers to a subject under the treatment of a clinician, e.g., physician. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.

As used herein, the term “neuropathy” defines a condition or disease involving dysfunction of one or more peripheral nerves, causing weakness, numbness, and pain from nerve damage, usually in the hands and feet. Non-limiting examples of neuropathy include peripheral neuropathy, diabetic neuropathy, diabetic polyneuropathy. “Peripheral neuropathy” is defined as neuropathy affecting the peripheral nervous system. “Diabetic neuropathy” is defined as neuropathy that results in a subject with diabetes (type I or type II diabetes, or prediabetic type II diabetes). “Diabetic polyneuropathy” is defined as diabetic neuropathy that affects multiple sites.

As used herein, the term “skin stroma cells” encompasses mesenchymal cells present in the dermis layer adjacent to the epidermis that release growth factors that promote cell division. For example, skin stroma cells include fibroblasts and pericytes.

As used herein, the term “fibroblast” refers to mesenchymal cell types in the dermis connective tissue. Non-limiting examples of fibroblasts include reticular, dermal sheath (DS), dermal papilla (DP), fascial, and papillary fibroblasts.

As used herein, the term “induced neuronal cell (iN)” refers to non-neuronal cells which have been converted into a neuronal lineage. In certain embodiments, the methods herein comprise converting skin fibroblasts into iN.

2. ASK Therapy

ASK therapy comprises the administration of: 1) an antisense oligonucleotide (ASO) against miR200b (ASO-miR200b), 2) nucleic acid encoding SOXIO, and 3) nucleic acid encoding KROX20. The present disclosure demonstrates methods for preventing and/or treating nerve damage using ASK therapy. In certain embodiments, the nerve damage is associated with diabetic polyneuropathy.

ASK comprises neurogenic and neurotrophic reprogramming of skin cells in vivo wherein skin fibroblasts are converted Schwann cells and induced neuronal cells (iN). ASK induces changes in the tissue microenvironment that can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from loss under conditions of diabetes. For example, ASK can maintain or increase sensation of the skin, e.g., reducing numbness or increasing sensitivity to touch.

2, 1 Nucleic acid encoding ASQ-miR200b

In certain embodiments, ASK comprises a nucleic acid molecule encoding ASO- miR200b or a functional fragment thereof. In certain embodiments, ASO-miR200b has a nucleic acid sequences that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/NCBI database accession no. NR 029639.1. In certain embodiments, the nucleic acid molecule encoding ASO-miR200b can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/NCBI database accession no. NR 029639.1, that do not significantly alter the function or activity of the ASO-miR200b.

In certain embodiments, the ASO-miR200b is a human ASO-miR200b. In certain embodiments, the nucleic acid molecule encoding ASO-miR200b comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 1, which is provided below.

CCAGCTCGGGCAGCCGTGGCCATCTTACTGGGCAGCATTGGATGGAGTCAGGTCT CTAATACTGCCTGGTAATGATGACGGCGGAGCCCTGCACG (SEQ ID NO.: 1)

In certain embodiments, ASO-miR200b has a nucleic acid sequences that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/NCBI database accession no. NR 029587.1. In certain embodiments, the nucleic acid molecule encoding ASO-miR200b can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/NCBI database accession no. NR 029587.1, that do not significantly alter the function or activity of the ASO-miR200b. In certain embodiments, the ASO-miR200b is a mouse ASO-miR200b.

In certain embodiments, the nucleic acid molecule encoding ASO-miR200b comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 2, which is provided below. GCCGTGGCCATCTTACTGGGCAGCATTGGATAGTGTCTGATCTCTAATACTGCCT GGTAATGATGACGGC (SEQ ID NO.: 2)

The ASO-miR200b can comprise modifications for preventing degradation following administration. In certain embodiments, ASO-miR200b comprises at least one locked nucleic acid (LN A).

In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount ranging from between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM.

In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount ranging from between about 0.5 ng/pL and about 100 ng/pL, between about 0.5 ng/pL and about 75 ng/pL, between about 0.5 ng/pL and about 50 ng/pL, between about 0.5 ng/pL and about 25 ng/pL, between about 0.5 ng/pL and about 10 ng/pL, between about 0.5 ng/pL and about 5 ng/pL, between about 0.5 ng/pL and about 1 ng/gL, between about 1 ng/gL and about 100 ng/gL, between about 1 ng/gL and about 75 ng/gL, between about 1 ng/gL and about 50 ng/gL, between about 1 ng/gL and about 25 ng/gL, between about 1 ng/gL and about 10 ng/gL, between about 1 ng/gL and about 5 ng/gL, between about 5 ng/gL and about 100 ng/gL, between about 5 ng/gL and about 75 ng/gL, between about 5 ng/gL and about 50 ng/gL, between about 5 ng/gL and about 25 ng/gL, between about 5 ng/gL and about 10 ng/gL, between about 10 ng/gL and about 100 ng/gL, between about 10 ng/gL and about 75 ng/gL, between about 10 ng/gL and about 50 ng/gL, between about 10 ng/gL and about 25 ng/gL, between about 25 ng/gL and about 100 ng/gL, between about 25 ng/gL and about 75 ng/gL, between about 25 ng/gL and about 50 ng/gL, between about 50 ng/gL and about 100 ng/gL, between about 50 ng/gL and about 75 ng/gL, or between about 75 ng/gL and about 100 ng/gL. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of at least about 0.5 ng/gL, at least about 1 ng/gL, at least about 2 ng/gL, at least about 3 ng/gL, at least about 4 ng/gL, at least about 5 ng/gL, at least about 6 ng/gL, at least about 7 ng/gL, at least about 8 ng/gL, at least about 9 ng/gL, at least about 10 ng/gL, at least about 25 ng/gL, at least about 50 ng/gL, at least about 75 ng/gL, or at least about 100 ng/gL. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount up to about 0.5 ng/gL, up to about 1 ng/gL, up to about 2 ng/gL, up to about 3 ng/gL, up to about 4 ng/gL, up to about 5 ng/gL, up to about 6 ng/gL, up to about 7 ng/gL, up to about 8 ng/gL, up to about 9 ng/gL, up to about 10 ng/gL, up to about 25 ng/gL, up to about 50 ng/gL, up to about 75 ng/gL, or up to about 100 ng/gL. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of about 0.5 ng/gL, about 1 ng/gL, about 2 ng/gL, about 3 ng/gL, about 4 ng/gL, about 5 ng/gL, about 6 ng/gL, about 7 ng/gL, about 8 ng/gL, about 9 ng/gL, about 10 ng/gL, about 25 ng/gL, about 50 ng/gL, about 75 ng/gL, or about 100 ng/gL.

In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng, between about 1 ng and about 400 ng, between about 1 ng and about 300 ng, between about 1 ng and about 200 ng, between about 1 ng and about 100 ng, between about 1 ng and about 50 ng, between about 50 ng and about 500 ng, between about 50 ng and about 400 ng, between about 50 ng and about 300 ng, between about 50 ng and about 200 ng, between about 50 ng and about 100 ng, between about 100 ng and about 500 ng, between about 100 ng and about 400 ng, between about 100 ng and about 300 ng, between about 100 ng and about 200 ng, between about 200 ng and about 500 ng, between about 200 ng and about 400 ng, between about 200 ng and about 300 ng, between about 300 ng and about 500 ng, between about 300 ng and about 400 ng, between about 400 ng and about 500 ng, In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of at least about 1 ng, at least about 5 ng, at least about 10 ng, at least about 15 ng, at least about 20 ng, at least about 25 ng, at least about 30 ng, at least about 35 ng, at least about 40 ng, at least about 45 ng, at least about 50 ng, at least about 55 ng, at least about 60 ng, at least about 65 ng, at least about 70 ng, at least about 75 ng, at least about 80 ng, at least about 85 ng, at least about 90 ng, at least about 95 ng, at least about 100 ng, at least about 125 ng, at least about 150 ng, at least about 175 ng, at least about 200 ng, at least about 225 ng, at least about 250 ng, at least about 275 ng, at least about 300 ng, at least about 325 ng, at least about 350 ng, at least about 375 ng, at least about 400 ng, at least about 425 ng, at least about 450 ng, at least about 475 ng, or at least about 500 ng. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount up to about 1 ng, up to about 5 ng, up to about 10 ng, up to about 15 ng, up to about 20 ng, up to about 25 ng, up to about 30 ng, up to about 35 ng, up to about 40 ng, up to about 45 ng, up to about 50 ng, up to about 55 ng, up to about 60 ng, up to about 65 ng, up to about 70 ng, up to about 75 ng, up to about 80 ng, up to about 85 ng, up to about 90 ng, up to about 95 ng, up to about 100 ng, up to about 125 ng, up to about 150 ng, up to about 175 ng, up to about 200 ng, up to about 225 ng, up to about 250 ng, up to about 275 ng, up to about 300 ng, up to about 325 ng, up to about 350 ng, up to about 375 ng, up to about 400 ng, up to about 425 ng, up to about 450 ng, up to about 475 ng, or up to about 500 ng. In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO- miR200b in an amount of about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 15 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 55 ng, about 60 ng, about 65 ng, about 70 ng, about 75 ng, about 80 ng, about 85 ng, about 90 ng, about 95 ng, about 100 ng, about 125 ng, about 150 ng, about 175 ng, about 200 ng, about 225 ng, about 250 ng, about 275 ng, about 300 ng, about 325 ng, about 350 ng, about 375 ng, about 400 ng, about 425 ng, about 450 ng, about 475 ng, or about 500 ng.

2,2 Nucleic acid encoding SOXIO

A second component of ASK is nucleic acid encoding SOXIO. In certain embodiments, the SOXIO is a human SOXIO. In certain embodiments, SOXIO has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/Gene ID no. 6663. In certain embodiments, the nucleic acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/Gene ID no. 6663, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank no. NM 006941.4. In certain embodiments, the nucleic acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank no. NM 006941.4, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, the nucleic acid molecule encoding SOXIO comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 3, which is provided below.

AGTCGCTCAGTCAGTCTCGGGCTGTCCGGCCAGGGTGGTTGGTGGTAAGGATTCA GGCTCCGTCCTAACGAGGCCGTGGCCTGAGGCTCAGGGCCCCCCGCCCCTCCCTC CCAGCCCACCAGCGTCACCTCCCAGCCCCGAGCTGGACCGCACACCTTGGGACA CGGTTTTCCACTTCCTAAGGACGAGCCCCAGACTGGAGGAGAGGTCCGAGGAGG TGGGCGTTGGACTCTTTGCGAGGACCCCGGCGGCTGGCCCGGGGGAGGCGGCCG AGGCGGCGGCGGCGGCGGCCGGGGGCGACATGGCGGAGGAGCAGGACCTATCG GAGGTGGAGCTGAGCCCCGTGGGCTCGGAGGAGCCCCGCTGCCTGTCCCCGGGG AGCGCGCCCTCGCTAGGGCCCGACGGCGGCGGCGGCGGATCGGGCCTGCGAGCC AGCCCGGGGCCAGGCGAGCTGGGCAAGGTCAAGAAGGAGCAGCAGGACGGCGA GGCGGACGATGACAAGTTCCCCGTGTGCATCCGCGAGGCCGTCAGCCAGGTGCT CAGCGGCTACGACTGGACGCTGGTGCCCATGCCCGTGCGCGTCAACGGCGCCAG CAAAAGCAAGCCGCACGTCAAGCGGCCCATGAACGCCTTCATGGTGTGGGCTCA GGCAGCGCGCAGGAAGCTCGCGGACCAGTACCCGCACCTGCACAACGCTGAGCT CAGCAAGACGCTGGGCAAGCTCTGGAGGCTGCTGAACGAAAGTGACAAGCGCCC

CTTCATCGAGGAGGCTGAGCGGCTCCGTATGCAGCACAAGAAAGACCACCCGGA

CTACAAGTACCAGCCCAGGCGGCGGAAGAACGGGAAGGCCGCCCAGGGCGAGG

CGGAGTGCCCCGGTGGGGAGGCCGAGCAAGGTGGGACCGCCGCCATCCAGGCCC

ACTACAAGAGCGCCCACTTGGACCACCGGCACCCAGGAGAGGGCTCCCCCATGT

CAGATGGGAACCCCGAGCACCCCTCAGGCCAGAGCCATGGCCCACCCACCCCTC

CAACCACCCCGAAGACAGAGCTGCAGTCGGGCAAGGCAGACCCGAAGCGGGAC

GGGCGCTCCATGGGGGAGGGCGGGAAGCCTCACATCGACTTCGGCAACGTGGAC

ATTGGTGAGATCAGCCACGAGGTAATGTCCAACATGGAGACCTTTGATGTGGCTG

AGTTGGACCAGTACCTGCCGCCCAATGGGCACCCAGGCCATGTGAGCAGCTACT

CAGCAGCCGGCTATGGGCTGGGCAGTGCCCTGGCCGTGGCCAGTGGACACTCCG

CCTGGATCTCCAAGCCACCAGGCGTGGCTCTGCCCACGGTCTCACCACCTGGTGT

GGATGCCAAAGCCCAGGTGAAGACAGAGACCGCGGGGCCCCAGGGGCCCCCAC

ACTACACCGACCAGCCATCCACCTCACAGATCGCCTACACCTCCCTCAGCCTGCC

CCACTATGGCTCAGCCTTCCCCTCCATCTCCCGCCCCCAGTTTGACTACTCTGACC

ATCAGCCCTCAGGACCCTATTATGGCCACTCGGGCCAGGCCTCTGGCCTCTACTC

GGCCTTCTCCTATATGGGGCCCTCGCAGCGGCCCCTCTACACGGCCATCTCTGAC

CCCAGCCCCTCAGGGCCCCAGTCCCACAGCCCCACACACTGGGAGCAGCCAGTA

TATACGACACTGTCCCGGCCCTAAAGGGGGCCCTGTCGCCACCACCCCCCGCCCA

GCCCCTGCCCCCAGCCTGTGTGCCCTGTTCCTTGCCCACCTCAGGCCTGGTGGTG

GCAGTGGAGGAGGCTGAGGAGGCTGAAGAGGCTGACAGGTCGGGGGGCTTTCTG

TCTGGCTCACTGCCCTGATGACCCACCCGCCCCATCCAGGCTCCAGCAGCAAAGC

CCCAGGAGAACAGGCTGGACAGAGGAGAAGGAGGTTGACTGTTGCACCCACACT

GAAAGATGAGGGGCTGCACCTTCCCCCAGGAATGACCCTCTATCCCAGGACCTG

AGAAGGGCCTGCTCACCCTCCTCGGGGAGGGGAAGCACCAGGGTTGGTGGCATC

GGAGGCCTTACCACTCCTATGACTCCTGTTTTCTCTCTCACAGATAGTGAGGGTCT

GACATGCCCATGCCACCTATGCCACAGTGCCTAAGGGCTAGGCCACCCAGAGAC

TGTGCCCGGAGCTGGCCGTGTCTCCCACTCAGGGGCTGAGAGTAGCTTTGAGGAG

CCTCATTGGGGAGTGGGGGGTTCGAGGGACTTAGTGGAGTTCTCATCCCTTCAAT

GCCCCCTCCCTTTCTGAAGGCAGGAAGGAGTTGGCACAGAGGCCCCCTGATCCA

ATTCTGTGCCAATAACCTCATTCTTTGTCTGAGAAACAGCCCCCAGTCCTCCTCCA

CTACAACCTCCATGACCTTGAGACGCATCCCAGGAGGTGACGAGGCAGGGGCTC

CAGGAAAGGAATCAGAGACAATTCACAGAGCCTCCCTCCCTGGGCTCCTTGCCA

GCTCCCTCTTCCCTTACTAGGCTCTATGGCCCCTGCTCAGTCAGCCCCACTCCCTG GGCTTCCCAGAGAGTGACAGCTGCTCAGGCCCTAACCCTTGGCTCCAGGAGACA CAGGGCCCAGCACCCAGGTTGCTGTCGGCAGGCTGAAGACACTAGAATCCTGAC CTGTACATTCTGCCCTTGCCTCTTACCCCTTGCCTCCCAGTGGTATTTGAATAAAG TATGTAGCTATATCTGCCCCTATTTTCCTGTTCTGCAGCCCCCCAAATCCACATGT AACTCATTACTGTCTCCTGTTATTTATCTCAGTAGTCCCCTCTCCTAGCCACTCTA GCCCCTATTAACTCTGCATTAAGCATTCCACATAATAAAATTAAAGGTTCCGGTT A (SEQ ID NO.: 3)

In certain embodiments, SOXIO has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank/Gene ID no. 6663. In certain embodiments, the amino acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank/Gene ID no. 6663, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank no. NP 008872.1. In certain embodiments, the amino acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank no. NP 008872.1, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 4, which is provided below.

MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKE QQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFM VWAQAARRI<LADQYPHLHNAELSI<TLGI<LWRLLNESDI<RPFIEEAERLRMQHI<I<D HPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSP MSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIG EISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWIS KPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFP SISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSP THWEQPVYTTLSRP (SEQ ID NO.: 4)

In certain embodiments, the SOXIO is a mouse SOXIO. In certain embodiments, SOXIO has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/Gene ID no. 20665. In certain embodiments, the nucleic acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/Gene ID no. 20665, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank/Gene ID no. 20665. In certain embodiments, the amino acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank/Gene ID no. 20665, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank no. NM 011437.1. In certain embodiments, the nucleic acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank no. NM 011437.1, that do not significantly alter the function or activity of the SOXIO. In certain embodiments, the nucleic acid molecule encoding SOXIO comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 5, which is provided below.

AGTCAGTCTCGGCTGTCCAGCCAGGGTGTTTGGTGGTGAGGATTCAGGCTCCGTC CAGACAAGGCAGTGGCCTGAGGCTCAGGGCCCCCCAGCCCCTCCCTCCCAGTCC ATCAGCGTCACTCCCCAGCCCCGAGCTGGACCGCACACCTTGGGACACGGTTTTC CACTTCCTCAGGACGAGCCCCAGACTGGAGGAGAGGTCGGAGGAGGTGGGCGTT GGGCTCTTCACGAGGACCCCGGCGGCGGGCCCGGGGGAGGCGGCCGAAGCGGC GGCGGCCGGGAGCGACATGGCCGAGGAACAAGACCTATCAGAGGTGGAGCTGA GCCCTGTGGGCTCGGAGGAACCCCGCTGCCTGTCCCCAGGCAGCGCGCCGTCGCT GGGACCCGACGGCGGCGGCGGTGGCTCGGGCTTGCGAGCCAGCCCGGGGCCCGG TGAACTGGGCAAGGTCAAGAAGGAACAGCAGGACGGCGAGGCGGACGATGACA AGTTCCCCGTGTGCATCCGCGAGGCGGTCAGCCAGGTGCTCAGCGGCTACGACTG GACGCTGGTGCCCATGCCCGTGCGCGTCAACGGTGCCAGCAAGAGCAAGCCGCA CGTCAAGAGGCCCATGAACGCCTTCATGGTGTGGGCACAGGCGGCACGCAGAAA GCTAGCCGACCAGTACCCTCACCTCCACAATGCTGAGCTCAGCAAGACACTAGG CAAGCTCTGGAGGTTGCTGAACGAAAGTGACAAGCGCCCCTTCATTGAGGAGGC TGAGAGGCTCCGGATGCAGCACAAAAAGGACCATCCGGACTACAAGTACCAACC TCGGCGGCGGAAGAACGGGAAGGCAGCCCAGGGGGAGGCAGAATGCCCAGGCG GGGAAGCCGAGCAAGGAGGGGCTGCTGCTATTCAGGCTCACTACAAGAGTGCCC ACCTGGACCACCGGCACCCAGAAGAAGGCTCCCCCATGTCAGATGGGAACCCAG AGCACCCCTCAGGCCAGAGCCATGGCCCCCCAACCCCTCCAACCACCCCAAAGA CAGAGCTGCAGTCCGGCAAGGCAGACCCCAAAAGGGATGGGCGCTCCTTGGGGG AGGGCGGGAAGCCCCACATCGACTTCGGCAACGTGGACATCGGGGAGATCAGCC ACGAGGTAATGTCCAACATGGAAACCTTTGATGTGACTGAGCTGGACCAATACCT GCCACCCAACGGGCACCCAGGCCATGTGGGTAGCTACTCGGCAGCTGGCTACGG GCTGGGCAGTGCCCTGGCTGTGGCCAGTGGACACTCTGCCTGGATCTCCAAGCCA CCAGGTGTGGCTCTGCCCACGGTCTCGCCCCCTGGTGTGGATGCCAAAGCCCAGG TGAAGACAGAGACCACAGGCCCCCAGGGACCCCCACACTACACCGACCAGCCGT CCACTTCCCAGATCGCCTACACTTCCCTCAGTCTGCCCCACTACGGCTCCGCCTTC CCCTCCATCTCACGACCCCAGTTTGACTATTCTGACCATCAGCCCTCAGGACCCT ATTATGGCCATGCAGGCCAGGCCTCTGGCCTCTATTCAGCCTTTTCTTACATGGG GCCCTCCCAGCGGCCCCTCTACACTGCCATCTCTGACCCCAGCCCCTCAGGGCCC CAATCCCACAGTCCCACACACTGGGAGCAGCCAGTATATACGACTCTATCCCGAC CTTAGAGGGGCCCTGTCACCACCAGTGCCCACAGGGCCTTGGGGCCCCTTCCCCC AGCCTGTGTGCCCTGCTCCTCATCAGCCTCAGGCTTGGCAGTGGAGGAGGCTGCA GAGGCTGACAGCAGCAGGAAGGCTTCTGCCAGCTCCTGCCCTGATGACCTCCACC CACCCCGGCTCTGGAGGCCAAGCCCTGACTGAGCTGGCAAAGGAAGTTTGGTGG CTCTCCCAGACTGAGGGTGAGGGGCCACCCTTCCCCAGAAATGACCCTCTATCCC AGGACCTGAGAGTCCTGCTCAGTCCTTCAGGGAGGCAGGAAGGGTTAGGGTAGG AGGCCTCACTGCTCCTGTTATCTCCTTGGAGAGCAAGGGGCCCGTGTGCTACTGT GTCATTGCCTAAGAGCTAAGCTGCCCAGGGACCTGCCTCAAAGCCTGGAGCTGGT TCTGTCCTGTCACTAAGGACGCACTGAGGACAGCTTTGAACAGCTTTGTAAAGAG CAGGGTGGGACACTCTGATCCTTTCTCCGCCCCCTCTTCACCTGAATGTGGGAAC TGGTCGAGGTCCCCAGATCCAGTTCCGTGTCAATAACCTCATCCCTTGCCTAACT GAGGAGAGTCCCCATGTTCTTCCCATCCCACCCCAGGTGAGGAGCCAGGGGCTCC AGGAAAGAGTCAGAGGCAATCCACCTAGCCTTCTTCTCGCCTTCTGTCAGCCTCA AGCCCCCAGGCTCAGAGTTAGCATGGCACGGCCCTAGCCCCTGGGTTGCTGGCA GCAGGCTGGACACTAAACCCCTGCCATGTACAGTCTACCTTTGCCTTGCACCCTT TGCTCCAGCGATACCTTAATAAAGTACGTAGCTCTGTCTCCCCATCTCCTGTCTGC AGCCCAGTCCACATGTAACACATCGCTGCCCCTTTATTTATCTCCATAGTCGCCAT GTTCCTATCCCTCTAGCTACTCCAGCCCCGATTAACTTGCATTAAGCACTCCAAGT AATAAAATCAATGCTTCTGGTTGCCTGCTTGGTGGCCTGA (SEQ ID NO.: 5)

In certain embodiments, SOXIO has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank/Gene ID no. 20665. In certain embodiments, the amino acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank/Gene ID no. 20665, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank no. NP 035567.1. In certain embodiments, the amino acid molecule encoding SOXIO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank no. NP 035567.1, that do not significantly alter the function or activity of the SOXIO.

In certain embodiments, SOXIO has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 6, which is provided below.

MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKE QQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFM VWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKD HPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGAAAIQAHYKSAHLDHRHPEEGSP MSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSLGEGGKPHIDFGNVDIGE ISHEVMSNMETFDVTELDQYLPPNGHPGHVGSYSAAGYGLGSALAVASGHSAWISK PPGVALPTVSPPGVDAKAQVKTETTGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSI SRPQFDYSDHQPSGPYYGHAGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPT HWEQPVYTTLSRP (SEQ ID NO.: 6)

In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM.

In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding SOX10 in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL.

In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 35 pg, up to about 40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about 60 pg, up to about 65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about 85 pg, up to about 90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding SOXIO in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 Hg-

2,3 Nucleic acid encoding KRQX20

A third component of ASK is nucleic acid encoding KROX20. In certain embodiments, the KROX20 is a human KROX20. In certain embodiments, KROX20 has a nucleic acid sequences that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/Gene ID no. 1959. In certain embodiments, the nucleic acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/NCBI database accession no. 1959, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank no. NM 000399.5. In certain embodiments, the nucleic acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank no. NM 000399.5, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, the nucleic acid molecule encoding KROX20 comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 7, which is provided below.

AACTGAGCGAGGAGCAATTGATTAATAGCTCGGCGAGGGGACTCACTGACTGTT ATAATAACACTACACCAGCAACTCCTGGCTTCCCAGCAGCCGGAACACAGACAG GAGAGAGTCAGTGGCAAATAGACATTTTTCTTATTTCTTAAAAAACAGCAACTTG

TTTGCTACTTTTATTTCTGTTGATTTTTTTTTCTTGGTGTGTGTGGTGGTTGTTTTTA

AGTGTGGAGGGCAAAAGGAGATACCATCCCAGGCTCAGTCCAACCCCTCTCCAA

AACGGCTTTTCTGACACTCCAGGTAGCGAGGGAGTTGGGTCTCCAGGTTGTGCGA

GGAGCAAATGATGACCGCCAAGGCCGTAGACAAAATCCCAGTAACTCTCAGTGG

TTTTGTGCACCAGCTGTCTGACAACATCTACCCGGTGGAGGACCTCGCCGCCACG

TCGGTGACCATCTTTCCCAATGCCGAACTGGGAGGCCCCTTTGACCAGATGAACG

GAGTGGCCGGAGATGGCATGATCAACATTGACATGACTGGAGAGAAGAGGTCGT

TGGATCTCCCATATCCCAGCAGCTTTGCTCCCGTCTCTGCACCTAGAAACCAGAC

CTTCACTTACATGGGCAAGTTCTCCATTGACCCTCAGTACCCTGGTGCCAGCTGCT

ACCCAGAAGGCATAATCAATATTGTGAGTGCAGGCATCTTGCAAGGGGTCACTTC

CCCAGCTTCAACCACAGCCTCATCCAGCGTCACCTCTGCCTCCCCCAACCCACTG

GCCACAGGACCCCTGGGTGTGTGCACCATGTCCCAGACCCAGCCTGACCTGGACC

ACCTGTACTCTCCGCCACCGCCTCCTCCTCCTTATTCTGGCTGTGCAGGAGACCTC

TACCAGGACCCTTCTGCGTTCCTGTCAGCAGCCACCACCTCCACCTCTTCCTCTCT

GGCCTACCCACCACCTCCTTCCTATCCATCCCCCAAGCCAGCCACGGACCCAGGT

CTCTTCCCAATGATCCCAGACTATCCTGGATTCTTTCCATCTCAGTGCCAGAGAG

ACCTACATGGTACAGCTGGCCCAGACCGTAAGCCCTTTCCCTGCCCACTGGACAC

CCTGCGGGTGCCCCCTCCACTCACTCCACTCTCTACAATCCGTAACTTTACCCTGG

GGGGCCCCAGTGCTGGGGTGACCGGACCAGGGGCCAGTGGAGGCAGCGAGGGA

CCCCGGCTGCCTGGTAGCAGCTCAGCAGCAGCAGCAGCCGCCGCCGCCGCCGCC

TATAACCCACACCACCTGCCACTGCGGCCCATTCTGAGGCCTCGCAAGTACCCCA

ACAGACCCAGCAAGACGCCGGTGCACGAGAGGCCCTACCCGTGCCCAGCAGAAG

GCTGCGACCGGCGGTTCTCCCGCTCTGACGAGCTGACACGGCACATCCGAATCCA

CACTGGGCATAAGCCCTTCCAGTGTCGGATCTGCATGCGCAACTTCAGCCGCAGT

GACCACCTCACCACCCATATCCGCACCCACACCGGTGAGAAGCCCTTCGCCTGTG

ACTACTGTGGCCGAAAGTTTGCCCGGAGTGATGAGAGGAAGCGCCACACCAAGA

TCCACCTGAGACAGAAAGAGCGGAAAAGCAGTGCCCCCTCTGCATCGGTGCCAG

CCCCCTCTACAGCCTCCTGCTCTGGGGGCGTGCAGCCTGGGGGTACCCTGTGCAG

CAGTAACAGCAGCAGTCTTGGCGGAGGGCCGCTCGCCCCTTGCTCCTCTCGGACC

CGGACACCTTGAGATGAGACTCAGGCTGATACACCAGCTCCCAAAGGTCCCGGA

GGCCCTTTGTCCACTGGAGCTGCACAACAAACACTACCACCCTTTCCTGTCCCTCT

CTCCCTTTGTTGGGCAAAGGGCTTTGGTGGAGCTAGCACTGCCCCCTTTCCACCT

AGAAGCAGGTTCTTCCTAAAACTTAGCCCATTCTAGTCTCTCTTAGGTGAGTTGA CTATCAACCCAAGGCAAAGGGGAGGCTCAGAAGGAGGTGGTGTGGGGACCCCTG GCCAAGAGGGCTGAGGTCTGACCCTGCTTTAAAGGGTTGTTTGACTAGGTTTTGC TACCCCACTTCCCCTTATTTTGACCCATCACAGGTTTTTGACCCTGGATGTCAGAG TTGATCTAAGACGTTTTCTACAATAGGTTGGGAGATGCTGATCCCTTCAAGTGGG GACAGCAAAAAGACAAGCAAAACTGATGTGCACTTTATGGCTTGGGACTGATTT GGGGGACATTGTACAGTGAGTGAAGTATAGCCTTTATGCCACACTCTGTGGCCCT AAAATGGTGAATCAGAGCATATCTAGTTGTCTCAACCCTTGAAGCAATATGTATT ATAAACTCAGAGAACAGAAGTGCAATGTGATGGGAGGAACATAGCAATATCTGC TCCTTTTCGAGTTGTTTGAGAAATGTAGGCTATTTTTTCAGTGTATATCCACTCAG ATTTTGTGTATTTTTGATGTACACTGTTCTCTAAATTCTGAATCTTTGGGAAAAAA TGTAAAGCATTTATGATCTCAGAGGTTAACTTATTTAAGGGGGATGTACATATAT TCTCTGAAACTAGGATGCATGCAATTGTGTTGGAAGTGTCCTTGGTGCCTTGTGT GATGTAGACAATGTTACAAGGTCTGCATGTAAATGGGTTGCCTTATTATGGAGAA AAAAAATCACTCCCTGAGTTTAGTATGGCTGTATATTTCTGCCTATTAATATTTGG AATTTTTTTTAGAAAGTATATTTTTGTATGCTTTGTTTTGTGACTTAAAAGTGTTA CCTTTGTAGTCAAATTTCAGATAAGAATGTACATAATGTTACCGGAGCTGATTTG TTTGGTCATTAGCTCTTAATAGTTGTGAAAAAATAAATCTATTCTAACGCAAAAC CACTAACTGAAGTTCAGATAATGGATGGTTTGTGACTATAGTGTAAATAAATACT TTTCAACAATA (SEQ ID NO.: 7)

In certain embodiments, KROX20 has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank/Gene ID no. 1959. In certain embodiments, the amino acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank/Gene ID no. 1959, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank no. NP 000390.2. In certain embodiments, the amino acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank no. NP 000390.2, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 8, which is provided below.

MMTAKAVDKIPVTLSGFVHQLSDNIYPVEDLAATSVTIFPNAELGGPFDQMNGVAG DGMINIDMTGEKRSLDLPYPSSFAPVSAPRNQTFTYMGKFSIDPQYPGASCYPEGIINI VSAGILQGVTSPASTTASSSVTSASPNPLATGPLGVCTMSQTQPDLDHLYSPPPPPPPY SGCAGDLYQDPSAFLSAATTSTSSSLAYPPPPSYPSPKPATDPGLFPMIPDYPGFFPSQ CQRDLHGTAGPDRKPFPCPLDTLRVPPPLTPLSTIRNFTLGGPSAGVTGPGASGGSEGP RLPGSSSAAAAAAAAAAYNPHHLPLRPILRPRKYPNRPSKTPVHERPYPCPAEGCDR RFSRSDELTRHIRIHTGHKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDYCGRKFA RSDERKRHTKIHLRQKERKS S AP S AS VP AP ST ASC SGGVQPGGTLC S SNS S SLGGGPL APCSSRTRTP (SEQ ID NO.: 8)

In certain embodiments, the KROX20 is a mouse KROX20. In certain embodiments, KROX20 has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank/Gene ID no. 13654. In certain embodiments, the nucleic acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank/Gene ID no. 13654, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank no. NM 001347458.1. In certain embodiments, the nucleic acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank no. NM 001347458.1, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, the nucleic acid molecule encoding KROX20 comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 9, which is provided below.

TACACCAGCAACTCCTGGCTCCCCAACAGCCGGATCACAGGCAGGAGAGACTGC CTGACAGCCTCTACCCGGTGGAAGACCTCGCCGCCTCGTCGGTGACCATCTTCCC CAATGGTGAACTGGGAGGCCCCTTTGACCAGATGAACGGAGTGGCGGGAGATGG CATGATCAACATTGACATGACTGGAGAGAAGAGACCCCTGGATCTCCCGTATCC GAGTAGCTTCGCTCCCATCTCTGCACCTAGAAACCAGACCTTCACTTACATGGGC AAATTCTCCATTGACCCACAGTACCCTGGTGCCAGCTGCTATCCAGAAGGTATCA TCAATATTGTGAGTGCGGGCATCTTGCAAGGGGTCACCCCTCCAGCTTCAACCAC AGCCTCCTCCAGCGTCACCTCCGCCTCCCCCAACCCACTGGCCACGGGACCCCTG GGTGTGTGTACCATGTCCCAGACTCAGCCTGAACTGGACCACCTCTACTCTCCGC CACCACCTCCTCCTCCTTATTCGGGCTGTACAGGAGATCTCTACCAGGATCCTTCA GCATTCTTATCGCCGCCATCCACCACTTCCACCTCCTCTCTGGCCTACCAGCCACC TCCTTCCTACCCATCCCCCAAGCCAGCTATGGACCCAGGTCTCATTCCTATGATCC CAGACTATCCTGGATTTTTTCCATCTCCGTGCCAGAGAGATCCACACGGTGCTGC TGGCCCAGATCGAAAGCCGTTTCCCTGTCCTCTGGACTCCCTGCGAGTGCCCCCT CCACTCACGCCACTCTCTACCATCCGTAATTTTACTCTGGGGGGTCCCGGTGCTG GAGTCACGGGACCAGGAGCAAGTGGAGGTGGTGAGGGACCTCGGCTGCCTGGCA GTGGGTCTGCAGCAGTGACTGCCACCCCTTATAATCCGCACCACCTGCCATTGCG GCCCATCCTGCGACCTCGAAAGTACCCTAACAGGCCCAGCAAAACGCCAGTGCA CGAAAGGCCCTATCCCTGCCCAGCAGAAGGTTGTGATAGGAGGTTCTCACGCTCT GATGAGCTGACCAGGCACATCCGAATCCACACGGGCCACAAGCCCTTCCAGTGT CGGATCTGCATGCGAAACTTCAGCCGAAGTGACCACCTTACTACTCACATCCGAA CCCACACCGGGGAGAAGCCCTTTGCCTGTGACTATTGTGGCCGCAAGTTTGCCAG GAGTGACGAAAGGAAGCGCCACACCAAGATCCACCTTCGGCAGAAGGAACGGA AGAGCAGTGCTCCCTCTGCACCTCCATCTGCCCAGTCTTCAGCCTCTGGTCCTGG GGGCTCGCAGGCCGGGGGCAGCCTGTGCGGTAACAGCGCCATTGGAGGACCACT GGCCTCCTGCACCTCTCGAACCAGGACACCGTGAGATGAAGCTCTGGCTGACAC ACCAGTTTCTCCAGGCCCCAGAGGCCCTCTGTCCGAGCTGCCAACACTACCACCC TTCCCTGTTCCTCCCCATGATCCCGTGATCTGGGCAAAGGACCTTGATGGAGCCC AGCTCTGTCCCACCTTCTCACGGACGGCCTTCCGAAAACTTAGGCCATTTGAAGG GAGTTGACTGTCACTCCAAGAAATGGGGGAGCAAAAAGAGGGCTGGGTGAGGG CCCCTGGCCTACAGGGCTGCGCTCTGACCCTGACTGAGAGATGTCTGACTATGGT CTGCTAGCCCTTTCCGTTGACCCTGGATGCCAGTTGTTCTGAGACTTTTTCTACAA TAGGTTGGGAGTTGCTGATTCCTTTGATCGAGGACAGCGGAAAAGACTAAATTA AAGCAAAACTGATGTGGCACTTTAATGGCTTGGGACTGACTTGGGGGTGGGGGT GGGGAGTTGTACAGTGAGCAGAGCCTCATCCTGGCTGCTGCACTCTGGCCCTAGA GCAGTGAATGGAGGTTTCTCTGGCCATCTCAACCCTTAAGCAATATGTCCTAGAA ACTCAAGAGAATGGAAGTGCAATGTCGGGCAGGACAAAGCAATATTGGCTCCTT TTTTTGTTGTAGTTGAGGAACAAAGATTATTTTTTCAGTGTATATCCATTTAGATT TTGTGTATTTTTGATGTGCACTGCTCTCCGAGTTCTGAACCTTCGGGAAAAAAAT GTAAAGCATTTATGATCTCTTGAAATGAGTCAAAGGTTAACTAACTTATTTAAAG GGGGACGTACATAGGATGCATGCAGTGAGTGGTGTTGCAAGTGTCCTCTGTGCCT TGTGTGATGTGGGCAGTGTTAACAGGGTCTGCATGTGTACAGGATGCCTTACTAT GGGAACAGAAAAATCACTCCTTGGGTTTAAGTATGGCTGTATATTTCTGCCTATT AATATTTGGAATTTTTTTAGAAAGTATATTTTTGTATGCTCTGTTTTGTGACTTAA AAGTGTTACCTTTGTAGCCAAATTTCAGGTGAGAATGTGCTTCAATGTCACTGCC GCTGATTTGTTTGGTTATTAGCTCTTAATAGTTGTGGAGAGAGAAACAATCTATT CTAACATAAAAAAACCACTAACTGGAGTTCAGATAATGGATGGCTTATTGACTAT GGTGTAAATAAATACTTTTCAACAATAAAAAAAAAAAAAAAAAAAAAAAAAAA A (SEQ ID NO.: 9)

In certain embodiments, KROX20 has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank/Gene ID no. 13654. In certain embodiments, the amino acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank/Gene ID no. 13654, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank no. NP 001334387.1. In certain embodiments, the amino acid molecule encoding KROX20 can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence set forth in GenBank no. NP 001334387.1, that do not significantly alter the function or activity of the KROX20.

In certain embodiments, KROX20 has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 10, which is provided below.

MNGVAGDGMINIDMTGEKRPLDLPYPSSFAPISAPRNQTFTYMGKFSIDPQYPGASC YPEGIINIVSAGILQGVTPPASTTASSSVTSASPNPLATGPLGVCTMSQTQPELDHLYSP PPPPPPYSGCTGDLYQDPSAFLSPPSTTSTSSLAYQPPPSYPSPKPAMDPGLIPMIPDYP GFFPSPCQRDPHGAAGPDRKPFPCPLDSLRVPPPLTPLSTIRNFTLGGPGAGVTGPGAS GGGEGPRLPGSGSAAVTATPYNPHHLPLRPILRPRKYPNRPSKTPVHERPYPCPAEGC DRRFSRSDELTRHIRIHTGHKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDYCGR KFARSDERKRHTKIHLRQKERKSSAPSAPPSAQSSASGPGGSQAGGSLCGNSAIGGPL ASCTSRTRTP (SEQ ID NO.: 10)

In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM.

In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 35 pg, up to about 40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about 60 pg, up to about 65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about 85 pg, up to about 90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 pg.

2,4 Compositions comprising ASK

As disclosed herein, in certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b and 2) a nucleic acid encoding SOX10. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 ranges from about 10:1 to about 1:10, from about 10:1 to about 1:1, or from about 1:1 to 1:10. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOXIOis about 10:1, about5:l, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10.

In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b and 2) a nucleic acid encoding KROX20. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10:1 to about 1:1, or from about 1 : 1 to 1 : 10. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10.

In certain embodiments, ASK comprises 1) a nucleic acid encoding SOX10 and 2) a nucleic acid encoding KROX20. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10:1 to about 1:1, or from about 1:1 to 1:10. In certain embodiments, the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10.

In certain embodiments, ASK comprises: 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b, 2) a nucleic acid encoding SOX10, and 3) a nucleic acid encoding KROX20. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO- miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1 : 10: 1 to about 10:1:10; from about 1 : 10: 1 to about 1:1:1, from about 1:1:1 to about 10:1:10, from about 1 : 1 : 10 to about 10:10:1, from about 1 : 1 : 10 to about 1 : 1 : 1, or from about 1:1:1 to about 10:10:1. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2: 1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1.

In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b and 2) a nucleic acid encoding SOX10 and KROX20. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 and KROX20 ranges from about 10: 1 to about 1:10, from about 10: 1 to about 1:1, or from about 1:1 to 1:10. In certain embodiments, the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 and KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10.

In certain embodiments, ASK comprises: 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b, 2) a nucleic acid encoding SOX10, and 3) a nucleic acid encoding KROX20. In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO- miR200b in an amount ranging from between about 25 nM and about 200 nM or between about 1 ng and about 500 ng; 2) a nucleic acid encoding SOX10 in an amount ranging from between about 1 nM and about 200 nM or between about 0.5 pg and about 100 pg; and 3) a nucleic acid encoding KROX20 in an amount ranging from between about 1 nM and about 200 nM or between about 0.5 pg and about 100 pg.

In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount ranging from between about 50 ng and about 300 ng; 2) a nucleic acid encoding SOX10 in an amount ranging from between about 1 pg and about 50 pg; and 3) a nucleic acid encoding KROX20 in an amount ranging from between about 1 pg and about 50 pg. In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng; 2) a nucleic acid encoding SOX10 in an amount ranging from between about 5 pg and about 10 pg; and 3) a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg. In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of about 150 ng; 2) a nucleic acid encoding SOX10 in an amount of about 8 pg; and 3) a nucleic acid encoding KROX20 in an amount ranging from between about 8 pg. In certain embodiments, ASK comprises 1) ASO-miR200b or a nucleic acid encoding ASO-miR200b in an amount of about 200 ng; 2) a nucleic acid encoding SOXIO in an amount of about 8 pg; and 3) a nucleic acid encoding KROX20 in an amount of about 8 pg.

2,4 Pharmaceutical compositions

The present disclosure further provides pharmaceutical compositions comprising ASK or compositions comprising ASK. In certain embodiments, the pharmaceutical composition described herein further includes a pharmaceutically acceptable carrier, e.g., an excipient. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredients and/or that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents and sterile solutions. Additional nonlimiting examples of pharmaceutically acceptable carriers include gels, bioadsorbable matrix materials, and any other suitable vehicle, delivery, or dispensing means or material.

In certain embodiments, the pharmaceutically acceptable carrier can be a buffering agent. Non-limiting examples of suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As a buffering agent, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide other calcium salts, and combinations thereof.

In certain embodiments, the pharmaceutical composition can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combinations thereof in oils, as liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combinations thereof.

3. Methods of Treatment

The present disclosure provides methods for preventing and/or treating nerve damage in subject in need thereof, using ASK. ASK can be used to treat a subject having a condition associated with nerve damage. Non-limiting examples of conditions associated with nerve damage include diabetic polyneuropathy, diabetic neuropathy, and peripheral neuropathy. In certain embodiments, the nerve damage is associated with diabetic polyneuropathy.

3.1 Diabetic Polyneuropathy

Diabetic polyneuropathy (DPN) is a serious diabetes complication that can affect as many as 50% of people with diabetes (type I or type II). High blood sugar (glucose) can damage nerves throughout the body, and most often damages nerves in an individual’s legs and feet. DPN symptoms can range from pain and numbness in the legs and feet to problems with the digestive system, urinary tract, blood vessels and heart.

Nerve damage associated with DPN can be observed in other related conditions, e.g., diabetic neuropathy or peripheral neuropathy.

3.2 Innervation of Grafted Skin

The ability of tissues to regulate and govern their functions is dependent on nerve innervation. Aspects of nerve innervation in a tissue include the robustness of nerves entering the tissue, patterning of nerves with respect to tissue morphologies, and integration of nerves with tissue structures (e.g. blood vessels). The importance of nerve innervation has been demonstrated in developing tissues and organs, as well as engineered tissues and organs which can be fabricated for engraftment. The methods disclosed herein, i.e., the use of TNTASK for the neurotrophic and neurogenic reprogramming of the skin, can additionally be applied to skin grafts, including engineered skin grafts, for improving nerve innervation and tissue functionality of skin grafts.

The present disclosure provides in vivo methods of preventing and/or treating nerve damage in a subject in need thereof, or enhancing nerve innervation in skin grafts in a subject in need thereof, comprising administering a therapeutically effective amount of ASK, wherein ASK comprises: ASO-miR200b or a nucleic acid encoding ASO-miR200b; a nucleic acid encoding SOXIO; and a nucleic acid encoding KROX20. In certain embodiments, the method comprises administering TNTASK to the skin graft. In certain embodiments, the skin graft comprises skin fibroblasts. In certain embodiments, ASK is administered to skin fibroblasts using lipid nanoparticles. In certain embodiments, ASO-miR200b is administered to skin fibroblasts using tissue nanotransfection. In certain embodiments, the subject has nerve damage. In certain embodiments, the nerve damage is associated with diabetic neuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or pre-diabetic type II diabetes.

In certain embodiments, ASK comprises a therapeutically effective amount of ASO- miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 0.5 ng/pL and about 100 ng/pL, between about 0.5 ng/pL and about 75 ng/pL, between about 0.5 ng/pL and about 50 ng/pL, between about 0.5 ng/pL and about 25 ng/pL, between about 0.5 ng/pL and about 10 ng/pL, between about 0.5 ng/pL and about 5 ng/pL, between about 0.5 ng/pL and about 1 ng/pL, between about 1 ng/pL and about 100 ng/pL, between about 1 ng/pL and about 75 ng/pL, between about 1 ng/pL and about 50 ng/pL, between about 1 ng/pL and about 25 ng/pL, between about 1 ng/pL and about 10 ng/pL, between about 1 ng/pL and about 5 ng/pL, between about 5 ng/pL and about 100 ng/pL, between about 5 ng/pL and about 75 ng/pL, between about 5 ng/pL and about 50 ng/pL, between about 5 ng/pL and about 25 ng/pL, between about 5 ng/pL and about 10 ng/pL, between about 10 ng/pL and about 100 ng/pL, between about 10 ng/pL and about 75 ng/pL, between about 10 ng/pL and about 50 ng/pL, between about 10 ng/pL and about 25 ng/gL, between about 25 ng/gL and about 100 ng/gL, between about 25 ng/gL and about 75 ng/gL, between about 25 ng/gL and about 50 ng/gL, between about 50 ng/gL and about 100 ng/gL, between about 50 ng/gL and about 75 ng/gL, or between about 75 ng/gL and about 100 ng/gL. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 0.5 ng/gL, at least about 1 ng/gL, at least about 2 ng/gL, at least about 3 ng/gL, at least about 4 ng/gL, at least about 5 ng/gL, at least about 6 ng/gL, at least about 7 ng/gL, at least about 8 ng/gL, at least about 9 ng/gL, at least about 10 ng/gL, at least about 25 ng/gL, at least about 50 ng/gL, at least about 75 ng/gL, or at least about 100 ng/gL. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b up to about 0.5 ng/gL, up to about 1 ng/gL, up to about 2 ng/gL, up to about 3 ng/gL, up to about 4 ng/gL, up to about 5 ng/gL, up to about 6 ng/gL, up to about 7 ng/gL, up to about 8 ng/gL, up to about 9 ng/gL, up to about 10 ng/gL, up to about 25 ng/gL, up to about 50 ng/gL, up to about 75 ng/gL, or up to about 100 ng/gL. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 0.5 ng/gL, about 1 ng/gL, about 2 ng/gL, about 3 ng/gL, about 4 ng/gL, about 5 ng/gL, about 6 ng/gL, about 7 ng/gL, about 8 ng/gL, about 9 ng/gL, about 10 ng/gL, about 25 ng/gL, about 50 ng/gL, about 75 ng/gL, or about 100 ng/gL. In certain embodiments, ASK comprises a therapeutically effective amount of ASO- miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 1 ng and about 500 ng, between about 1 ng and about 400 ng, between about 1 ng and about 300 ng, between about 1 ng and about 200 ng, between about 1 ng and about 100 ng, between about 1 ng and about 50 ng, between about 50 ng and about 500 ng, between about 50 ng and about 400 ng, between about 50 ng and about 300 ng, between about 50 ng and about 200 ng, between about 50 ng and about 100 ng, between about 100 ng and about 500 ng, between about 100 ng and about 400 ng, between about 100 ng and about 300 ng, between about 100 ng and about 200 ng, between about 200 ng and about 500 ng, between about 200 ng and about 400 ng, between about 200 ng and about 300 ng, between about 300 ng and about 500 ng, between about 300 ng and about 400 ng, between about 400 ng and about 500 ng, In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 1 ng, at least about 5 ng, at least about 10 ng, at least about 15 ng, at least about 20 ng, at least about 25 ng, at least about 30 ng, at least about 35 ng, at least about 40 ng, at least about 45 ng, at least about 50 ng, at least about 55 ng, at least about 60 ng, at least about 65 ng, at least about 70 ng, at least about 75 ng, at least about 80 ng, at least about 85 ng, at least about 90 ng, at least about 95 ng, at least about 100 ng, at least about 125 ng, at least about 150 ng, at least about 175 ng, at least about 200 ng, at least about 225 ng, at least about 250 ng, at least about 275 ng, at least about 300 ng, at least about 325 ng, at least about 350 ng, at least about 375 ng, at least about 400 ng, at least about 425 ng, at least about 450 ng, at least about 475 ng, or at least about 500 ng. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO up to about 1 ng, up to about 5 ng, up to about 10 ng, up to about

15 ng, up to about 20 ng, up to about 25 ng, up to about 30 ng, up to about 35 ng, up to about

40 ng, up to about 45 ng, up to about 50 ng, up to about 55 ng, up to about 60 ng, up to about

65 ng, up to about 70 ng, up to about 75 ng, up to about 80 ng, up to about 85 ng, up to about

90 ng, up to about 95 ng, up to about 100 ng, up to about 125 ng, up to about 150 ng, up to about 175 ng, up to about 200 ng, up to about 225 ng, up to about 250 ng, up to about 275 ng, up to about 300 ng, up to about 325 ng, up to about 350 ng, up to about 375 ng, up to about 400 ng, up to about 425 ng, up to about 450 ng, up to about 475 ng, or up to about 500 ng. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 15 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 55 ng, about 60 ng, about 65 ng, about 70 ng, about 75 ng, about 80 ng, about 85 ng, about 90 ng, about 95 ng, about 100 ng, about 125 ng, about 150 ng, about 175 ng, about 200 ng, about 225 ng, about 250 ng, about 275 ng, about 300 ng, about 325 ng, about 350 ng, about 375 ng, about 400 ng, about 425 ng, about 450 ng, about 475 ng, or about 500 ng.

In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM.

In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOX 10 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 35 pg, up to about 40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about 60 pg, up to about 65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about 85 pg, up to about 90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding SOXIO in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 pg.

In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, ASK comprises a nucleic acid encoding KROX20 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about200 nM. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about

35 pg, up to about 40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about

60 pg, up to about 65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about

85 pg, up to about 90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments,

ASK comprises a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 pg.

The present disclosure provides in vivo methods of preventing and/or treating nerve damage in a subject in need thereof, or enhancing nerve innervation in skin grafts in a subject in need thereof, comprising administering a therapeutically effective amount of: a. ASO- miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOX10; and c. a nucleic acid encoding KROX20. In certain embodiments, the ASO-miR200b or a nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOX10, and the nucleic acid encoding KROX20 are administered sequentially. In certain embodiments, the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered using tissue nanotransfection. In certain embodiments, the nucleic acid encoding SOX10 is administered using tissue nanotransfection. In certain embodiments, the nucleic acid encoding KROX20 is administered using tissue nanotransfection. In certain embodiments, the nerve damage is associated with diabetic polyneuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or prediabetic type II diabetes.

In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO- miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 0.5 ng/pL and about 100 ng/pL, between about 0.5 ng/pL and about 75 ng/pL, between about 0.5 ng/pL and about 50 ng/pL, between about 0.5 ng/pL and about 25 ng/pL, between about 0.5 ng/pL and about 10 ng/pL, between about 0.5 ng/pL and about 5 ng/pL, between about 0.5 ng/pL and about 1 ng/pL, between about 1 ng/pL and about 100 ng/pL, between about 1 ng/pL and about 75 ng/pL, between about 1 ng/pL and about 50 ng/pL, between about 1 ng/pL and about 25 ng/pL, between about 1 ng/pL and about 10 ng/pL, between about 1 ng/pL and about 5 ng/pL, between about 5 ng/pL and about 100 ng/pL, between about 5 ng/pL and about 75 ng/pL, between about 5 ng/pL and about 50 ng/pL, between about 5 ng/pL and about 25 ng/pL, between about 5 ng/pL and about 10 ng/pL, between about 10 ng/pL and about 100 ng/pL, between about 10 ng/pL and about 75 ng/pL, between about 10 ng/pL and about 50 ng/pL, between about 10 ng/pL and about 25 ng/pL, between about 25 ng/pL and about 100 ng/pL, between about 25 ng/pL and about 75 ng/pL, between about 25 ng/pL and about 50 ng/pL, between about 50 ng/pL and about 100 ng/pL, between about 50 ng/pL and about 75 ng/pL, or between about 75 ng/pL and about 100 ng/pL. In certain embodiments, ASK comprises a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 0.5 ng/pL, at least about 1 ng/pL, at least about 2 ng/pL, at least about 3 ng/pL, at least about 4 ng/pL, at least about 5 ng/pL, at least about 6 ng/pL, at least about 7 ng/pL, at least about 8 ng/pL, at least about 9 ng/pL, at least about 10 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, or at least about 100 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO- miR200b or a nucleic acid encoding ASO-miR200b up to about 0.5 ng/pL, up to about 1 ng/pL, up to about 2 ng/pL, up to about 3 ng/pL, up to about 4 ng/pL, up to about 5 ng/pL, up to about 6 ng/pL, up to about 7 ng/pL, up to about 8 ng/pL, up to about 9 ng/pL, up to about 10 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, or up to about 100 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 0.5 ng/pL, about 1 ng/pL, about 2 ng/pL, about 3 ng/pL, about 4 ng/pL, about 5 ng/pL, about 6 ng/pL, about 7 ng/pL, about 8 ng/pL, about 9 ng/pL, about 10 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, or about 100 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b ranging from between about 1 ng and about 500 ng, between about 1 ng and about 400 ng, between about 1 ng and about 300 ng, between about 1 ng and about 200 ng, between about 1 ng and about 100 ng, between about 1 ng and about 50 ng, between about 50 ng and about 500 ng, between about 50 ng and about 400 ng, between about 50 ng and about 300 ng, between about 50 ng and about 200 ng, between about 50 ng and about 100 ng, between about 100 ng and about 500 ng, between about 100 ng and about 400 ng, between about 100 ng and about 300 ng, between about 100 ng and about 200 ng, between about 200 ng and about 500 ng, between about 200 ng and about 400 ng, between about 200 ng and about 300 ng, between about 300 ng and about 500 ng, between about 300 ng and about 400 ng, between about 400 ng and about 500 ng, In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of at least about 1 ng, at least about 5 ng, at least about 10 ng, at least about 15 ng, at least about 20 ng, at least about 25 ng, at least about 30 ng, at least about 35 ng, at least about 40 ng, at least about 45 ng, at least about 50 ng, at least about 55 ng, at least about 60 ng, at least about 65 ng, at least about 70 ng, at least about 75 ng, at least about 80 ng, at least about 85 ng, at least about 90 ng, at least about 95 ng, at least about 100 ng, at least about 125 ng, at least about 150 ng, at least about 175 ng, at least about 200 ng, at least about 225 ng, at least about 250 ng, at least about 275 ng, at least about 300 ng, at least about 325 ng, at least about 350 ng, at least about 375 ng, at least about 400 ng, at least about 425 ng, at least about 450 ng, at least about 475 ng, or at least about 500 ng. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO up to about 1 ng, up to about 5 ng, up to about 10 ng, up to about 15 ng, up to about 20 ng, up to about 25 ng, up to about 30 ng, up to about 35 ng, up to about 40 ng, up to about 45 ng, up to about 50 ng, up to about 55 ng, up to about 60 ng, up to about 65 ng, up to about 70 ng, up to about 75 ng, up to about 80 ng, up to about 85 ng, up to about 90 ng, up to about 95 ng, up to about 100 ng, up to about 125 ng, up to about 150 ng, up to about 175 ng, up to about 200 ng, up to about 225 ng, up to about 250 ng, up to about 275 ng, up to about 300 ng, up to about 325 ng, up to about 350 ng, up to about 375 ng, up to about 400 ng, up to about 425 ng, up to about 450 ng, up to about 475 ng, or up to about 500 ng. In certain embodiments, the method comprises administering a therapeutically effective amount of ASO-miR200b or a nucleic acid encoding ASO-miR200b of about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 15 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 55 ng, about 60 ng, about 65 ng, about 70 ng, about 75 ng, about 80 ng, about 85 ng, about 90 ng, about 95 ng, about 100 ng, about 125 ng, about 150 ng, about 175 ng, about 200 ng, about 225 ng, about 250 ng, about 275 ng, about 300 ng, about 325 ng, about 350 ng, about 375 ng, about 400 ng, about 425 ng, about 450 ng, about 475 ng, or about 500 ng.

In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM.

In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 35 pg, up to about

40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about 60 pg, up to about

65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about 85 pg, up to about

90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding SOX10 in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 1 nM and about 200 nM, between about 1 nM and about 175 nM, between about 1 nM and about 150 nM, between about 1 nM and about 125 nM, between about 1 nM and about 100 nM, between about 1 nM and about 75 nM, between about 1 nM and about 50 nM, between about 1 nM and about 25 nM, between about 25 nM and about 200 nM, between about 25 nM and about 175 nM, between about 25 nM and about 150 nM, between about 25 nM and about 125 nM, between about 25 nM and about 100 nM, between about 25 nM and about 75 nM, between about 25 nM and about 50 nM, between about 50 nM and about 200 nM, between about 50 nM and about 175 nM, between about 50 nM and about 150 nM, between about 50 nM and about 125 nM, between about 50 nM and about 100 nM, between about 50 nM and about 75 nM, between about 75 nM and about 200 nM, between about 75 nM and about 175 nM, between about 75 nM and about 150 nM, between about 75 nM and about 125 nM, between about 75 nM and about 100 nM, between about 100 nM and about 200 nM, between about 100 nM and about 175 nM, between about 100 nM and about 150 nM, between about 100 nM and about 125 nM, between about 125 nM and about 200 nM, between about 125 nM and about 175 nM, between about 125 nM and about 150 nM, between about 150 nM and about 200 nM, between about 150 nM and about 175 nM, or between about 175 nM and about 200 nM. In certain embodiments, the method comprises administering a nucleic acid encoding KROX20 in an amount of at least about 1 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 125 nM, at least about 150 nM, at least about 175 nM, or at least about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of up to about 1 nM, up to about 10 nM, up to about 25 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 125 nM, up to about 150 nM, up to about 175 nM, or up to about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 1 ng/pL and about 500 ng/pL, about 1 ng/pL and about 400 ng/pL, about 1 ng/pL and about 300 ng/pL, about 1 ng/pL and about 200 ng/pL, about 1 ng/pL and about 100 ng/pL, between about 100 ng/pL and about 500 ng/pL, about 100 ng/pL and about 400 ng/pL, about 100 ng/pL and about 300 ng/pL, about 100 ng/pL and about 200 ng/pL, between about 200 ng/pL and about 500 ng/pL, about 200 ng/pL and about 400 ng/pL, about 200 ng/pL and about 300 ng/pL, between about 300 ng/pL and about 500 ng/pL, about 300 ng/pL and about 400 ng/pL, or between about 400 ng/pL and about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of at least about 1 ng/pL, at least about 25 ng/pL, at least about 50 ng/pL, at least about 75 ng/pL, at least about 100 ng/pL, at least about 125 ng/pL, at least about 150 ng/pL, at least about 175 ng/pL, at least about 200 ng/pL, at least about 225 ng/pL, at least about 250 ng/pL, at least about 275 ng/pL, at least about 300 ng/pL, at least about 325 ng/pL, at least about 350 ng/pL, at least about 375 ng/pL, at least about 400 ng/pL, at least about 425 ng/pL, at least about 450 ng/pL, at least about 475 ng/pL, or at least about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount up to about 1 ng/pL, up to about 25 ng/pL, up to about 50 ng/pL, up to about 75 ng/pL, up to about 100 ng/pL, up to about 125 ng/pL, up to about 150 ng/pL, up to about 175 ng/pL, up to about 200 ng/pL, up to about 225 ng/pL, up to about 250 ng/pL, up to about 275 ng/pL, up to about 300 ng/pL, up to about 325 ng/pL, up to about 350 ng/pL, up to about 375 ng/pL, up to about 400 ng/pL, up to about 425 ng/pL, up to about 450 ng/pL, up to about 475 ng/pL, or up to about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 1 ng/pL, about 25 ng/pL, about 50 ng/pL, about 75 ng/pL, about 100 ng/pL, about 125 ng/pL, about 150 ng/pL, about 175 ng/pL, about 200 ng/pL, about 225 ng/pL, about 250 ng/pL, about 275 ng/pL, about 300 ng/pL, about 325 ng/pL, about 350 ng/pL, about 375 ng/pL, about 400 ng/pL, about 425 ng/pL, about 450 ng/pL, about 475 ng/pL, about 500 ng/pL. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg, between about 0.5 pg and about 80 pg, between about 0.5 pg and about 60 pg, between about 0.5 pg and about 40 pg, between about 0.5 pg and about 20 pg, between about 0.5 pg and about 10 pg, between about 0.5 pg and about 5 pg, between about 0.5 pg and about 1 pg, between about 1 pg and about 100 pg, between about 1 pg and about 80 pg, between about 1 pg and about 60 pg, between about 1 pg and about 40 pg, between about 1 pg and about 20 pg, between about 1 pg and about 10 pg, between about 1 pg and about 5 pg, between about 5 pg and about 100 pg, between about 5 pg and about 80 pg, between about 5 pg and about 60 pg, between about 5 pg and about 40 pg, between about 5 pg and about 20 pg, between about 5 pg and about 10 pg, between about 10 pg and about 100 pg, between about 10 pg and about 80 pg, between about 10 pg and about 60 pg, between about 10 pg and about 40 pg, between about 10 pg and about 20 pg, between about 20 pg and about 100 pg, between about 20 pg and about 80 pg, between about 20 pg and about 60 pg, between about 20 pg and about 40 pg, between about 40 pg and about 100 pg, between about 40 pg and about 80 pg, between about 40 pg and about 60 pg, between about 60 pg and about 100 pg, between about 60 pg and about 80 pg, or between about 80 pg and about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of at least about 0.5 pg, at least about 1 pg, at least about 5 pg, at least about 10 pg, at least about 15 pg, at least about 20 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, at least about 40 pg, at least about 45 pg, at least about 50 pg, at least about 55 pg, at least about 60 pg, at least about 65 pg, at least about 70 pg, at least about 75 pg, at least about 80 pg, at least about 85 pg, at least about 90 pg, at least about 95 pg, or at least about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of up to about 0.5 pg, up to about 1 pg, up to about 5 pg, up to about 10 pg, up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 35 pg, up to about 40 pg, up to about 45 pg, up to about 50 pg, up to about 55 pg, up to about 60 pg, up to about 65 pg, up to about 70 pg, up to about 75 pg, up to about 80 pg, up to about 85 pg, up to about 90 pg, up to about 95 pg, or up to about 100 pg. In certain embodiments, the method comprises administering a therapeutically effective amount of a nucleic acid encoding KROX20 in an amount of about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, or about 100 pg.

In any of the methods disclosed herein, a therapeutically effective amount of ASO- miR200b or a nucleic acid encoding ASO-miR200b can be an amount ranging from between about 1 ng and about 500 ng. In certain embodiments, a therapeutically effective amount of a nucleic acid encoding SOX10 can be an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, a therapeutically effective amount of a nucleic acid encoding KROX20 can be an amount ranging from between about 0.5 pg and about 100 pg. In certain embodiments, the therapeutically effective amounts of the ASO-miR200b or a nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOX10, and the nucleic acid encoding KROX20 can be formulated into a single composition for administration. In certain embodiments, the therapeutically effective amounts of the ASO-miR200b or a nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOXIO, and the nucleic acid encoding KROX20 can be administered sequentially.

3.3 Methods for Administration

Administering ASK to the skin in vivo promotes neurogenic and neurotrophic reprogramming of skin fibroblasts to Schwann cells and induced neuronal cells (iN). In certain embodiments, ASK comprises ASO-miR200b or a nucleic acid encoding ASO-miR200b. ASO-miR200b can be administered to skin fibroblasts using transfection methods known in the art. In certain embodiments, administration of ASK comprises simultaneous administration of ASO-miR200b or a nucleic acid encoding ASO-miR200b, a nucleic acid encoding SOXIO, and a nucleic acid encoding KROX20. In certain embodiments, administration of ASK comprises sequential administration of ASO-miR200b or a nucleic acid encoding ASO- miR200b, a nucleic acid encoding SOXIO, and a nucleic acid encoding KROX20.

Non-limiting methods of administering ASK to skin fibroblasts include tissue nanotransfection, electroporation, lipid nanoparticles, viral transduction. In certain embodiments, compositions disclosed herein can be administered via topical, transcutaneous, transdermal, intra-joint, intradermal, intralesional, via an implanted reservoir, or subcutaneous routes. In certain embodiments, ASK is administered to skin fibroblasts using lipid nanoparticles. In certain embodiments, ASO-miR200b is administered to skin fibroblasts using tissue nanotransfection. Tissue nanotransfection is an electroporation-based technique capable of delivering nucleic acid sequences and proteins into the cytosol of cells at nanoscale. In certain embodiments, the expression of the nucleic acid molecule encoding ASO-miR200b is operably linked to a promoter. As used herein, “operably linked” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid locus to control transcriptional initiation and/or expression of that locus. In certain embodiments, at least one nucleic acid molecule encoding ASO-miR200b, SOXIO, and/or KROX20 is integrated into the genome of a virus, where the expression of the nucleic acid molecule is operably linked to a promoter that is active or activatable in the virus infected cell. Further details regarding methods for administration have been described in international published patent application no. WO2021222491, the disclosure of which is incorporated herein by reference.

3.4 Methods for Transplantation

The present disclosure further provides methods for transplanting reprogrammed tissue. Following administration of ASK to subject, reprogrammed cells can be transplanted from the site of ASK administration to another location of the subject which exhibits nerve damage but has not received ASK treatment. Transplanted tissue can comprise reprogrammed cells, i.e., skin fibroblasts which have been converted to Schwann cells or iN, and cells of the skin which have not been reprogrammed.

The present disclosure provides methods of treating nerve damage, e.g., diabetic polyneuropathy, comprising administering ASK to skin tissue of the subject. In certain embodiments, the skin tissue comprises skin fibroblasts. In certain embodiments, the skin fibroblasts are reprogrammed to a cell population comprising Schwann cells. In certain embodiments, skin tissue administered with ASK is transplanted to another location of the subject.

The present disclosure provides a pharmaceutical composition comprising skin tissue administered with a therapeutically effective amount of ASK, wherein ASK comprises: ASO- miR200b or a nucleic acid encoding ASO-miR200b; a nucleic acid encoding SOXIO; and a nucleic acid encoding KROX20. In certain embodiments, the skin tissue comprises skin fibroblasts that are reprogrammed to a cell population comprising Schwann cells. In certain embodiments, the skin tissue comprises non-reprogrammed skin fibroblasts. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

EXAMPLES

The present disclosure will be better understood by reference to the following Examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

EXAMPLE 1: TNTASK BASED NEUROGENIC AND NEUROTROPHIC REPROGRAMMING OF THE SKIN

The present example demonstrates ASK as a new cocktail consisting of three transcription factors - ASO, SoxlO, Krox20 (ASK) - for direct neurogenic and neurotrophic reprogramming of the skin in vivo. ASK is delivered using tissue nanotransfection (TNT) technology which is an electroporation-based technique capable of delivering nucleic acid sequences and proteins into the cytosol of cells at nanoscale. The successful neurogenic conversion of cells by ASK using TNT (TNTASK) caused neurotrophic enrichment of the skin stroma which can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Thus, ASK directly promotes functional rescue of diabetic peripheral neuropathy (DPN). TNT ASK spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. This tissue engineering process that delivers ASK to skin enables the generation of autologous neural cells that can be transplanted to other parts of the body.

Experiments were conducted as shown in the schematic in Figure 1A. db/db diabetic mice (BKS.Cg-Dock7m+/+Leprdb/J strain) or db/+ heterozygous littermate control mice were administered via TNT either 1) ASK comprising SoxlO, Krox20, and LNA anti-miR-200b, or 2) control plasmid and LNA-oligonucleotide. Confirmation of the delivery of reprograming factors to mouse skin was evaluated 72 hours following TNT (Figures IB and 1C).

Reprograming of the skin by TNTASK resulted in neurogenic fate change of dermal fibroblasts as indicated by the expression of NF200 in dermal papillary fibroblasts (Figures 2A-2C). TNTASK increased NGF production in fibroblasts from dermal sheath origin (MYL9+/MEF2C+) in skin samples db/db mice collected two weeks post TNT, which persisted through 21 weeks post TNT (Figures 4A-4D and 25B), and accompanied by increased expression of NGF (Figures 4C-4D) and NP2 (Figure 5).

Functional recovery of DPN-compromised allodynia and thermal hyperalgesia was evaluated for db/db diabetic mice following TNTASK treatment, db/db diabetic mice which received a mock treatment exhibited increased withdrawal latency in response to mechanical thermal stimuli versus db/+ heterozygous littermate control mice (Figures 6A-6D). However, db/db diabetic mice which received TNTASK exhibited withdrawal latency which was significantly reduced versus db/db diabetic mice which received the mock treatment and similar to that observed for db/+ heterozygous littermate control mice. Transmission electron microscopy was used to conduct morphometric analysis of the sciatic nerve following ASK treatment. Remak bundles and myelination of A-fibers were reduced in db/db diabetic mice (Figures 20A-20F). In addition, the percentage of nerves having myelin infoldings was increased. In db/db mice that received ASK treatment, the Remak bundles, myelination, and infoldings were rescued to levels observed in db/+ heterozygous littermates. Nerve conductivity was evaluated 11 weeks following TNTASK treatment using a synaptic transistor sensor (Figures 7A-7D). In db/db diabetic mice, nerve conductance was increased; however, TNTASK treatment reduced nerve conductance to levels observed in db/+ heterozygous littermates (Figures 7E-7G). PGP9.5 fiber density was evaluated in footpads collected 21 weeks following TNTASK treatment. In db/db diabetic mice, the density of PGP9.5 -expressing nerve fibers was decreased; however, TNTASK treatment increased the density of PGP9.5- expressing nerve fibers to levels observed in db/+ heterozygous littermates (Figures 8).

Cytokines sensitive to TNTASK were identified in a protein array. The array identified several differentially expressed cytokines, including PTX2 (NP2) and IL7 (Figure 10A). Differential expression of PTX2 (NP2) was confirmed by ELISA (Figure 10B). NP2 was further evaluated in vivo for mice two weeks following TNT ASK treatment. In db/db diabetic mice, NP2 expression was decreased; however, TNT ASK treatment increased NP2 expression to levels observed in db/+ heterozygous littermates (Figures 11 A-l 1C).

EXAMPLE 2: DERMAL FIBROBLAST SUBSETS IN NEUROTROPHIC REPROGRAMMING OF DIABETIC SKIN

The skin exhibits marked tissue plasticity [1-3], Numerous studies have relied on skin cells for the generation of iPSCs which were then used to generate a wide range of neural cells including neurogenic progenitor cells and neurons [20,21], However, in settings where these cells were to be re-introduced into the body it was noted that the use of pluripotent-derived cells posed the risk of cancer as a side-effect [22,23], To address such risk, an approach for direct conversion of skin fibroblasts to functional dopaminergic neurons by delivering Ascll/Nurrl/Lmxla (ANL) was investigated [24], Such direct conversion of skin cells to mouse and human neural cells quickly picked up momentum as several papers reported numerous “direct” approaches to achieve the production of neural progenitor cells [25], functional neurons [26,27] and Schwann cells [28], It was reported that old neurons have been generated from the aged donor skin [29], Until 2017, studies on direct reprogramming to neural cells were done on cells isolated from the skin tissue under laboratory conditions.

For neurogenic outcomes, one-time delivery of ABM plasmid cocktail (Ascll/Bm2/Mytll) to skin using a nanoelectroporation technique called tissue nanotransfection (TNT) was employed [14,15], Such TNTABM reprogramming of the skin in vivo resulted in neural cell development from dermal fibroblasts. In addition, other dermal fibroblasts surrounding the neurogenic cells produced high levels of neurotrophic factors which can be leveraged to rescue pre-existing nerve fibers of the skin from DPN [16], Additional studies showed that while TNTABM was effective in sparing nerve fibers of the diabetic skin as detected by immunohistochemistry (IHC), it was not effective in rescuing sensory function.

Diabetic neuropathy (DPN) affects 50% of all people with diabetes. Effective management of DPN helps avoid neuropathic pain and foot ulcers which frequently lead to septicemia, amputation and death [30], Reduced neurotrophic stimulation is one factor underlying DPN [31], Absence of mechanistic-based treatment in routine clinical practice represents a current void in DPN management [32], There is insufficient evidence to demonstrate that improved glycemic control alone delays the progression of DPN in type-2 diabetes (T2D) [33], Efforts to employ neurotrophic factors to manage DPN have therefore been undertaken clinically. Two sets of phase II clinical trials showed that recombinant human NGF (rhNGF) administration was effective for ameliorating the symptoms associated with both DPN and HIV-related neuropathy [69], Over two decades ago, a large-scale phase III clinical trial testing rhNGF failed to confirm the earlier indications of efficacy. Among the explanations offered for the discrepancy between the two sets of trials was inadequate dosage and changes to the formulation of rhNGF for the phase III trial [69], However, in both animals and patients with DPN, recent literature recognized that NGF deficiency is linked to neuronal death and impaired nerve repair [31], NGF plays an important role in regulating axon growth and guidance, resulting in neuroprotective and regenerative effects [70], In 2024, new research revived interest in productizing neurotrophic factor, including NGF, based treatment of diabetic neuropathy [71], In another approach for neurotrophic factor based DPN therapy, the power of neurogenic reprogramming of the skin is harnessed to enrich the diabetic skin with a neurotrophic niche that nature induces in order to support the maturation of nascent neural cells.

Evaluation of dermal fibroblast subsets in neurotrophic reprograming of the diabetic skin. Dermal fibroblasts are a diverse heterogeneous population of cells representing the major mesenchymal cell type in the dermis connective tissue [44], Dermal fibroblasts include reticular, dermal sheath (DS), dermal papilla (DP), fascial, and papillary fibroblasts (Table 1, Figure 12) among others [17,37,93], TNTABM induced NGF and Nt3 in the skin stroma and spared the loss of cutaneous PGP9.5+ mature nerve fibers in skin of db/db mice [16], While such rescue improved functional response to thermal stimuli, nerve conductivity was not improved. The lack of functional rescue (i.e., nerve conductivity and thermoception recovery) motivated the development of TNT ASK. TNT ASK of db/db mouse skin (Figure 13) rescued the loss of PGP9.5+ nerve fibers in the skin, by spatial proteomics analysis (Figures 14 and 15). TNT ASK rescued db/db mouse sciatic epineural MPZ (myelin P0) and SI 00(3 (Figure 16) indicative of myelin sheath rescue. Myelin protein zero is expressed by Schwann cells (SC) and accounts for over 50% of all proteins in the peripheral nervous system, making it the most common protein expressed in the PNS [94], TNT ASK rescued nerve bundles up to 21 wks posttreatment as evaluated by spatial proteomics analysis (Figure 17). This was associated with recovery of regenerative (GAP43), nociceptor (Navi.6) and mechanoceptor (TRPV4) markers. Functional characterization of the effects of TNTASK exhibited recovery of mechanical allodynia (Von Frey) and thermoception (thermal plantar) (Figure 18). Mechanisms of both demyelinating neuropathy and axonopathy implicate the impairment of SC [95], TNTASK of db/db skin rescued Remak bundles or multiple small-diameter C fiber axons (Figure 19). Futhermore, myelinated A-fibers were restored post- TNT ASK (Figure 19). Myelinated A fibers, essential for saltatory conduction in axons, showed significantly improved nerve conductance following TNT ASK (Figure 20). Elevated TUJ1 (immature neurons or mature sensory neurons) and NF200 (mature neurons) markers following TNTASK indicate successful neurogenic reprograming of the diabetic skin (Figures 21 and 22). The ability of skin fibroblasts to convert to functional neurons is well known [96-99], Consistently, TNTASK of db/db skin showed enhanced co-localization of TUJ1 and NF200 with specific fibroblasts subsets (Figures 21 and 22).

Table 1. Fibroblast markers.

Neurogenic state-change of DP fibroblasts following TNTASK will be characterized employing DNA barcoded antibodies (Figure 14) detected using Phenocycler Fusion 2.0 (Akoya Biosciences). Such spatial proteomic approach provides advantages over traditional IHC by allowing simultaneous detection of up to 50-60 antibodies in situ, without spectral overlap concerns [101,102], Phenocycler-fusion in situ multiplex imaging will be performed at single cell resolution in fresh frozen and formalin fixed & paraffin embedded tissue sections (FFPE) (Figure 14). Antibody panel: neurogenic markers: NF200, TUJ1, MAP2, DAPI, and fibroblast markers (Table 1): CollA2 and MEF2C barcoded antibodies for multiplexed imaging. Spatial proteomics analysis will be performed using PhenoChart software (Akoya) [103,104], As needed other markers of dermal fibroblast subsets (Table 1) will be included in the analysis to simultaneously look for potential neurogenic state-change of fibroblasts other than DP. Image processing and quantitation will be performed using the CODEX Toolkit uploader & CODEX toolkit segmenter [105,106], Standard (FCS) files will be generated from each tissue microarray (TMA) spot and used for further downstream analysis to detect fibroblasts co-expressing neurogenic markers. For VF detection, as reported [17], fibroblasts (Table 1) will co-express the following endothelial genes: vWF, Cd31, Cdh5, Nos3(eNOS), Glut, Vegfa, Kdr, Junb. (any 4 out of 8) [17],

To identify multiple cell types and cellular responses integral to a mechanism based on spatial and biological signaling, in situ Xenium spatial transcriptomics will be employed (Figure 23). Xenium custom panels (360 genes) including dermal cell identifying genes (including all fibroblast subsets, Table 1) as landmarks and a collection of all possible neural cell (developing, regenerative and mature) markers has been produced. Examples of such markers and interpretation are in Table 2. This Xenium approach will also enable mapping of the detected NF to fibroblast subsets (data in Seurat platform) detected via scRNASeq (Fig.1 , Table 1). Next, for candidate genes of interest identified from Xenium studies, Akoya Phenocycler Fusion spatial proteomics (Figure 23) will be employed to look at corresponding proteins and determine causal network (Ingenuity Pathway Analysis, IP A). To accomplish the integration of spatial proteomics data with the identified fibroblast subsets from Xenium, “label transfer” approach Seurat [108], LIGER [109] or GLUER [110] will be applied. These data will be further enhanced by tissue segmentation which will be performed using “DeepLabV3” neural network of Visiopharm Al pathology tool [111-116], Table 2. Neural Markers.

The strategy of neurogenic reprogramming of the skin results in neurotrophic enrichment of multiple factors in the skin, including NGF (Figure 24). The neurotrophic enrichment caused by TNT ASK is effective in “nurturing” nascent NF for several weeks to maturity such that they acquire electrophysiological activity (Figure 20) [107], TNTASK of db/db skin will be followed by the study of neurotrophic enrichment of the diabetic skin. NGF, transcription factors and co-regulated neurotrophic factors will be studied. TNTASK treated db/db skin will be harvested 1, 2, 4 and 21 weeks after TNT procedure and analyzed in tandem employing in situ Xenium spatial transcriptomics and Akoya Phenocycler Fusion spatial proteomics to look at corresponding proteins. Early time points (weeks 1 and 2) will primarily look for signaling mediators participating in causing neurotrophic enrichment outcomes recorded in wk21 (Figures 14-23). Anti-NGF, anti-SOXlO, anti-KROX20, anti- SP1, anti- SNAIL and anti-C/EBPa along with fibroblast markers anti-Col 1 A2 and anti-MEF2C barcoded antibodies will be used for multiplexed imaging. As needed other markers of dermal fibroblast subsets (Table 1) will be included in the analysis. ASK-treated tissue serial sections will be harvested for ELISA quantification analysis (Biosensis# BEK2213) of NGF and transcription factors (SOX 10, KROX20, SP1, C/EBPa, SNAIL). Quantification and colocalization analysis of transcription factors predicted to regulate NGF within ASK-treated db/db dermal fibroblast populations will be performed. To accomplish the integration of spatial proteomics data with the identified fibroblast subsets from Xenium, “label transfer” approach Seurat [108], LIGER [109] or GLUER [110] will be applied. These data will be further enhanced by tissue segmentation which will be performed using “DeepLabV3” neural network of Visiopharm Al tool [111-116],

Evaluation of neurotrophic cargo of fibroblast-originating exosomes Exofib). Exosomal mechanisms play a central role in both TNT-induced vasculogenic as well as neurogenic murine skin reprogramming. Exosomes harvested from TNT -treated skin were able to induce vasculogenesis and neurogenesis respectively [14], Subsequent studies revealed a critical role of exosomal mechanisms in cell-cell crosstalk which are expected to be involved in in vivo skin reprograming [76,135], In peripheral neural regeneration, Schwann cell exosomes are internalized by peripheral nerve axons thus enhancing neurite outgrowth [136,137], Daily injections of SC exosomes into the distal segment resulted in 2x increase in axon growth following nerve injury [136], Exosome therapy shows early signs of being promising for the management of DPN [138-140], TNTASK increased NGF abundance with increase in neural cells in db/db skin (Figures 15, 21, 22, and 24).

4 weeks after TNTASK, fibroblast-targeted Coll Al promoter-driven CD plasmids with “in frame” mNeonGFP reporter (Figure 25) will be delivered via TNT on the same (site of TNTASK) spot marked with the tattoo [76], Mice will be euthanized 24-48h post-TNT_mNeonGFP and skin site harvested. Exoab will be isolated [76], Size, concentration and morphology of isolated fibroblast-specific exosomes will be recorded using Nanosight™ and TEM. The Exofib will then be immunostained for NGFAF64 as well as exosome markers CD63 AF488, CD81AF488, CD9AF488 as described by us [76], dSTORM imaging will be performed using super-resolution Nanoimager S (ONI, resolution 20 nm) (Figures 26 and 27). The image will be acquired using NimOS software and will be analyzed using CODI software for quantitative distribution of NGF within the Exoab. The three murine Coll Al promoter-driven plasmids encoding CD9, CD63 and CD81 with “in frame” mNeonGFP reporter will be delivered via TNT to hindlimb skin of db/db and littermate m+/db mice (n=16). 4h after such TNT, the sciatic nerve will be collected in OCT. Uptake of Exoab by SC will be evaluated from the colocalization of mNeonGFP and SC, two antibodies will be used for SC specificity, MPZ and MBP using Airyscan SRCM (Zeiss). For ex vivo mechanistic studies, the sciatic nerve will be harvested [144], Isolated SC will be seeded in a glass bottom petridish (0.5xl0⁶ cells in p-Dish 35 mm, Ibidi) and cultured in RPMI-1640 media under standard cell culture conditions with 10% exosome-depleted FBS (Gibco). Isolated Exoab (3xl0⁶) will be added. The uptake kinetics (0-4h) of exosome will be studied using dSTORM super-resolution (20 nm) microscopy fitted with live cell imaging (Oxford Nano Imager, UK).

A “self-peptide” (CD47) approach will be adopted to test whether Exoab enriched NGF and co-regulated factors, following TNTASK, improve nerve fiber conductivity in DPN [153- 157], CollAl promoter-driven plasmids were designed encoding murine CD63, CD9 and CD81 with “in frame” mNeon-GFP reporter connected by IRES with CD47 sequence (Figure 29). Delivery of such plasmid on murine skin will result in the release of Exonb which will also express the “eat me not” signal because of the self-peptide (CD47). Such self-peptide tagged Exoab will not be taken up by Schwann cells [153-157], This approach will attenuate the beneficial effects of TNT ASK on DPN outcomes. This study will be conducted in both male and female diabetic mice (n=30). db/db mice will be randomly divided into three groups (n=10). The first two groups (Group 1& 2) will be transfected with Coll Al promoter driven tetraspanins plasmids with “in frame” mN-GFP reporter for labeling Exofib. The third group (Group 3) will be transfected with Coll Al promoter driven tetraspanins plasmids with “in frame” mN-GFP reporter connected by IRES with CD47 sequence. One day following delivery of these plasmids, TNTEP and TNTASK will be performed on G1 and G2-3, respectively. Assessment of mechanical allodynia and thermoception will be performed at 2, 6, and 18 weeks post- TNTASK using a Dynamic Plantar Aesthesiometer and Thermo Plantar systems (Ugo Basile). Mechanical sensitivity of the hind paw will be analyzed using a thin steel filamentous rod against the plantar surface of the paw from beneath with a constantly increasing force from 0 to 5 g in 10 seconds (ramp 0.5 g/s) and then holds 5 g for an additional 10 s [158-161], For thermoception, mice will be placed unrestrained in individual compartments. After an acclimation period, an infrared source will be positioned under the glass floor directly beneath the hind paw of the animals and paw withdrawal latency will be measured [162], Paw withdrawal latency will be calculated as the mean of 4-6 measurements with at least 20 s interval between each reading. Peripheral nerve conductivity will be quantified at 10 and 18 weeks post-TNTASK using an EMStat3 potentiostat (PalmSens, Netherlands) with a minimum current resolution of IpA and an approximate rise time of 100 ps and conducting polymer microelectrodes. A bias voltage is applied to partially reduce the conducting polymer and data will be collected at 1000 Hz. A 14G needle-catheter will be inserted intramuscularly in the tibialis anterior muscles in such a way that the catheter is in close proximity to the peroneal nerve. The needle will be withdrawn leaving the catheter in place. A 1.85 mm probe with a PPy(DBS) conducting polymer working electrode [107] (Figure 20), capable of measuring in situ cation concentration, will be inserted. Capacity transients will be mitigated and series resistance compensated by adjusting the bias voltage according to the in vivo cyclic voltammetry response. Dynamic changes in cation concentration in the vicinity (<2 mm) of the partially reduced conducting polymer membrane will be recorded and will be plotted as a function of neuronal excitability. Prior to insertion, the sensor will be equilibrated via cyclic voltammetry in saline solution that has a concentration of 154 mM Na+ ions. A partial reduction potential (ca. - 400 mV) will be selected and applied to the polymer, the current response will be measured utilizing chronoamperometry (CA). The mouse will be electrically grounded on a temperature regulated base plate and the muscle will be mechanically stimulated using sharp forceps. Laser speckle imaging will be done at 1, 2, 4, and 8 weeks post- TNT to assess blood flow (Figure 30) [164], Color-coded perfusion maps of each time-point (as above) post-wounding will be acquired. Perfusion will be scored using PimSoft vl.4 software (Perimed Inc).

Mice will be euthanized at 18 weeks post-measurement of peripheral nerve conduction. The sciatic nerve will be exposed and 0.5 cm will be excised. The excised sciatic nerve will be fixed with 1 ml of 4% paraformaldehyde and 3% glutaraldehyde in 0.1 M PBS. Following 24- 48 h fixation, samples will be washed 3x with 5 ml PBS before secondary fixation with osmium tetroxide for 1 h at room temperature. Samples will be washed and subjected to sequential dehydration and embedding in epoxy resin in molds and baked at 60 °C for 48 h to solidify. Samples will be sectioned and TEM imaging performed (JEOL). Morphometric analysis of ‘myelinated fibers’ (spherical cross section) or ‘myelin infoldings’ (spherical cross section with a pronounced concavity on at least one side) and presence of Remak bundles in the sciatic nerve trunk will be detected [169, 170], Myelin fiber density (#myelin fibers/FOV), myelinated area (%myelin thickness/FOV), and ratio of fibers with myelin infoldings will be calculated using AxonDeepSeg program. Remak bundles will be visually identified as clusters of small C-fibers without a myelin sheath. Additionally, the average number of axons present in Remak bundles will be quantified for each experimental “Eat Me Not” Exosomes to Block Cellular Uptake condition. To assess the PGP9.5 fiber density following neuropathy, the plantar footpad skin will be harvested, fixed in formalin, and embedded in paraffin (FFPE). Akoya Phenocycler Fusion spatial proteomics will be performed from the FFPE sections using antibodies against, PGP9.5, MEF2C, MGP, MYL9, (and DAPI) and fluorescence imaging will be performed on a Zeiss Axioscan.Zl (Figures 15, 22, and 24).

TNT ASK reprogramming of the skin induces neuronal pentraxin (NP2) to rescue DPN by improving neuronal sensory signal transduction. NP2 is a member of the pentraxin family, identified as a neuronal activity-regulated pentraxin, and is expressed in many tissues (skin, brain, testis, pancreas, liver, skeletal muscle, and heart) [173], In skin, NP2 is expressed in dermal fibroblast populations including DP and papillary fibroblasts (Human Protein Atlas). NP2 is an extracellular scaffolding protein that regulates homeostatic synaptic plasticity [174,175] and synaptogenesis [176], Cytokine array (Proteome Profiler Mouse XL Cytokine Array; Biotechne) studies on TNTASK treated db/db skin identified differentially expressed genes: NP2, IL7, DKK-1, EGF, CD93, and MMP2. Thus, TNTASK restores NP2 in the diabetic skin and promotes colocalization with NGF (Figure 31). Skin NP2 induces SNAIL (RNA and protein), and interacts with METTL3 to promote the m6A methylation of SNAIL [177], Of note, SNAIL is a transcriptional regulator predicted to bind and regulate NGF through its promoter region. In published scRNASeq data, NP2 is upregulated in fibroblast populations (by descending order of expression): dermal sheath>papillary>dermal papilla>fascia>pericytes>hypodermal [178], TNTASK of db/db skin inhibited AMP ARI and mGLURl pointing towards potential mechanisms of neurotransmission rescue (Figure 32).

Mice (db/db, 6 weeks) will be treated as described. Quantifiable changes in NP2 and NGF expression will be quantified 2, 6, and 21 week post- TNTASK by ELISA (Biosensis# BEK2213) and spatial proteomics (Figure 23H and 31 A). Spatial proteomic samples will be fixed in formalin, embedded in paraffin blocks, cut into 5 pm sections, and analyzed using AKOYA PhenoCycler-Fusion2.0. Staining with Abeam barcoded antibodies for anti-NP2, anti-NGF, anti-Synapsinl, anti-Synaptophysin, anti-mGlURla, anti-p38MAPK14, anti- PSD95, anti-NMDA, anti-AMPARl, anti-Navl.6, anti TRPV4 anti-BDNF, anti-GAP43, anti- MAP2, anti-TUIl, anti-NF200, and anti-CollA2 will be performed. Spatial proteomics analysis will be performed using PhenoChart software, as described previously (Figures 14 and 23). The impact of NP2 expression on excitatory peripheral neurotransmission in DPN (db/db) mice will be assessed using NP2 knockdown and NP2 activation CRISPR interference (CRISPRi) strategies. TNT of CRISPRi plasmids will be performed on two groups of mice using our published method [179], in 6 wk old db/db mice (and db/m+ heterozygous controls) with the following exceptions. A different promoter, Coll A2 (fibroblast promoter) will be used to identify cell-specific and gene-specific changes. Group 1 (n=8) will receive inhibitory plasmid pHR-UCOE-SFFV-dCas9-mCherry-ZIM3-KRAB (Addgene# 154473) and CollA2- Np2-gRNA (NovoPro) or control CollA2-Scrambled-Np2-gRNA (NovoPro). Group 2 (n=8) will receive an activator plasmid dCas9-p300 RFP (Millipore# DCAS9P300RFP-1EA) with the same Np2 gRNA construct or scrambled control as Group 1. Guide RNA (gRNA) plasmids will be designed to target Np2 CpG Exon 2 or scrambled control, both with the promoter replaced by a fibroblast-specific CollA2 promoter. Post-TNTASK (2 weeks) 6 mm skin punch biopsies will be assessed for NP2 and NGF using spatial proteomic imaging. Over a time course of 2, 6, and 21 weeks post-TNTASK treatment, mice will be assessed for conductivity, mechanical allodynia (Von Frey) and thermoception (thermal plantar paw withdrawal latency) as described.

EXAMPLE 3: RESCUE OF METABOLICALLY ACQUIRED PERIPHERAL NEUROPATHY (PN) INDUCED BY HIGH-FAT DIET

The present example demonstrates the impact of TNT ASK on rescue of PN in a mouse model of neuropathy induced by high-fat diet, i.e., mice with diet-induced obesity (DIO) (Figure 33). DIO mice exhibit phenotypes that mimic human metabolic disorders such as obesity and type II diabetes; phenotypes include increased body weight, elevated blood glucose, impaired glucose tolerance, leptin resistance, and insulin resistance. As such, DIO mice serve as a model for obesity or pre-diabetic type II diabetes.

DIO was initiated by feeding mice with a high-fat diet (60 kcal% fat) starting at 5 weeks of age. Mice fed with a standard diet (SD) were used as a control.

Functional recovery of PN-compromised allodynia and thermal hyperalgesia was evaluated for DIO mice following TNTASK treatment. DIO mice which received a mock treatment exhibited increased withdrawal latency in response to mechanical or thermal stimuli versus SD control mice (Figures 34A-34D). However, DIO mice which received TNTASK exhibited withdrawal latency which was significantly reduced versus DIO mice which received the mock treatment and similar to that observed for SD control mice.

Functional recovery of PN-associated thermal hypothesia was separately tested using a hot plate apparatus to generate heat stimuli. Latency of several behaviors was measured: licking of the hind paw, rearing, and jumping. Similar to previous results, DIO mice which received a mock treatment exhibited increased latency in response to thermal stimuli from the hot plate versus SD control mice (Figures 35A-35D). DIO mice which received TNTASK exhibited behavior latency which was significantly reduced versus DIO mice which received the mock treatment and similar to that observed for SD control mice.

Laser speckle imaging was done at baseline or 2 weeks post- TNT to assess blood flow (Figure 36A-36C). Perfusion was significantly reduced in DIO mice in comparison to SD control mice. However, TNTASK significantly improved perfusion in feet of DIO mice; perfusion of feet in DIO mice was similar to that observed for SD control mice.

Nerve conductivity was measured. Nerve conductivity was significantly reduced in DIO mice in comparison to SD control mice (Figure 37). TNTASK significantly improved nerve conductivity in DIO mice; nerve conductivity in DIO mice was similar to that observed for SD control mice. Comparable results were obtained when evaluating PGP9.5 fiber density. PGP9.5 fiber density was significantly reduced in DIO mice in comparison to SD control mice (Figures 38A-38B); TNTASK significantly improved nerve fiber density in DIO mice.

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* * *

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and compositions of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, this present disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of preventing and/or treating nerve damage in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOXIO; and c. a nucleic acid encoding KROX20.

2. The method of claim 1, wherein ASK comprises ASO-miR200b.

3. The method of claim 1, wherein ASK comprises a nucleic acid encoding ASO- miR200b.

4. The method of claim 1, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

5. The method of claim 4, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

6. The method of claim 5, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

7. The method of claim 6, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO : 1 or SEQ ID NO.: 2.

8. The method of any one of claims 1-7, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng.

9. The method of claim 8, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng.

10. The method of any one of claims 1-7, wherein ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 0.5 pg and about 100 pg.

11. The method of claim 10, wherein ASK comprises a nucleic acid encoding SOXIO in an amount ranging from between about 5 pg and about 10 pg.

12. The method of any one of claims 1-7, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg.

13. The method of claim 12, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg.

14. The method of any one of claims 1-13, wherein ASK is administered using tissue nanotransfection.

15. The method of any one of claims 1-14, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOXIO to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1:10:1 to about 10:1:10; from about 1:10:1 to about 1:1:1, from about 1 : 1 : 1 to about 10:1:10, from about 1 : 1 : 10 to about 10:10:1, from about 1:1:10 to about 1 : 1 : 1, or from about 1:1:1 to about 10:10:1.

16. The method of any one of claims 1-15, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2: 1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1.

17. The method of any one of claims 1-16, wherein the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10: 1 to about 1 : 1, or from about 1:1 to 1:10.

18. The method of any one of claims 1-17, wherein the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 10:1, about5:l, about2:l, about 1:1, about 1:2, about 1:5, or about 1:10.

19. The method of any one of claims 1-18, wherein the molar ratio of nucleic acid encoding SOXIO to nucleic acid encoding KROX20 is about 1:1.

20. The method of any one of claims 1-19, wherein the nerve damage is associated with diabetic polyneuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or pre-diabetic type II diabetes.

21. The method of any one of claims 1-20, wherein ASK is administered to skin tissue of the subject.

22. The method of any one of claims 1-21, wherein the skin tissue comprises skin fibroblasts.

23. The method of claim 22, wherein the skin fibroblasts are reprogrammed to a cell population comprising Schwann cells.

24. The method of claim 21, further comprising transplanting skin tissue administered with ASK to another location of the subject.

25. A pharmaceutical composition comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOXIO; and c. a nucleic acid encoding KROX20.

26. The pharmaceutical composition of claim 25, wherein ASK comprises ASO-miR200b.

27. The pharmaceutical composition of claim 25, wherein ASK comprises a nucleic acid encoding ASO-miR200b.

28. The pharmaceutical composition of claim 25, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

29. The pharmaceutical composition of claim 28, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

30. The pharmaceutical composition of claim 29, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

31. The pharmaceutical composition of claim 30, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

32. The pharmaceutical composition of any one of claims 25-31, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng.

33. The pharmaceutical composition of claim 32, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng.

34. The pharmaceutical composition of any one of claims 25-31, wherein ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 0.5 pg and about 100 pg.

35. The pharmaceutical composition of claim 34, wherein ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 5 pg and about 10 pg.

36. The pharmaceutical composition of any one of claims 25-31, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg.

37. The pharmaceutical composition of claim 36, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg.

38. The pharmaceutical composition of any one of claims 25-37, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1:10:1 to about 10:1:10; from about 1:10:1 to about 1:1:1, from about 1:1:1 to about 10:1:10, from about 1:1:10 to about 10:10:1, from about 1:1:10 to about 1:1:1, or from about 1:1:1 to about 10:10:1.

39. The pharmaceutical composition of any one of claims 25-38, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2:1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5: 1 :5, about 10: 1 : 10, about 1 : 1 : 10, about 1: 1 :5, about 1 : 1 :2, about 2:2: 1, about 5:5: 1, or about 10: 10: 1.

40. The pharmaceutical composition of any one of claims 25-39, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10: 1 to about 1 : 10, from about 10: 1 to about 1 : 1, or from about 1 : 1 to 1 : 10.

41. The pharmaceutical composition of any one of claims 25-40, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10: 1, about 5: 1, about 2: 1, about 1 : 1, about 1 :2, about 1 :5, or about 1 : 10.

42. The pharmaceutical composition of any one of claims 24-41, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 1 : 1.

43. The pharmaceutical composition of any one of claims 25-42, further comprising a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of any one of claims 25-43, wherein the skin tissue comprises skin fibroblasts that are reprogrammed to a cell population comprising Schwann cells.

45. The pharmaceutical composition of any one of claims 25-44, wherein the skin tissue comprises non-reprogrammed skin fibroblasts.

46. An in vivo method of enhancing nerve innervation in a skin graft in a subject in need thereof, comprising administering to the skin graft a therapeutically effective amount of a composition comprising ASK, wherein ASK comprises: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOX10; and c. a nucleic acid encoding KROX20.

47. The method of claim 46, wherein ASK comprises ASO-miR200b.

48. The method of claim 46, wherein ASK comprises a nucleic acid encoding ASO- miR200b.

49. The method of claim 46, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

50. The method of claim 49, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

51. The method of claim 50, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

52. The method of claim 51, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO : 1 or SEQ ID NO.: 2.

53. The method of any one of claims 46-52, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 1 ng and about 500 ng.

54. The method of claim 53, wherein ASK comprises ASO-miR200b or nucleic acid encoding ASO-miR200b in an amount ranging from between about 150 ng and about 200 ng.

55. The method of any one of claims 46-52, wherein ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 0.5 pg and about 100 pg.

56. The method of claim 55, wherein ASK comprises a nucleic acid encoding SOX10 in an amount ranging from between about 5 pg and about 10 pg.

57. The method of any one of claims 46-52, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 0.5 pg and about 100 pg.

58. The method of claim 57, wherein ASK comprises a nucleic acid encoding KROX20 in an amount ranging from between about 5 pg and about 10 pg.

59. The method of any one of claims 46-58, wherein ASK is administered using tissue nanotransfection.

60. The method of any one of claims 46-59, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1:1 to about 1:10:10, from about 10:1:1 to about 1:1:1, from about 1:1:1 to about 1:10:10, from about 1:10:1 to about 10:1:10; from about 1:10:1 to about 1:1:1, from about 1 : 1 : 1 to about 10:1:10, from about 1 : 1 : 10 to about 10:10:1, from about 1:1:10 to about 1 : 1 : 1, or from about 1:1:1 to about 10:10:1.

61. The method of any one of claims 46-60, wherein the molar ratio of ASO-miR200b or nucleic acid encoding ASO-miR200b to nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1:1, about 5:1:1, about 2: 1:1, about 1:1:1, about 1:2:2, about 1:5:5, about 1:10:10, about 1:10:1, about 1:5:1, about 1:2:1, about 2:1:2, about 5:1:5, about 10:1:10, about 1:1:10, about 1:1:5, about 1:1:2, about 2:2:1, about 5:5:1, or about 10:10:1.

62. The method of any one of claims 46-61, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 ranges from about 10:1 to about 1:10, from about 10: 1 to about 1 : 1, or from about 1:1 to 1:10.

63. The method of any one of claims 46-62, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, or about 1:10.

64. The method of any one of claims 46-63, wherein the molar ratio of nucleic acid encoding SOX10 to nucleic acid encoding KROX20 is about 1:1.

65. The method of claim 46, wherein the skin graft comprises skin fibroblasts.

66. A method of preventing and/or treating nerve damage in a subject in need thereof, comprising administering a therapeutically effective amount of: a. ASO-miR200b or a nucleic acid encoding ASO-miR200b; b. a nucleic acid encoding SOX10; and c. a nucleic acid encoding KROX20.

67. The method of claim 66, wherein the ASO-miR200b or a nucleic acid encoding ASO- miR200b, the nucleic acid encoding SOX10, and the nucleic acid encoding KROX20 are administered sequentially.

68. The method of claim 66 or claim 67, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

69. The method of claim 68, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

70. The method of claim 69, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 2.

71. The method of claim 70, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b comprises a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO : 1 or SEQ ID NO.: 2.

72. The method of any one of claims 66-71, the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered in an amount ranging from between about 1 ng and about 500 ng-

73. The method of claim 72, wherein the ASO-miR200b or the nucleic acid encoding ASO- miR200b is administered in an amount ranging from between about 150 ng and about 200 ng.

74. The method of any one of claims 66-71, wherein the nucleic acid encoding SOX10 is administered in an amount ranging from between about 0.5 pg and about 100 pg.

75. The method of claim 74, wherein the nucleic acid encoding SOX10 is administered in an amount ranging from between about 5 pg and about 10 pg.

76. The method of any one of claims 66-71, wherein the nucleic acid encoding KROX20 is administered in an amount ranging from between about 0.5 pg and about 100 pg.

77. The method of claim 76, wherein the nucleic acid encoding KROX20 is administered in an amount ranging from between about 5 pg and about 10 pg.

78. The method of any one of claims 66-77, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b is administered using tissue nanotransfection.

79. The method of any one of claims 66-77, wherein the nucleic acid encoding SOX10 is administered using tissue nanotransfection.

80. The method of any one of claims 66-77, wherein the nucleic acid encoding KROX20 is administered using tissue nanotransfection.

81. The method of any one of claims 66-80, wherein the nerve damage is associated with diabetic polyneuropathy, peripheral neuropathy, obesity, type I diabetes, type II diabetes, or pre-diabetic type II diabetes.

82. The method of any one of claims 66-81, wherein the ASO-miR200b or the nucleic acid encoding ASO-miR200b, the nucleic acid encoding SOXIO, and the nucleic acid encoding KROX20 are administered to skin tissue of the subject.

83. The method of any one of claims 66-82, wherein the skin tissue comprises skin fibroblasts.

84. The method of claim 83, wherein the skin fibroblasts are reprogrammed to a cell population comprising Schwann cells.