Methods and compositions for treating neuropathy

Abstract

Disclosed herein are gene therapy vectors that are adipocyte-tropic (AAVRec2-BDNF or AAVRec2-NGF, for example) which can improve measurements of tissue innervation in neuropathic adipose tissue as well as platforms and kits for delivering the same. The disclosed platforms and kits can effectively deliver AAV-containing fluids subdermally across the subcutaneous adipose depot.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING NEUROPATHY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 558,518, filed February' 27, 2024, which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT SUPPORT CLAUSE

[0005] This invention was made with government support under grant / contract numbers DK076169 and U24 DK115255, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] The present disclosure relates to methods, platforms, and kits using gene therapy vectors that are adipocyte-tropic (AAVRec2-BDNF or AAVRec2-NGF, for example) and the uses thereof to treat Peripheral Neuropathy.

[0008] BACKGROUND

[0009] Diabetes is the leading cause of peripheral neuropathy (PN), which results in loss of innervation in tissues like skin and muscle, and as was demonstrated previously, also in adipose tissues. PN is a chronic condition of progressive nerve degeneration that affects an estimated 20 million people in the U.S. alone. This estimate is considered low as many individuals with PN symptoms have not been tested, and there is a lack of effective diagnosis of small fiber PN. PN often begins in the most distal long axons and progresses proximally. Therefore, most PN patients present with initial symptoms in extremities (hands / feet). However, PN can also occur in small fiber nerves of various internal tissues or organs, thereby impairing their function. Although there are over 30 causes of PN, the major underlying cause of PN is diabetes (which includes pre-diabetes). Given that the number of diabetic patients continues to rise worldwide, increased cases of diabetic (D)PN will inevitably follow. PN is a painful, debilitating disease, burdened by high healthcare costs and no effective treatment beyond pain control. Followed by DPN, idiopathic PN (which has its highest prevalence in the aging population) is the second most common form of PN, followed by chemotherapy -induced (CI)PN. DPN can occur in more than half of patients with diabetes, often presenting as painful (p)DPN. pDPN is particularly challenging to manage clinically, in large part due to the risk of dependency on opioid pain medications and the low number of other reliable treatment options. While evidence now indicates that neuropathy onset can occur as early as the pre-diabetic stage, sensitive diagnoses and effective treatments for small fiber neuropathies like these types have remained elusive. However, unlike the nerves of the central nervous system (CNS), peripheral nervous system (PNS) axons are more capable of regeneration under certain conditions, providing a target for PN treatment development. Peripheral nerves are abundant in adipose tissue and provide a means of communication with the CNS to coordinate the regulation of energy balance. For decades, surgical and chemical denervation studies in adipose tissue have demonstrated the importance of sympathetic and sensory nerves for proper tissue function and metabolic control. In previous studies, it has been demonstrated that nerve density is reduced in subcutaneous white adipose tissue (scWAT) in states of obesity / diabetes or aging in mice and humans (a condition called “adipose neuropathy”) and that cold or exercise can promote neural plasticity in adipose.

[0010] Thus, there is a need to address the aforementioned problems and other shortcomings associated with traditional interv entions to stimulate nerve regeneration while treating peripheral neuropathy. These needs and others are at least partially satisfied by the present disclosure.

[0011] SUMMARY

[0012] The present invention relates to a method of treating peripheral neuropathy or peripheral nerve degeneration or dysfunction in a subject in need thereof, the method comprising delivering a vector to the subj ect, wherein the vector comprises one or more neurotrophic factor gene(s). wherein said one or more neurotrophic factor gene(s) improves and / or supports tissue innervation in the subject.

[0013] Also disclosed is a platform for direct deliver}' of a vector to adipose tissue, wherein said vector can treat peripheral neuropathy, wherein the vector comprises one or more gene(s) encoding at least one neurotrophic factor, wherein said the at least one neurotrophic factor improves and / or supports tissue innervation in the subject.

[0014] Accordingly, in one aspect, disclosed herein is a method of treating peripheral neuropathy (such as, for example, adipose neuropathy) in a subject in need thereof, the method comprising delivering a vector (such as, for example, adenovirus-associated virus (AAV)) to the subject, wherein the vector comprises one or more neurotrophic factor gene(s) (such as, for example. Brain- Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs)), wherein said one or more neurotrophic factor gene(s) treats peripheral neuropathy in the subject. In some embodiments, the peripheral neuropathy is adipose neuropathy. In some embodiments, the peripheral neuropathy is obesity / diabetes-associated. In some embodiments, peripheral neuropathy is related to drug or environmental toxicity, aging, or idiopathy, is related to viral or immune issues, or is caused by injury / surgery, is medically related, or is genetically related.

[0015] In some embodiments, peripheral neuropathy is treated by directly or indirectly improving tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nerve function.

[0016] In some embodiments, the vector is delivered directly to a dermal tissue, subdermal tissue or a tissue of the subject. In some embodiments, the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue. In some embodiments, the vector comprises adipose-tropic, a neurotropic vector, a hepatotropic vector, a myotropic vector, a cardiotropic vector, a pulmotropic vector, a renotropic vector, a hematopoietic tropic vector, a pancreatic tropic vector and an osteotropic vector. In some embodiments, the vector comprises adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle. In some embodiments, the AAV vector is AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO and Rec2. In some embodiments, the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF). Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s). In some embodiments, the dosage of the neurotrophic factor gene is about IxlO13vg per fat depot. In some embodiments, the vector is delivered by syringe injection. In some embodiments, the vector is delivered through an adipose tissue targeting device. In some embodiments, the device is a DEN- TEN device.

[0017] In some embodiments, the vector can be combined with at least one additional form of treatment or therapy. In some embodiments, the additional form of treatment inhibits signals that prevent or block nerve regrowth or regeneration. In some embodiments, the additional form of treatment comprises tissue nanotransfection. In some embodiments, said tissue nanotransfection takes place before or during the delivery of the vector. In some embodiments, the additional form of treatment comprises an additional gene or genes for therapeutic purposes. In some embodiments, the additional gene or genes are within the same vector. In some embodiments, the genes are in different vectors or other delivery vehicles. In some embodiments, the additional form of treatment comprises one or more therapeutic composition(s). In some embodiments, the additional form of therapy comprises lifestyle modification. In some embodiments, said lifestyle modification comprises a blood sugar regulation, calorie-restricted diets / exercise, cholesterol-free diet, low sugar diet, and / or low-fat diet. In one aspect, disclosed herein is a platform for direct delivery of a vector (such as, for example, adenovirus-associated virus (AAV)) to adipose tissue, wherein said vector can treat peripheral neuropathy (such as. for example, adipose neuropathy), wherein the vector comprises one or more gene(s) encoding at least one neurotrophic factor (such as, for example, Brain-Derived Neurotrophic Factor (BDNF), Nerve Grow th Factor (NGF), Bone Morphogenic Proteins (BMPs)), wherein said the at least one neurotrophic factor supports and / or improves tissue innervation in the subject. In some embodiments, the peripheral neuropathy is adipose neuropathy.

[0018] In some embodiments, peripheral neuropathy is treated by improving tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nen e function.

[0019] In some embodiments, the vector is delivered directly to a dermal tissue, subdermal tissue or a tissue of the subject. In some embodiments, the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue. In some embodiments, the vector comprises adipose-tropic, a neurotropic vector, a hepatotropic vector, a myotropic vector, a cardiotropic vector, a pulmotropic vector, a renotropic vector, a hematopoietic tropic vector, a pancreatic tropic vector and an osteotropic vector. In some embodiments, the vector comprises an adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle. In some embodiments, the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhl 0 and Rec2. In some embodiments, the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s). In some embodiments, the dosage of the neurotrophic factor gene is about 1x1013vg per fat depot. In some embodiments, the vector is delivered by syringe injection. In some embodiments, the vector is delivered through an adipose tissue targeting device. In some embodiments, the device is a DEN-TEN device.

[0020] In some embodiments, the vector can be combined with at least one additional form of treatment or therapy. In some embodiments, the additional form of treatment comprises an additional gene or genes for therapeutic purposes. In some embodiments, the additional gene or genes are within the same vector. In some embodiments, the genes are in different vectors. In some embodiments, the additional form of treatment comprises one or more therapeutic composition(s). In some embodiments, the one or more therapeutic composition(s) comprise an immune- and / or inflammation-modulatory’ treatment and / or a growth factor. In some embodiments, the additional form of treatment is delivered to a different tissue of the subject than the vector (e.g., skin or subdermal tissue), is targeted to a different tissue of the subj ect than the vector, and / or has a different treatment timescale (i.e., short-term vs. long-term) than the vector. In some embodiments, the additional form of treatment comprises gene deletion / modification / knock-down with a gene overexpression.

[0021] In some embodiments, a first vector comprising a first neurotrophic factor is delivered to the subject at a first time point, and wherein a second vector comprising a second neurotrophic factor is delivered to the subject at a second time point later than the first time point. In some embodiments, the first time point and the second time point are from about 1 week to about 1 year apart.

[0022] In one aspect, disclosed herein is a kit for direct delivery of a vector to adipose tissue, wherein the vector (such as, for example, adenovirus-associated virus (AAV)) to adipose tissue, wherein said vector can treat peripheral neuropathy (such as, for example, adipose neuropathy) in a subject, wherein the kit comprises the vector and an adipose tissue-targeting device, wherein the vector comprises one or more neurotrophic factor gene(s) (such as, for example, Brain-Derived Neurotrophic Factor (BDNF), Nerve Grow th Factor (NGF), Bone Morphogenic Proteins (BMPs)), wherein said one or more neurotrophic factor gene(s) supports and / or improve innervation in the subject. In some embodiments, the peripheral neuropathy is adipose neuropathy. In some embodiments, the peripheral neuropathy is obesity / diabetes-associated. In some embodiments, peripheral neuropathy is related to drug or environmental toxicity, aging, or idiopathy, is related to viral or immune issues, or is caused by injury / surgery, is medically related, or is genetically related. In some embodiments, peripheral neuropathy is treated by improving tissue innervation, synaptic contacts, nen e and / or axonal plasticity, or nerve function.

[0023] In some embodiments, the vector is delivered directly to a dermal tissue, subdermal tissue or a tissue of the subject. In some embodiments, the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue. In some embodiments, the vector comprises adipose-tropic, a neurotropic vector, a hepatotropic vector, a myotropic vector, a cardiotropic vector, a pulmotropic vector, a renotropic vector, a hematopoietic tropic vector, a pancreatic tropic vector and an osteotropic vector. In some embodiments, the vector comprises an adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle. In some embodiments, the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhl 0 and Rec2. In some embodiments, the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s). In some embodiments, the dosage of the neurotrophic factor gene is about 1x1013vg per fat depot. In some embodiments, the vector is delivered by syringe injection. In some embodiments, the vector is delivered through an adipose tissue targeting device. In some embodiments, the device is a DEN-TEN device.

[0024] In some embodiments, the vector can be combined with at least one additional form of treatment or therapy. In some embodiments, the additional form of treatment inhibits signals that prevent or block nerve regrowth or regeneration. In some embodiments, the additional form of treatment comprises tissue nanotransfection. In some embodiments, said tissue nanotransfection takes place before or during the delivery of the vector. In some embodiments, the additional form of treatment comprises an additional gene or genes for therapeutic purposes. In some embodiments, the additional gene or genes are within the same vector. In some embodiments, the genes are in different vectors or other delivery vehicles. In some embodiments, the additional form of treatment comprises one or more therapeutic composition(s). In some embodiments, the one or more therapeutic composition(s) comprise an immune- and / or inflammation-modulatory treatment and / or a grow th factor. In some embodiments, the additional form of treatment is delivered to a different tissue of the subject than the vector (e.g., skin or subdermal tissue), is targeted to a different tissue of the subject than the vector, and / or has a different treatment timescale (i.e. , short-term vs. long-term) than the vector. In some embodiments, the additional form of treatment comprises gene deletion / modification / knock-down with a gene overexpression.

[0025] In some embodiments, a first vector comprising a first neurotrophic factor is delivered to the subject at a first time point, and wherein a second vector comprising a second neurotrophic factor is delivered to the subject at a second time point later than the first time point. In some embodiments, the first time point and the second time point are from about 1 week to about 1 year apart.

[0026] Additional aspects and advantages of the disclosure will be set forth, in part, in the detailed description and any claims which follow, and in part will be derived from the detailed description or can be learned by practice of the various aspects of the disclosure. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and, together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0029] FIGS. 1 A-1F depicts a study overview and AAV -BDNF and AAV -NGF delivery to adipose of obese mice. Figure 1A shows experimental designs for all studies. Figure IB shows a timeline for dietary interv ention (left panel) and AAV administration (right panel). BTBRo6 / o6male mice at 16 weeks of age (neuropathic age) received a bolus injection of IxlO10vg of AAVRec2-BDNF unilaterally to the ing-scWAT. with contralateral depot receiving virus buffer as vehicle control. Figure 1C shows the change in body weight and weights of ing-scWAT and pgWAT of AAVRec2- BDNF treated versus vehicle-treated depots at final disposition. Figures 1D-1E show protein expression of pan-neuronal marker PGP9.5 (Figure ID) and sympathetic nen e marker tyrosine hydroxylase (TH) (Figure IE) between AAVRec2-BDNF treated versus vehicle-treated depots. Figure IF shows the protein expression of BDNF in AAVRec2-BDNF -treated versus vehicle- treated depots. Western blot data normalized to indicated housekeepers, band intensities quantified in Image J, and analyzed by two-tailed Student’s t-test. Error bars are SEMs.

[0030] FIGS. 2A-2B depicts protein expression of BDNF in the hypothalami of mice administered AAVRec2-BDNF or AAVRec2 -Empty vector via injection into ing-scWAT, highlighting that overexpression of BDNF in the peripheral tissue (scWAT) does not alter BDNF expression in the CNS. Figure 2B depicts the protein expression of PGP9.5 of hind paw skin (left panel) and ing- scWAT (right panel) in mice at 17 weeks of HFND. Western blot data normalized to indicated housekeepers, band intensities quantified in Image J, and analyzed by two-tailed Student’s t-test. Error bars are SEMs. * Denotes data removed from analysis due to protein not running properly or lack of expression of loading control.

[0031] FIGS. 3A-3H depicts AAV -BDNF delivery to adipose of mice with shorter-durationHFND. Figure 3A shows a timeline for dietary intervention (left panel) and AAV administration (right panel). C57BL / 6J male mice at 12-13 weeks old were fed with HFND for 17 weeks; AAV was administered unilaterally, as described in Figure 1. Figure 3B shows the change in body weight and weights of ing-scWAT and pgWAT of AAVRec2-BDNF treated and vehicle-treated depots at the terminal time point. Figures 3C-3H show' protein expressions of PGP9.5 (Figure 3C), TH (Figure 3D), GAP43 (Figure 3E), VASP (Figure 3F), MPZ (Figure 3G), and BDNF (Figure 3H) between vehicle and AAVRec2-BDNF treated scWAT depots were shown. Western blot data were normalized to indicated housekeepers, and band intensities w ere quantified in Image J and analyzed using a tw o-tailed Student’s t-test. Error bars are SEMs.

[0032] FIGS. 4A-4H depicts AAV -BDNF delivery’ to adipose of mice with longer-duration HFND. Figure 4A shows a timeline for dietary intervention (left panel) and AAV administration (right panel). C57BL / 6J male mice at 10-12 weeks old were fed a high-fat neuropathy diet. After 41-43 weeks, mice received either AAVRec2-empty vector or AAVRec2-BDNF bilaterally to the ing- scWATs. Change in body weight within 4 weeks post-injection is shown in Figure 4B. Figure 4C shows the change in body weight and weights of ing-scWAT of AAVRec2-BDNF injected and empty vector injected mice at a terminal time point. Figures 4D-4H show protein expressions of PGP9.5 (Figure 4D), TH (Figure 4E), GAP43 (Figure 4F), MPZ (Figure 4G), and Vasp (Figure 4H) of ing-scWAT in AAVRec2-empty vector vs AAVRec2-BDNF treated mice at the endpoint. Western blot data were normalized to indicated housekeepers, and band intensities were quantified in Image J and analyzed using a two-tailed Student’s t-test. Error bars are SEMs.

[0033] FIG. 5 depicts changes in body composition pre- and post-AAV-BDNF treatment. The body composition of the AAVRec2-empty vector and AAVRec2-BDNF injected mice was measured by echoMRI pre- and 4 weeks post- AAV injection.

[0034] FIGS. 6A-6I depicts AAV-NGF delivery to adipose of mice on shorter-duration dietary intervention. Figure 6A shows a timeline of 60%HFD / 10% LFD feeding and AAV-mediatedNGF treatment. C57BL / 6J male mice at 6 weeks old were fed with either HFD or LFD. After 14-16 weeks, mice received either AAVRec2-empty vector or AAVRec2-NGF bilaterally to the ing- scWATs (Figure 6A, right panel). Figure 6B shows the protein expression of NGF in ing-scWAT 8 weeks post- AAV treatment versus pre-AAV treatment, which was measured via ELISA. Figure 6C shows a glucose tolerance test (GTT) for all groups 8 weeks post AAV treatment. Figure 6D shows energy expenditure measured by metabolic cages (CLAMS) 9 weeks post-AAV treatment for HFD groups only. Figures 6E-6I show protein expressions of PGP9.5 (Figure 6E), TH (Figure 6F), GAP43 (Figure 6G), Vasp (Figure 6H), and MPZ (Figure 61), in ing-scWAT of AAVRec2-NGF treated mice versus empty vector controls in HFD group only at 10 weeks post AAV treatment.

[0035] FIGS. 7A-7F shows body weights monitored following AAV treatment. Figure 7B shows that the body weight of each group at 8 weeks post AAV injection when performing the GTT test showed no difference. Figure 7C shows the weights of ing-scWAT, pgWAT, BAT, and ratios of ing-scWAT / BW, pgWAT / BW, BAT / BW of AAVRec2-NGF treated versus AAVRec2-empty vector controls in either LFD or HFD-fed mice. Figure 7D shows whole body composition measured both pre- and 9-weeks post-AAV injection in LFD and HFD-fed groups. Figures 7E-7F show skin surface temperate above the ingscWAT (Figure 7E) and BAT (Figure 7F) 8 weeks post-AAV treatment versus pre-AAV treatment measured via FLIR thermal imaging.

[0036] FIGS. 8A-8C depicts the Detecting Early Neuropathy to Treat Early Neuropathy (DEN- TEN) microneedle array for localized subdermal tissue delivery’ of AAV-mediated gene therapy. Figure 8A shows the DEN-TEN 3x3 microneedle array constructed using 32G stainless steel hollow needles to allow for AAV delivery directly to scWAT in a diffuse manner. The sharp beveled ends of needles penetrated the skin above the ing-scWAT to a depth of 2mm; Tygon tubing was attached to the blunt ends of needles, which were fitted with PEEK sleeves to ensure a tight leakless fit (i). 3D- printed needle holders and protective bases were designed to maintain needles at equal spacing and protect tips from damage (ii-iv). AAVRec2-BDNF, AAVRec2-NGF, or empty vector control were delivered to isoflurane-anesthetized mice, as illustrated in Figure 8B. The flank skin above the ing- scWAT was shaved, and the microneedle array was inserted through the skin with needles penetrating the fat pad. The DEN-TEN device was secured in place with medical tape. Figure 8B shows a multiple micro-syringe pump used to deliver the treatment at a slow rate of 0.03mL / min. Figure 8C shows Evans blue (EB) dye delivery to inguinal scWAT with tubing attached to 1, 2, or 3 needles within the DEN-TEN array. Arrow points to dye diffusion in inguinal scWAT following delivery of EB dye through a single needle (i) inset is a digital zoom in of scWAT (ii). The whole inguinal scWAT depot was resected to show the diffusion of EB dye injected through 2 needles of the array (iii). The whole inguinal scWAT depot was resected to show the diffusion of EB dye injected through 3 needles of the array (iv).

[0037] FIGS. 9A-9H depicts the physiological relevance of using an adipose tropic AAV for neurotrophic factor cargo delivery to scWAT to improve adipose neuropathy. Figure 9A show's a timeline of HFND feeding and AAV-mediated BDNF or NGF treatment. AAVRec2-BDNF, AAVRec2-NGF, or empty vector control w as delivered via microneedle array to bilateral ing- scWATs (at a dose of IxlO10vg per fat pad) to adult male mice following 25 weeks of HFND feeding. Figure 9B shows skin surface temperature above the ing-scWAT at 7 weeks post-treatment compared to pre-treatment by thermal imaging in all three groups. Figure 9C show's the body weights of each group at 7 weeks post- AAV treatment. Figures 9D-9H show' protein expression of PGP9.5 (Figure 9D). TH (Figure 9E), GAP43 (Figure 9F), Vasp (Figure 9G), and MPZ (Figure 9H) were measured at the endpoint.

[0038] FIGS. 10A-10C shows body weights following AAV treatment (left panel) and body weight, weights of ing-scWAT, and the ratio of ing-scWAT / BW at the endpoint (right panels). Figure 10B shows whole body composition changes in pre-AAV versus 6 w eeks post-AAV injection. Figure IOC shows brightfield imaging of axillary scWAT following Hemalum staining (left panels) and average cell size area and perimeter used to evaluate axillary scWAT cellularity (right panels).

[0039] FIG. 11 depicts ing-scWAT excised from adult male PGP9.5-EGFP mice following 25 weeks of HFND and AAV-mediated NGF treatment. Due to the large size of obese ing-scWAT depots, each tissue was bisected longitudinally, with the bottom tissue piece (containing the sub-iliac lymph node) processed for whole-mount imaging. Tissues were immunostained against the sympathetic nerve marker tyrosine hydroxylase (TH, red) and micrographs of the entire tissues were generated by tiling 10X objective magnification images.

[0040] FIG. 12 depicts AAVRec2-NGF increases targeted innervation and nerve terminal structures in ing-scWAT. ing-scWAT excised from adult male mice following 25 weeks ofHFND and AAV- mediated NGF treatment. Clusters of neuro-adipose nexus (NAN) with insets also display adipocyte autofluorescence. Representative image of axon density at NAN and diagram summarizing these observations. All micrographs are displayed as z-maximum intensity projections.

[0041] FIG. 13 depicts a conceptual model. Based on current literature and additional data, it is proposed that neurotrophic factor type and their cellular sources determine which nerves “wdre-up” to specific cell ty pe targets in tissues. In this adipose-specific model, BDNF secreted from immune cells (and other stromal vascular cells) maintains and promotes plasticity of TrkB-expressing nerve endings; adipocyte-secreted NGF maintains innervation of adipocytes and promotes NAN formation with TrkA-expressing nerve endings, endothelial cell-secreted NT3 maintains vascular innervation with TrkC-expressing nerve endings, and BDNF, NGF, and GDNF all contribute to bidirectional neural communication with Schwann cells.

[0042] DETAILED DESCRIPTION

[0043] Definitions

[0044] In this specification and in the claims w hich follow', reference will be made to a number of terms which shall be defined to have the following meanings:

[0045] Throughout the description and claims of this specification, the word “comprise7’ and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0046] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0047] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another 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 embodiment. 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. It is also understood that when a value is disclosed that is “less than or equal to” the value, “greater than or equal to the value,” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed, the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats. This data represents endpoints, starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises.” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0049] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0050] The term “administer.” “administering,” or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intrajoint, parenteral, intra- arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0051] “Comprising” is intended to mean that the compositions, methods, etc., include the recited elements but do not exclude others. “Consisting essentially of’ when used to define compositions and methods shall mean including the recited elements but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0052] As used herein, “diagnose,” “diagnosed,” “diagnosing,” and any grammatical variations thereof as used herein refer to the act or process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms. A “decrease” can 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, or composition in a statistically significant amount. For example, a decrease can mean in protein expression, such as of PSD-95. 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 so long as the decrease is statistically significant.

[0053] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0054] “Expression,” as used herein, refers to the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce a peptide / protein end product and ultimately affect a phenotype as the final effect.

[0055] A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein may be used to identity7larger fragments or full-length coding sequences of the gene with which they are associated. This term also includes genes with sequence modifications, such as deletions, insertions, replacements, and the like.

[0056] An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity7. An increase can be any individual, median, or average increase in a condition, symptom, activity, or composition in a statistically significant amount. For example, a decrease can mean in protein expression, such as of PSD-95. Thus, the increase 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% increase so long as the increase is statistically significant.

[0057] “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity7, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0058] A “neurodegenerative disease” is caused by the progressive loss of structure or function of neurons or glial cells, which make up the nervous system. These diseases include but are not limited to Peripheral Neuropathy, Amyotrophic Lateral Sclerosis (ALS). multiple sclerosis, Parkinson's disease. Alzheimer's disease. Huntington’s disease, and prion diseases. Neurodegenerative diseases can lead to cognitive and physical impairments, neuroinflammation (inflammation of the brain and spinal cord), and deterioration of brain and spinal cord tissues.

[0059] A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and makes up the cellular genetic material. Nucleic acids comprise nucleotides, which are monomers made of a 5 -carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0060] A “protein,” "polypeptide", or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.

[0061] By “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.

[0062] A “receptor” is a cellular protein whose activation causes a cell to modify its present functions or actions.

[0063] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., PSD-95 levels or symptoms of peripheral neuropathy). It is understood that this is typically in relation to some standard or expected value; in other words, it is relative, but it is not always necessary for the standard or relative value to be referred to. For example, “reduces PSD-95 levels” means reducing the level of PSD-95 compared to a standard or a control.

[0064] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0065] “Therapeutic composition” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., anon-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic composition” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0066] The term “therapeutically effective” refers to the amount of the composition used that is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0067] The term “therapeutic purposes” refers to the execution of a method of treatment, administration of a therapeutic composition, or another such action for the amelioration of one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0068] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as an ophthalmological disorder. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of ophthalmological disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e.. mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment", as used herein, can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subj ects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating" can include inhibiting the disease, disorder, or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. Treatments are administered to a subject prior to onset (e.g., before obvious signs of peripheral neuropathy), during early onset (e.g. , upon initial signs and symptoms of peripheral neuropathy), or after an established development of peripheral neuropathy.

[0069] As used herein, the term “vector” refers to any moiety which can deliver a nucleic acid sequence into a cell or virus so that the nucleic acid sequence can be replicated and / or expressed by the cell or virus. A “vector” refers to a DNA composition used as a vehicle to artificially carry foreign genetic material into another cell or tissue. A vector can be a bacterial plasmid, a viral vector, or a nanoparticle. A “bacterial plasmid” is a small extrachromosomal DNA molecule that can be incorporated into another cell that is physically separated from the chromosomal DNA and is easily replicated. A viral vector is a vehicle originally from a virus used to artificially carry foreign genetic material into another cell or tissue.

[0070] A “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid is flanked by two ITRs. A “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When arAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. A rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, e.g., an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”. An “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0071] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g.. a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0072] The term “transgene” refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.

[0073] The terms “genome particles (gp),” “genome equivalents,” or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002)A o / . Ther., 6:272-278.

[0074] The term “vector genome (vg)” as used herein may refer to one or more polynucleotides comprising a set of the polynucleotide sequences of a vector, e.g.. a viral vector. A vector genome may be encapsidated in a viral particle. Depending on the particular viral vector, a vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or doublestranded RNA. A vector genome may include endogenous sequences associated with a particular viral vector and / or any heterologous sequences inserted into a particular viral vector through recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a stiffer, a sequence of interest (e.g., an RNAi), and a polyadenylation sequence. A complete vector genome may include a complete set of the polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector may be measured in terms of vg / mL. Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

[0075] The terms “infection unit (iu),” “infectious particle,” or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0076] The term “transducing unit (tu)” as used in reference to a viral titer, refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in functional assays such as described in Examples herein, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0077] An “inverted terminal repeat" or “ITR” sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.

[0078] An “AAV inverted terminal repeat (ITR)'’ sequence, a term well-understood in the art, is an approximately 145 -nucleotide sequence that is present at both termini of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of selfcomplementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.

[0079] A “terminal resolution sequence” or “trs” is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.

[0080] A “helper virus” for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A number of such helper viruses have been identified, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Ban viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). “Percent (%) sequence identity” with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987). Supp. 30, section 7.7.18. Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity7to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / , where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B . It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0081] Method of Treatment

[0082] As used herein, “peripheral neuropathy” generally refers to dysfunction caused by damage or disease affecting nerves (e.g., peripheral nerves) leading to symptoms such as numbness, tingling, pain, and muscle weakness, often in the hands and feet. PN is a clinical diagnosis that is a progression / worsening of peripheral nerve degeneration or dysfunction. Peripheral neuropathy may be associated with decreased nen e conductance or aberrations in nerve electrical activity in an affected portion of the body. Peripheral neuropathy may be idiopathic (i.e., no known cause) or caused by, for example, a disease (e.g., diabetes), physical trauma, genetic background, and / or an infection. In some aspects, peripheral neuropathy is diabetic peripheral neuropathy (DPN), chemotherapy -induced peripheral neuropathy (CIPN), HIV or AIDS-induced peripheral neuropathy, and / or idiopathic peripheral neuropathies (IPN) (i.e., peripheral neuropathy with no identifiable known cause, such as with aging. In some aspects, the peripheral neuropathy is adipose neuropathy. In some aspects, the peripheral neuropathy is obesity / diabetes-associated. Peripheral neuropathy can also be related to drug or environmental toxicity, aging, or idiopathy, is related to viral or immune issues, is caused by injury / surgery, or is medically related or genetically related.

[0083] Maintaining functional adipose innervation is critical for metabolic health. Subcutaneous white adipose tissue (scWAT) undergoes peripheral neuropathy (PN) with obesity, diabetes, and aging (reduced small fiber innervation, nerve / synaptic / growth-cone / vesicle markers, altered nerve activity). Current therapeutic methods focus on managing the underlying cause (such as, for example, controlling diabetes), relieving symptoms with medications (such as. for example, analgesics) or therapies (such as. for example, physical therapy, acupuncture, transcutaneous electrical nerve stimulation (TENS)), and making lifestyle changes (such as, for example, maintaining a healthy diet, avoiding alcohol, quitting smoking, exercising regularly and managing blood sugar) to prevent further nerve damage. However, unlike peripheral nerve injuries, peripheral nerves don't regenerate with PN. Therefore, new therapies are needed to treat this condition that affects 20-30 million Americans.

[0084] Accordingly, disclosed herein is a gene therapy approach using a vector to deliver neurotrophic factors directly to adipose tissue to improve tissue-specific PN. Vector intra-adipose deliver}’ can support and / or improve tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nerve function in subjects with PN. This gene therapy approach can be used as a method to treat peripheral neuropathy in a subj ect in need thereof. The method comprises delivering a vector (such as, for example, adenovirus-associated virus (AAV)) to the subj ect, wherein the vector comprises one or more neurotrophic factor gene(s) (such as, for example, Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs)), wherein said one or more neurotrophic factor gene(s) treats peripheral neuropathy in the subject.

[0085] In some aspects, the vector is delivered directly to a dermal tissue, subdermal tissue or a tissue of the subject. This can be any type of tissue, such as, for example, subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nen es, liver tissue kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue, but in one specific example, it is adipose tissue. In some aspects, the adipose tissue is subcutaneous white adipose tissue (scWAT). In some aspects, the adipose tissue is inguinal subcutaneous white adipose tissue (ig-scWAT). In some aspects, the adipose tissue is dermal white adipose tissue (dWAT). In some aspects, the adipose tissue is perigonadal white adipose tissue (pgWAT). In some aspects, the adipose tissue is brown adipose tissue. In some aspects, the vector is delivered directly to the visceral adipose tissue of the subject. In some embodiments, the vector is adipose-tropic, neurotropic vector, hepatotropic vector, myotropic vector, cardiotropic vector, pulmotropic vector, renotropic vector, hematopoietic tropic vector, pancreatic tropic vector or osteotropic vector. In some embodiments, the vector is an adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, or an extracellular vesicle. In some embodiments, the vector is an adenovirus-associated virus (AAV). In some embodiments, the AAV vector is AAV 1 , AAV 2, AAV 5, AAV 6, AAV 8, AAV 9, AAV rh 10 or Rec2. In some embodiments, the AAV vector is Rec2.

[0086] Several compositions and methods can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral-based delivery systems and non-viral-based delivery systems. For example, the nucleic acids can be delivered through several direct delivery systems such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or phy si comechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, SISSIS, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. The methods can be modified to function with large DNA molecules in certain cases. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

[0087] Nucleic acid-based delivery systems

[0088] Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid) or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0089] As used herein, plasmid or viral vectors transport the disclosed nucleic acids, such as one or more neurotrophic factor gene(s), into the cell without degradation and include a promoter-yielding gene expression in the cells into which it is delivered. In some embodiments, the vector comprising one or more neurotrophic factor gene(s) is derived from either a virus or a retrovirus. Viral vectors are. for example. Adenovirus, Adeno-associated virus, Herpes virus. Vaccinia virus. Polio virus, AIDS vims, neuronal trophic vims, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families that share these viruses' properties, making them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia vims, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors can carnal arger genetic payload, i.e., atransgene or marker gene, than other viral vectors, andfor this reason, are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes; they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector that has been engineered to suppress the immune response of the host organism elicited by the viral antigens. Preferred vectors of this type carry coding regions for Interleukin 8 or 10.

[0090] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed, and a gene or gene / promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines that have been engineered to express the gene products of the early genes in trans.

[0091] Retroviral Vectors

[0092] A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M. Retroviral vectors for gene transfer.

[0093] A retrovirus is essentially a package that has been packed into its nucleic acid cargo. The nucleic acid cargo carries a packaging signal, ensuring that the replicated daughter molecules are efficiently packaged within the package coat. In addition to the package signal, a number of molecules are needed in cis for the replication and packaging of the replicated virus. Typically, a retroviral genome contains the gag, pol, and env genes, which are involved in making the protein coat. It is the gag, pol, and env genes that are typically replaced by the foreign DNA that is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence that signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine-rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. Removing the gag, pol. and env genes allows about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and. upon replication, be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the deliver}’ of one to many genes, depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0094] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line that has been transfected or transformed wi th a retrovirus that contains the replication and packaging machinery but lacks any packaging signal. When the vector carry ing the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0095] Adenoviral Vectors

[0096] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61: 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986): Davidson et al.. J. Virology 61: 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types since they can replicate within an initial infected cell but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high-efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Moullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4 461-416 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis in the same manner as wild-ty pe or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386- 396 (1973); Svensson and Persson, J. Ezro / ogy 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth et al., Mol. Cell. Biol. 4: 1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0097] A viral vector can be one based on an adenovirus that has had the El gene removed, and these virions are generated in a cell line such as the human 293 cell line. In another preferred embodiment, both the El and E3 genes are removed from the adenovirus genome.

[0098] Adeno-associated viral vectors

[0099] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV -type vectors can transport about 4 to 5 kb, and wild-type AAV is known to insert into chromosome 19 stably. Vectors that contain this site-specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0100] In some aspects, the vector is an adenovirus-associated virus (AAV). As used herein, the terms “adeno-associated virus (AAV) vector,” “AAV gene therapy vector,” and “gene therapy vector” refer to a vector having functional or partly functional ITR sequences and transgenes. As used herein, the term “ITR” refers to inverted terminal repeats (ITR). Adeno-associated viral vectors (AAV) can be constructed using known techniques to provide at least the operatively linked components of control elements, including a transcriptional initiation region, an exogenous nucleic acid molecule, a transcriptional termination region, and at least one post-transcriptional regulatory' sequence. The control elements are selected to be functional in the targeted cell. The resulting construct containing the operatively linked components is flanked at the 5' and 3' region with functional AAV ITR sequences. Herein, the ITRs flank at least one cassette containing a promoter that directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene that is not native to the AAV or B19 parvovirus. Typically, the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity7and site-specific integration but not cytotoxicity7and the promoter directs cell-specific expression. United States Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0101] In some aspects, the vector is selected from the serotype of one or more of AAV-1. AAV-2, AAV-3, AAV-4, AAAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV- 10. In some aspects, the vector is any human or non-human primate isolate, variant, recombinant, chimeric, or AAV capsid, including mutations, substitutions, deletions, or additions. In some aspects, the AAV vector is AAV- 2, a modified form of AAV-2 with an altered tropism. In some aspects, the vector is derived from AAV serotype 1 (AAV-1). In some aspects, the vector is selected according to the disclosure of U.S. Patent No. 9,265.843, which is hereby incorporated by reference in its entirety.

[0102] In some aspects, the vector is adipose-tropic. In some aspects, the vector is Rec2. Rec2 is a hybrid AAV serotype generated through capsid domain exchange or shuffling among AAV8, cy5 (cynomolgus macaque-variant 5), rh20 (rhesus macaque-variant 20), and rh39 (rhesus macaquevariant 39). Rec2 shows high transduction of both brown adipose tissue and white adipose tissue. The Rec2 serotype is detailed in Huang, W. et al. (2017). Targeting Visceral Fat by Intraperitoneal Delivery of Novel AAV Serotype Vector Restricting Off-Target Transduction in Liver. Molecular therapy. Methods & clinical development, 6, 68-78 and in Bates, R. et al. (2020). Adipose Tissue: An Emerging Target for Adeno-associated Viral Vectors. Molecular therapy. Methods & Clinical Development, 19, 236-249, both of which are hereby incorporated by reference in their entirety. In some aspects, the vector includes a combination of any of the above serotypes and / or modifications.

[0103] The disclosed vectors thus provide DNA molecules that are capable of integration into a mammalian chromosome without substantial toxicity'.

[0104] The inserted genes in viral and retroviral usually contain promoters and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location regarding the transcription start site. A promoter contains core elements required for the basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0105] Large payload viral vectors

[0106] Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated, and established in cells permissive to infection with herpesviruses (Sun et al., Nature Genetics 8: 33-41, 1994; Cotter and Robertson, Curr OpinMol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr vims (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells, such as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable. Maintaining these episomes requires a specific EBV nuclear protein, EBNA1 , constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0107] Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

[0108] Non-nucleic acid-based systems

[0109] The disclosed compositions can be delivered to the target cells in various ways. For example, the compositions can be delivered through electroporation, through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring, for example, in vivo or in vitro.

[0110] Thus, the compositions can comprise, in addition to the disclosed vector comprising one or more neurotrophic factor(s) or vectors, for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE. DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory' tract. Regarding liposomes, see. e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4, 897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0111] In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via various mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTION, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD). SUPERFECT (Qiagen, Inc. Hilden. Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA), as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0112] The materials may be in solution or suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell t pe via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins in tumor tissue (Senter et al., Bioconjugate Chem.. 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe et al., Br. J. Cancer, 58:700-703, (1988); Senter et al., Bioconjugate Chem., 4:3-9, (1993); Battelli et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colonic carcinoma), receptor-mediated targeting of DNA through cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220. (1989): and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in endocytosis pathways, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0113] Nucleic acids that are delivered to cells, which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral-related sequences, particularly when viral-based systems are used. These viral integration systems can also be incorporated into nucleic acids, which are to be delivered using a non-nucleic acid-based delivery system, such as a liposome, so that the nucleic acid contained in the delivery system can be integrated into the host genome.

[0114] Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary’ to promote homologous recombination are known to those of skill in the art.

[0115] In vivo / ex vivo

[0116] As described above, the compositions can be administered in a pharmaceutically acceptable carrier. They can be delivered to the subj ect cells in vivo and / or ex vivo by various mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis, and the like).

[0117] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate-mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard cell or tissue type methods. Standard methods are known for transplanting or infusing various cells into a subject.

[0118] Neurotrophic factors are proteins that support the growth, survival, and function of neurons. They play a crucial role in nerve development, regeneration, and protection against neurodegenerative diseases. In some embodiments, the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with neurotrophic factors. The genes encoding neurotrophic factors disclosed herein can be obtained from a variety of sources and can be naturally occurring, synthetically derived, or can be engineered. In the peripheral nervous system (PNS), “neurotrophic factors,’" along with their receptors, control axon survival as well as axon outgrowth / remodeling. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are the most well-characterized neurotrophic factors of the neurotrophin family, which also includes neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5). Neurotrophins are tissue-derived proteins that perform their actions through membrane-bound receptors on their target axons. These are the tyrosine kinase receptors or tropomyosin receptor kinases, Trks. While NGF and NT3 selectively bind to one Trk receptor (Trk A or Trk C, respectively), both BDNF and NT4 / 5 can signal through TrkB, and all neurotrophins can signal through the structurally unrelated p75 neurotrophin receptor (NTR). Upon neurotrophin binding to Trk receptors expressed on axon terminals, there is stimulation of a retrograde signaling cascade that promotes nerve survival or plasticity, while signaling via p75NTR can result in apoptosis. Although neurotrophins can signal individually via Trk or p75NTR receptors, Trk / p75NTR heterodimerization increases ligand binding affinity and augments growth and survival functions. Activated Trk receptors are internalized alone or along with their bound ligand and are retrogradely transported along the axon to the nerve cell body (which in the PNS is housed in the ganglia and not the tissue), where they activate gene expression changes necessary for nerve survival or axon outgrowth. In some aspects, the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), or a combination thereof. In some aspects, the neurotrophic factor is neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), or a combination thereof. In some aspects, the neurotrophic factor is BDNF, NGF, NT-3, NT-4 / 5, or any combination thereof. In some aspects, the neurotrophic factor is another suitable neurotrophin. In some aspects, the neurotrophic factor is a Bone Morphogenic Protein (BMP), such as BMP7. The BMP can also be used in combination with the neurotrophic factors discussed above.

[0119] In some aspects, the dosage of the neurotrophic factor gene is about IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, or IxlO14vg per fat depot (a discretely defined fat tissue), or any amount above, below, or in between these values. In one specific embodiment, the dosage is IxlO13vg per fat depot of approximately 0. 1-1.0 gram weight.

[0120] In some embodiments, the vector is delivered by syringe injection. In some embodiments, the vector is delivered through an adipose tissue targeting device. In some embodiments, the device is a DEN-TEN device. In some aspects, the DEN-TEN device is provided according to the disclosure of U.S. Patent Application Publication No. 2023 / 0233822. which is hereby incorporated by reference in its entirety.

[0121] In some aspects, the vector can be combined with at least one additional form of treatment or therapy. In some aspects, the additional form of treatment inhibits signals that prevent or block nene regrowth or regeneration. In some aspects, the additional form of treatment comprises tissue nanotransfection. As used herein, the term "‘tissue nanotransfeclion” or “TNT” refers to the delivery of cargo, particularly genes or peptides, into a cell using hollow microneedles and, typically, electroporation. Tissue nanotransfection is known to be one of the skills in the art and is described, for example, in Xuan, Y. et al. (2023). Tissue Nanotransfection in Regenerative Medicine. In MicroRNA in Regenerative Medicine (Second Edition) (pp. 1051-1074), which is hereby incorporated by reference in its entirety. In some aspects, said tissue nanotransfection takes place before or during the delivery of the vector. In some aspects, said tissue nanotransfection is used for short-acting gene therapy (i.e., over days or weeks, to prime the tissue for nerve regrowth), and the vector is used for long-acting gene therapy (i.e., over months or years, to generate maintain nerve regrowth). In some aspects, the additional form of treatment comprises an alternative short-acting gene therapy.

[0122] In some aspects, the additional form of treatment comprises an additional gene or genes for therapeutic purposes. In some aspects, the additional gene or genes are within the same vector. In some aspects, the genes are in different vectors or other delivery vehicles. In some aspects, the additional gene or genes are configured to knock down inhibitory signals that prevent nerve regeneration or regrowth. Some such exemplary genes are PTEN (Phosphatase and Tensin Homolog), S0CS3 (Suppressor of Cy tokine Signaling 3), RHOA (Ras Homolog Family Member A), ROCK1 / ROCK2 (Rho- Associated Kinase 1 and 2). RTN4 (Nogo- A) (Reticulon 4). NgRl (Nogo-66 Receptor 1), LINGO-1 (Leucine-Rich Repeat and Immunoglobulin-Like Domain-Containing Nogo Receptor-Interacting Protein 1), p75NTR (Neurotrophin Receptor p75), PTPo (Protein Tyrosine Phosphatase Sigma), KLF4 (Kriippel-Like Factor 4), KLF9 (Kriippel-Like Factor 9), KLF7 (Krtippel-Like Factor 7), DLK (MAP3K12) (Dual Leucine Zipper Kinase), and TP53 (p53) (Tumor Protein p53).

[0123] In some aspects, the additional form of treatment comprises one or more therapeutic composition(s). In some aspects, one or more therapeutic composition(s) can comprise viral and non- viral vectors, including those used for both short-term and long-term gene expression, combinations of various AAV serotypes to target multiple tissues, combinations of neurotrophic factors with immune / inflammation-modulatory treatments, combinations with other growth factors, or combinations of systemic treatments which are targeted to skin / subdermal areas, or combinations of immediate or delayed treatments, or combinations of gene deletion / modification / knock-down technology, combined with gene overexpression.

[0124] In some aspects, the additional form of therapy comprises lifestyle modification. Lifestyle modifications play a crucial role in managing peripheral neuropathy (PN) by promoting nen e health, reducing symptoms, and preventing further damage. In some aspects, said lifestyle modification comprises a calorie-restricted diet / exercise, a cholesterol-free diet, and / or a low-fat diet. In particular, modifications to the diet include balancing nutrition, controlling sugar, increasing hydration, and limiting alcohol. In some embodiments, including regular exercise involving low- impact activities (such as, for example, walking, swimming, and cycling), building muscle strength helps improve circulation and reduce nerve pain. Balance & flexibility exercises can help prevent falls and improve coordination. Quitting smoking can improve circulation and slow disease progression. Chronic stress can exacerbate pain and inflammation. Techniques such as deep breathing, meditation, mindfulness, and counseling can help manage stress and reduce neuropathic symptoms. For diabetic neuropathy, checking feet daily for wounds or infections is crucial, as numbness may prevent early detection of injuries. Protecting hands and feet from extreme heat or cold can prevent further nerve damage. Poor sleep can increase nerve pain and fatigue. Establishing a regular sleep routine, reducing caffeine intake, and using relaxation techniques can improve sleep quality. Alternative therapies such as acupuncture, massage therapy, and transcutaneous electrical nerve stimulation (TENS) may help reduce pain, enhance circulation, relieve muscle tension, and improve nerve function.

[0125] Methods of Administration

[0126] When one or more of the disclosed compositions is used in combination with a second therapeutic agent the dose of each compound can be either the same as or differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. The term “administration’’ and variants thereof (e.g., “administering’’ a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention is provided in combination with one or more other active agents, “administration” and its variants are each understood to include concurrent and sequential introduction of the composition thereof and other agents.

[0127] In vivo application of the disclosed compositions can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically -acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0128] The compositions comprising them can also be administered utilizing nanoparticle and / or liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.

[0129] The compositions disclosed herein can be formulated according to known methods for prepanng pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically- acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99%, and especially, 1 and 15% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0130] “Pharmaceutically acceptable excipient7’ refers to an excipient that is conventionally useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0131] A “pharmaceutically acceptable carrier” is a carrier, such as a solvent, suspending agent or vehicle, for delivering the disclosed compounds to the patient. The carrier can be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutical carrier. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0132] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, ater for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile pow der, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the t pe of formulation in question.

[0133] Compositions disclosed herein can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivery' of compounds and compositions disclosed herein to a cell comprises attaching the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Compounds can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p- carboxyphenoxy) propane: sebacic acid] in a 20: 80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.

[0134] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile inj ectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glycery l esters, and suitable mixtures thereof. The proper fluidity' can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0135] Sterile injectable solutions are prepared by incorporating a composition disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0136] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver a compound to the skin are disclosed in U.S. Patent No. 4,608.392; U.S. Patent No. 4,992,478; U.S. Patent No. 4,559,157; and U.S. Patent No. 4,820,508.

[0137] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949. Also disclosed are pharmaceutical compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of a compound constitute a preferred aspect. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

[0138] The vector comprising one or more neurotrophic factor gene(s) may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of one or more neurotrophic factor gene(s) will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the neuropathy, the particular neurotrophic factor gene, its mode of administration, its mode of activity, and the like. The vector comprising one or more neurotrophic factor gene(s) is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the vector comprising one or more neurotrophic factor gene(s) will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including the neuropathy type being treated and the severity of the peripheral neuropathy; the activity of the vector comprising one or more neurotrophic factor gene(s) employed; the specific neurotrophic factor employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific neurotrophic factor employed; the duration of the treatment; drugs used in combination or coincidental with the specific neurotrophic factor employed; and like factors well known in the medical arts.

[0139] In one aspect, disclosed herein is a vector comprising one or more neurotrophic factor gene(s) of any preceding aspect and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream. One or more active agent(s) (e.g., neurotrophic factor) can be administered in the “native” form or, if desired, in the form of salts, esters, amides, prodrugs, or a pharmacologically suitable derivative. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry' and described, for example, by March (1992) A dvanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4thEd. N.Y. Wilev-Interscience. In some embodiments, the neurotrophic factor can be prepared as a “concentrate,” e.g., in a storage container of a premeasured volume and / or a predetermined amount ready for dilution or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.

[0140] In some embodiments, the neurotrophic factor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0141] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

[0142] 39, 40, 41, 42, 43, 44. 45. 46, 47, 48, 49, 50, 51, 52. 53. 54, 55, 56, 57, 58, 59, 60. 61. 62, 63, 64, 65,

[0143] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

[0144] 93, 94, 95, 96, 97, 98, 99, 100, or more times. In some embodiments, the neurotrophic factor is administered daily. In some embodiments, the neurotrophic factor is administered every day, every72 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the neurotrophic factor is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some embodiments, the neurotrophic factor is administered every month, every72 months, every 3 months, every74 months, every75 months, every 6 months, every77 months, every 8 months, every79 months, every 10 months, every 11 months, every712 months, or more. In some embodiments, the neurotrophic factor is administered every7year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0145] In some aspects, a first vector comprising a first neurotrophic factor is delivered to the subj ect at a first time point, and a second vector comprising a second neurotrophic factor is delivered to the subject at a second time point later than the first time point. In some aspects, the first time point and the second time point are from about 1 week to about 6 months apart, or from about 1 week to about 1 months apart, or from about 1 week to about 2 months apart, or from about 1 week to about 3 months apart, or from about 1 week to about 4 months apart, or from about 1 week to about 5 months apart, or from about 1 week to about 7 months apart, or from about 1 week to about 8 months apart, or from about 1 week to about 9 months apart, or from about 1 week to about 10 months apart, or from about 1 week to about 11 months apart, or from about 1 week to about 1 year apart, or 2 weeks to about 1 months apart, or from about 2 weeks to about 2 months apart, or from about 2 weeks to about 3 months apart, or from about 2 weeks to about 4 months apart, or from about 2 weeks to about 5 months apart, or from about 2 weeks to about 6 months apart, or from about 2 weeks to about 7 months apart, or from about 2 weeks to about 8 months apart, or from about 2 weeks to about 9 months apart, or from about 2 weeks to about 10 months apart, or from about 2 weeks to about 11 months apart, or from about 2 weeks to about 1 year apart, or 3 weeks to about 1 months apart, or from about 3 weeks to about 2 months apart, or from about 3 weeks to about 3 months apart, or from about 3 weeks to about 4 months apart, or from about 3 weeks to about 5 months apart, or from about 3 weeks to about 6 months apart, or from about 3 weeks to about 7 months apart, or from about 3 weeks to about 8 months apart, or from about 3 weeks to about 9 months apart, or from about 3 weeks to about 10 months apart, or from about 3 weeks to about 11 months apart, or from about 3 weeks to about 1 year apart, or from about 1 month to about 2 months apart, or from about 1 month to about 3 months apart, or from about 1 month to about 4 months apart, or from about 1 month to about 5 months apart, or from about 1 month to about 6 months apart, or from about 1 month to about 7 months apart, or from about 1 month to about 8 months apart, or from about 1 month to about 9 months apart, or from about 1 month to about 10 months apart, or from about 1 month to about 11 months apart, or from about 1 month to about 1 year apart, or . In some aspects, the first neurotrophic factor is BDNF, and the second neurotrophic factor is NGF. In other aspects, the first neurotrophic factor is NGF, and the second neurotrophic factor is BDNF.

[0146] As used herein, the term ’‘improve” or “improving,” in the specific example of improving tissue innervation, refers to either partially or fully restoring damaged nen es in a tissue. This improvement can be, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% compared to a subject which is non-treated, or a control.

[0147] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4thed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols inMolecular Biology: (F. M. AusubeL et al. eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2; A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R. I. Freshney, 6thed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods inMolecular Biology:, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.. J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds.. 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al.. eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Press. 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 2011).

[0148] Platforms and Kits

[0149] In an aspect, provided is a platform for direct delivery of a vector to adipose tissue, wherein said vector can treat peripheral neuropathy, wherein the vector comprises one or more gene(s) encoding at least one neurotrophic factor, wherein said one or more neurotrophic factor(s) improves tissue innervation in the subj ect. This platform is defined as a tool for treating peripheral neuropathy, and can include one or more additional components for treating peripheral neuropathy or the symptoms thereof. Examples of such components are given above.

[0150] The platform can also include means for delivering treatment to a subject in need thereof. These are disclosed in more detail below.

[0151] Kits for practicing the methods of the invention are further provided. By “kit” is intended any manufacture (e g., a package or a container) comprising at least one reagent, e g., anyone of the compounds described herein. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use. Any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers or pouches.

[0152] To provide for the administration of such dosages for the desired therapeutic treatment, in some embodiments, pharmaceutical compositions disclosed herein can comprise between about 0.1% and 45%, and especially, 1 and 15%, by weight of the total of one or more of the compounds based on the weight of the total composition including carrier or diluents. Illustratively, dosage levels of the administered active ingredients can be intravenous intraperitoneal, subcutaneous, intramuscular, or orally.

[0153] Also disclosed are kits that comprise a composition comprising a compound disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In another embodiment, a kit includes one or more anti-PN agents, such as those agents described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and / or agent disclosed herein in liquid or solution form.

[0154] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0155] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0156] EXAMPLES

[0157] To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of process conditions that can be used to optimize product quality and performance. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0158] Example 1

[0159] Local delivery of adipocyte-tropic A A VRec2-BDNF increases innervation of scWAT in genetic and HFD-induced obesity mouse models.

[0160] It was previously reported that obesity results in loss of adipose tissue innervation 12 in both genetic (BTBRo6 / o6) and diet-induced obesity mouse models, as well as obese human adipose tissues. It has also been demonstrated that BDNF levels in adipose tissue decrease in states of adipose neuropathy (aging, diabetes, obesity) and that loss of BDNF from myeloid lineage cells resulted in adipose ‘denervation’. Here a study was conducted which sought to determine if restoring local BDNF to the adipose tissue could restore innervation in the depot. An adipocyte-tropic AAV serotype (Rec2) was used to locally deliver BDNF to ing-scWAT, since this is the cell type with the highest contribution to tissue mass, via two distinct injection approaches. The Rec2 serotype developed by Lei Cao highly transduces adipocytes without off target transduction in liver or heart. To determine if BDNF delivery to ing-scWAT adipocytes could attenuate adipose neuropathy. 6-week-old male BTBR" "hmice were injected unilaterally with 1x1010vg per fat depot of AAVRec2-BDNF, and the virus was allowed to transduce for 2 weeks before assessments were performed with the contralateral depot used as an intraanimal control (FIG. IB). At 2 weeks post-injection, 2 out of 3 mice showed a decrease in body w eight (FIG. 1C left panel). How ever, no differences were seen in ing-scWAT tissue weights between the AAVRec2-BDNF injected ing-scWAT depots versus the contralateral vehicle injected depots (FIG. 1C middle panel). No changes were seen in perigonadal (pg)WAT weight (FIG. 1C right panel).

[0161] Two weeks post-AAVRec2-BDNF treatment, the injected depots showed an increase in PGP9.5 protein expression compared to vehicle injected contralateral control tissues (FIG. ID). PGP9.5 is a pan-neuronal marker, its protein expression levels correspond to total innervation within a tissue, and it is a common clinical marker for PN in skin biopsies through measuring intra-epidermal nerve fiber (IENF) densify. Protein expression of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis and a typical marker of sympathetic nerve activation and innervation, was not significantly altered in the AAVRec2-BDNF injected depot compared to contralateral vehicle injected control (FIG. IE). These data suggest that AAVRec2-BDNF treatment increased sensory innervation specifically, although the protein expression of TH in the AAVRec2-BDNF injected tissue w as variably expressed and is also known to mark a subset of sensory fibers (FIGS. 1D-1E). Protein expression of BDNF was only detectible in AAVRec2-BDNF-treated ing-scWAT (FIG. IF) and confirmed the effective transduction of virus. No BDNF protein expression was seen in vehicle treated ing-scWAT, which was not surprising considering these were obese BTBR"” mice which exhibit severe symptoms of PN and an associated decline in BDNF levels. Since each animal served as its own control for this initial study, it was confirmed that the AAV serotype and delivery approach remained site specific with no off-target transduction in non- AAV injected contralateral adipose (FIG. IF). Previous characterization of the AAV Rec2 serotype reported that, along with adipoc te specificity, the Rec2 virus did not produce off-target transduction in the brain. To confirm these findings, the protein levels of BDNF in the mouse brains that received AAVRec2-BDNF or AAVRec2-Empfy vector into ing-scWAT were analyzed. Analysis revealed no discernible variation between the two groups (FIG. 2A), again confirming no central targeting of this vector.

[0162] The study next sought to determine the effectiveness of AAVRec2-BDNF in improving ing- scWAT innervation in mice fed a 58% high-fat neuropathy -inducing (HFND) diet. Previous studies have demonstrated that by 16 weeks of this diet, which is 58% fat with cornstarch and no sucrose, mice develop PN. At 17 weeks of feeding, decreased hind paw skin innervation IENF was observed, as evidenced by decreased protein expression of PGP9.5 (FIG. 2A left panel), as well as decreased protein expression of PGP9.5 in ing-scWAT (FIG. 2A right panel), fitting with prior reports. Male C57BL / 6J mice at 12-13 weeks of age were placed on an HFND for 17 weeks and then received a unilateral transdermal injection of IxlO10vg AAVRec2-BDNF into their ing-scWAT depot, while the contralateral depot received an equal volume of vehicle. The timeline and approach for this experiment are shown in FIG. 3A. Body weight, ing-scWAT weight, and pgWAT weight were not impacted by this AAV treatment at 2 weeks post-injection (FIG. 3B). There was no difference in PGP9.5 protein expression of AAVRec2-BDNF versus vehicle-injected ing-scWAT after 2 weeks of transduction (FIG. 3C). However, protein expression of TH was robustly increased in AAVRec2-BDNF versus vehicle-injected ing-scWAT (FIG. 3D), suggesting that AAVRec2-BDNF treatment increased activation of sympathetic nerves and / or density of sympathetic neurites in ing-scWAT. In this study, there were no differences in protein expression of the axon growth cone markers growth-associated protein (GAP)43 and vasodilator-stimulated phosphoprotein (Vasp) in AAVRec2-BDNF treated versus vehicle-treated ing-scWAT (FIGS. 3E-3F). As some peripheral neuropathies are demyelinating, including DPN, protein expression of myelin protein zero (MPZ, a PNS myelin marker) was also measured in AAVRec2-BDNF versus contralateral vehicle-treated ing-scWAT, and no differences were seen (FIG. 3G). As in the BTBRoh / oftmodel (FIG. 1), protein expression of BDNF was significantly increased in the AAVRec2-BDNF versus vehicle-injected ing-scWAT after 2 weeks of transduction (FIG. 3H). Here, however, BDNF protein expression was detectible on the vehicle- injected ing-scWAT, which may be attributed to the less severe model of obesity and peripheral neuropathy in this cohort.

[0163] Taken together, after just 2 weeks of transduction - a relatively short duration for AAV therapies, AAVRec2-BDNF treatment increased nerve markers in both the BTBR°* and C57BL / 6J diet-induced neuropathy mouse models, but with different patterns of impact between the two models that may be due to differences in leptin-deficiency (pb / ob) versus leptin resistance (diet-induced) or severity of associated insulin resistance.

[0164] Effectiveness of A A VRec2-BDNFin improving scWAT innervation is hindered by longer duration of HFND.

[0165] The study next investigated whether AAVRec2-BDNF treatment was effective after longer- term HFND feeding when metabolic perturbations are more pronounced and neuropathy is more severe. Male mice were placed on HFND at 10-12 weeks of age and received AAVRec2-BDNF or vehicle after 41 -43 weeks on HFND; evaluations began 4 weeks after the treatment commenced (FIG. 4A, left panel). For this experiment, mice received bilateral injections of 1x1010vg / depot of either AAVRec2-BDNF or AAVRec2-Empty vector directly to ing-scWAT (FIG. 4A. right panel). Body weight was monitored weekly post-injection and there were no changes between BDNF and empty vector groups (FIG. 4B). There were no differences in terminal body weight, ing-scWAT, or pgWAT tissue weights between AAVRec2-BDNF and AAVRec2-Empty vector control mice (FIG. 4C). Body composition was evaluated by EchoMRI prior to commencement of the treatment and 4 weeks after virus injection. There were no changes in body weight, adipose mass, or lean mass between AAVRec2-BDNF and AAVRec2-Empty vector mice (FIG. 5A).

[0166] Four weeks post- AAV treatment, ing-scWAT tissues were collected to evaluate innervation. Notably, protein expression of PGP9.5, TH, MPZ, and Vasp were not changed in AAVRec2-BDNF injected mice versus AAVRec2-Empty vector controls, while GAP43 was decreased in AAVRec2- BDNF treated mice (FIGS. 4D-4H), indicating that AAVRec2-BDNF treatment was no longer able to improve adipose innervation following the longer duration HFND feeding, even after 4 weeks of AAV infection time. Taken together, it was concluded that there is a critical window early in disease progression when BDNF gene therapy is effective in improving diet-induced PN.

[0167] Local delivery of AAVRec2-NGF improved innervation of scWAT in HFD-induced neuropathy mouse model.

[0168] NGF is a neurotrophic factor that is endogenously expressed in adipose tissue and secreted from mature adipocytes in WAT of mice and human. To determine if NGF can attenuate peripheral neuropathy and improve adipose tissue innervation, delivered AAVRec2-NGF was locally delivered to ing-scWAT bilaterally with IxlO10vg / fat depot in mice fed either 10% low-fat diet (LFD) or 60% high-fat diet with sucrose and no cornstarch (HFD) for 14-16 weeks. Sex- and age-matched mice received bilateral injections of IxlO10vg of empty vector as control (FIG. 6A). NGF ELISA confirmed effective transfection of AAVRec2-NGF in ing-scWAT (FIG. 6B). There were no changes to body weight between mice that received AAVRec2-NGF compared to AAVRec2-Empty vector at any time throughout the 10-week post-injection assessment period (FIGS. 7A-7B). By 10 weeks post-treatment, all AAVRec2-NGF -injected and AAVRec2-Empty vector-injected mice had similar ing-scWAT, pgWAT, and BAT tissue weights in both dietary intervention groups (FIG. 7C). To assess whole body composition, EchoMRI was performed prior to commencing the treatment and 9 weeks after AAV injections. There were no changes in body weight, total fat mass, or total lean mass between animals that received AAVRec2-NGF vs AAVRec2-Empty vector for either the LFD or HFD groups (FIG. 7D).

[0169] Thermal imaging (FLIR thermography) was used to assess skin surface temperature overlying thermogenic adipose depots as a means of approximating thermogenic energy expenditure in a low- stress and non-invasive way since increased adipose innervation accompanies thermogenic states such as cold stimulation. In the HFD groups, there were no differences in skin surface temperatures above the ing-scWAT between mice that received AAVRec2-NGF versus those that received empty vector control (FIG. 7E). Similarly, AAVRec2-NGF treatment did not affect skin surface temperature above the interscapular brown adipose tissue (BAT) in either dietary' group (FIG. 7F).

[0170] Unexpectedly, at 8 weeks post-injection, glucose tolerance was impaired in AAVRec2-NGF treated mice compared to AAVRec2-Empty vector in the HFD group, as indicated by a glucose tolerance test (GTT) (FIG. 6C), despite no differences in body w eight between groups (FIG. 7B). This suggests that overexpression of NGF may have a negative impact on glucose control in scWAT of obese mice, potentially through NGF actions that are independent of neurite survival / outgrowth. These data correspond with a clinical study that found a strong positive correlation between increased circulating NGF levels and increased glucose levels in patients with gestational diabetes. Metabolic assessments for HFD-fed mice showed no difference in energy expenditure, quantified as heat production, betw een AAVRec2-NGF and AAVRec2-Empty vector-treated mice (FIG. 6D. top panel). AAVRec2-NGF did have a higher respiratory exchange ratio (RER) compared to control animals, indicating a difference in metabolism between the groups (FIG. 6D, bottom panel). However, both groups had an RER close to 1.0 indicative of carbohydrates being the predominant fuel source.

[0171] At 10 weeks post-treatment, adipose tissue was assessed for changes in innervation. Protein expression of PGP9.5, TH, GAP43, and MPZ were all increased in ing-scWAT of AAVRec2-NGF treated mice compared to empty vector controls in the HFD group, indicating increased innervation and axon outgrowth in the NGF-treated mice even in the obese / neuropathic state (FIGS. 6E-6G, FIG. 61). No difference in V asp expression was seen between AAVRec2-Empty vector or AAVRec2-NGF treated mice for HFD groups (FIG. 6H). These findings suggest that local delivery of AAVRec2-NGF into ing-scWAT at early stages of diet-induced adipose neuropathy improved innervation by promoting new' axon outgrowth and myelination in the tissue.

[0172] Local delivery of gene therapy treatments through a theragnostic device confirmed AAVRec2- NGF is capable of increasing innervation in scWAT even after long-term HFND

[0173] As previously described, a medical theragnostic device has been designed, developed, and validated for small fiber PN, which can measure nerve electrical activity in the skin and subdermal tissues. This technology, called the DEN-TEN (Detecting Early Neuropathy to Treat Peripheral Neuropathy), includes an electrically conductive needle array capable of measuring nerve electrical activity in the skin and subdermal tissues for screening, diagnosis and monitoring of PN. The DEN- TEN theragnostic platform features hollow needles for tissue / fluid sampling and targeted delivery of therapies, such as AAV-mediated gene delivery, to transdermal tissues impacted by PN. The DEN- TEN is disclosed in U.S. Patent Application Publication No. 2023 / 0233822 Al, which is hereby incorporated by reference in its entirety. In this permutation for targeted tissue delivery of liquescent therapies, the DEN is fitted with pressure-resistant tubing attached to an infusion pump (FIG. 8A). The DEN-TEN was utilized for the delivery of AAVRec2-BDNF and AAVRec2-NGF to target the mouse ing-scWAT (FIG. 8B), ensuring application of the treatment across the entire depot without the need for a bolus single-needle injection that can cause more tissue damage. To demonstrate the differences in tissue diffusion between the DEN-TEN and a conventional bolus injection, Evans Blue dye was injected in the ing-scWAT (FIG. 8C). Using the DEN-TEN, amore uniform distribution of dye in the tissue was achieved compared with a bolus inj ection, as compared at 5min post-inj ection. Most importantly, DEN-TEN injections eliminated the capsule formation in the tissue that formed from bolus injections (FIG. 8C vii-viii).

[0174] To demonstrate the efficiency of the DEN-TEN device for targeted gene therapy delivery, either AAVRec2-NGF or AAVRec2-BDNF was injected into inguinal scWAT bilaterally with IxlO10vg per depot. The flank skin above the ing-scWAT was shaved, and the microneedle array was inserted through the skin with needles penetrating the ing-scWAT fat pad. The DEN-TEN device was secured in place with medical tape, and a multiple micro-syringe pump was used to deliver the treatment at a rate of 0.03mL / min (FIGS. 8A-8B). This allowed for slower diffusion of AAVs through the tissue compared to insulin syringe injection and improved tissue coverage compared to bolus syringe delivery (FIG. 8C). Using the DEN-TEN, AAV-based treatments were delivered to adult mice on HFND for 25 weeks, and after 7 weeks were euthanized for endpoint assessments (FIG. 9A). Mice that received AAVRec2-NGF treatment had increased skin surface temperature above the inguinal scWAT at 7 w eeks post-treatment as evidenced by thermal imaging (FIG. 9B). This suggested increased thermogenic acti vity the ing-scWAT depot, which is known to be controlled by the tissue nerve activity. Weekly body weight monitoring showed that AAV-Rec2- NGF injected mice lost weight in the first week compared to the empty' vector control group, while AAVRec2-BDNF injected mice gained weight 4 w eeks post-injection compared to controls (FIG. 10A, left panel). By 7 weeks post-treatment, no differences in body weight and ing-scWAT tissue weight were seen in either AAVRec2-BDNF or AAVRec2-NGF treated mice versus the empty vector injected group (FIG. 9C and FIG. 10A, right panels). Whole body composition measured 6 weeks post-injection by EchoMRI showed similar fat mass and lean mass across all three groups (FIG. 10B). Compensatory' effects were explored in axillary scWAT, which has direct vascular connections to the ing-scWAT AAV-inject depots, and no difference in cellularity (FIG. 10C, left panels) or cell size (FIG. 10C, right panels) was observed between groups indicating that the virus remained localized to the inguinal fat pad.

[0175] Seven weeks post-treatment, the injected scWAT depots were collected. PGP9.5 expression trended upward in both AAVRec2-BDNF and AAVRec2-NGF treated mice compared to empty vector treated mice (FIG. 9D). Interestingly, protein expression levels of TH, GAP43, and Vasp were significantly elevated only in AAVRec2-NGF injected mice (FIGS. 9E-9G right panels), while AAVRec2-BDNF mice showed similar protein levels compared to empty vector treated mice (FIGS. 9D-9G. left panels). Protein expression for myelin protein zero (MPZ) in both AAVRec2-BDNF and AAVRec2-NGF treated ing-scWAT were similar to empty-vector controls (FIG. 9H). Taken together, these data indicated that only AAVRec2-NGF treatment increased adipose tissue innervation after long-term diet-induced neuropathy, either because of the tropism of the virus to adipocytes (the endogenous source of NGF) or because NGF is a more effective therapy in these models. These findings also confirmed the DEN-TEN device is competent for local delivery of virus into ing-scWAT.

[0176] AAVRec2-NGF treatment increases the density of neuro-adipose nexus (NANs) in scWAT.

[0177] The ing-scWAT from mice on 16+ weeks of HFND is technically challenging to image due to the size of the depots (~1 ,5g) and the autofluorescence from lipofuscin and fibrosis that result from obesity46. Therefore, whole-mount immunostaining and imaging were performed on tissues from obese (25 weeks HFND) mice 7 weeks post- AAV injection (FIG. 11) by bisecting ing-scWAT longitudinally to preserve the integrity of the sub-iliac lymph node, an area of dense innervation. Compared to tissues from AAVRec2 -Empty vector animals, ing-scWAT from the AAVRec2-NGF treated animals did not exhibit any major anatomical changes in large axon bundle innervation patterns, as demonstrated by PGP9.5-EGFP and TH staining (FIG. 11). However, there was a striking increase in the density of single axons (‘small fibers’), and specifically an increase in density of the nerve terminal structures46, the neuro-adipose-nexus (NANs) in ing-scWAT of AAVRec2- NGF treated obese animals (FIG. 12). NANs are single axons that wrap around individual adipocytes, which express markers of presynaptic proteins and contain synaptic vesicles42. Previous reports have show n that NANs are present in ing-scWAT as highly varicose axons wrapping around one or two adipocytes dispersed throughout the depot42,46’47. NANs densely wrapping around clusters of adipocytes like they do in the AAVRec2-NGF treated tissues have not been previously observed (FIG. 12, bottom panels). These data support suppositions that NGF is an adipocyte-secreted neurotrophic factor that maintains neural communication between the brain and individual adipocytes and can promote the formation of the NAN terminal structures (FIG. 13).

[0178] Discussion

[0179] Retrograde signaling via p75NTR, or failure of retrograde axonal transport of neurotrophin / Trk, results in neuronal cell apoptosis, while insufficient or impaired retrograde signaling has been proposed as a mechanism of neurodegenerative diseases, including the axon dieback in PN. It is currently unknow n if inflammatory damage, gluco- or lipotoxicity from diet, or other insults lead to this lack of neurotrophic factor signal efficacy in DPN. However, there is evidence that loss of neurotrophic factors could be a critical mechanism underlying the etiology’ of PN. For example, depletion of NGF is associated with development of CIPN in humans, and studies from human diabetic patients suggest that decreased neurotrophic factor availability contributes to pathogenesis of DPN. In adipose specifically, it has been previously demonstrated that local production of BDNF is indispensable in maintaining healthy innervation of inguinal subcutaneous (ing-sc)WAT of mice. It was found that BDNF is decreased in adipose w ith neuropathic states and that deletion of BDNF from myeloid lineage immune cells resulted in a ‘genetic denervation' of scWAT that led to lack of UCP1 induction with cold and worsened response to high-fat diet. Studies have also shown that certain BDNF single nucleotide polymorphisms (SNPs) are associated with obesity in humans. Others have shown that NGF also plays a critical role in maintaining adipose tissue innervation. Knockdown of the NGF-specific receptor TrkA resulted in decreased sympathetic nerve fiber density in ing-scWAT and inhibition of NGF signaling during cold exposure reduced cold-induced arborization in ing-scWAT.

[0180] NGF has been a target therapy for neurodegenerative diseases since its discovery7as the first neurotrophic factor in the 1950s by Nobel -prize winner Rita Levi Montalcini. Importantly, there is a marked reduction in endogenous NGF in DPN in some reports, while other studies show that high levels of NGF persist in the skin of DPN patients, but these differences may be due to disease duration or severity. In rodent models of DPN, NGF administration ameliorated symptoms of sensory’ neuropathy34, which led to several subsequent clinical trials of NGF in the treatment of DPN. Some Phase 2 trials saw- promising results in the treatment of DPN with recombinant NGF delivery at a well-tolerated dose of 0.3ug / kg given three times per week. Yet in a Phase 3 clinical trial, subcutaneous inj ection of recombinant NGF (at the dose of 0.1 ug / kg three times per week) failed to show7significant benefit to patients w ith DPN based on nen e function assessments, despite patients in the NGF treatment group reporting improvements in pain, as seen in the prior Phase 2 trials.

[0181] At this point in history, interest in NGF as a potential PN therapy waned. It could be argued that better endpoint outcomes and dosing choices, a different means of delivering NGF, or a neurotrophic factor combination therapy could have resulted in more positive results. However, newer treatment approaches for the delivery of NGF (or other neurotrophic factors) and bioavailability enhancement have underscored a renewed potential for NGF treatment in neurodegenerative disease3. For example, it has been suggested that another approach to NGF treatment would be to induce endogenous NGF production in tissues that are already responsive to NGF. Furthermore, previous approaches in NGF therapy have not been designed to overexpress / replenish NGF via gene therapy, a clinically viable chronic treatment approach, which may provide a more physiologically relevant mechanism to reinnervate neuropathic tissue, with the added benefit of targeting NGF expression to the appropriate endogenous cell source by taking advantage of viral tropism and serotypes.

[0182] Most neurotrophic factors, including BDNF and NGF. have a short half-life endogenously and poorly penetrate through the tissue, acting instead near the source of their release - a clear requirement for nen e remodeling in order to finely tune axons to their cell targets. As evidenced by the NGF clinical trials, systemic delivery of neurotrophic factors can also have unwanted side effects. Cellspecific, locally targeted adeno-associated virus (AAV)-mediated gene therapy may circumvent many of the hurdles previously reported in neurotrophic factor treatment of PN. AAVs are FDA-approved and do not cause a significant immune response, making them ideal targeting vectors for gene therapy. Viral tropism and promotor design can allow for targeted transduction of specific cell types, minimizing off-target expression, and they can be used to deliver several gene therapies at once.

[0183] In this study, both genetic and diet-induced mouse models of DPN were used (as outlined in FIG. 1A), mouse models which were previously demonstrated to develop adipose neuropathy, and the adipocyte-tropic AAVRec2 serotype was employed to deliver neurotrophic factors directly intraadipose - thus providing a tissue- and cell-specific gene therapy. These studies revealed that AAVRec2-BDNF and AAVRec2-NGF improved measures of innervation in neuropathic adipose tissue of male mice. It was also demonstrated that the theragnostic device platform, the microneedle array called Detecting Early Neuropathy to Treat Early Neuropathy (DEN-TEN), can effectively deliver AAV -based gene therapy fluids to the subcutaneous adipose depot through a microneedle array with pump delivery, thus leading to increased tissue innervation.

[0184] In this study, it was demonstrated that both AAVRec2-BDNF and AAVRec2-NGF delivery to ing-scWAT increased aspects of adipose innervation or nerve activity as early as 2 weeks after treatment. It was interesting to observe that the BTBR°6 obmodel of DPN had a robust increase of PGP9.5 in the AAVRec2-BDNF injected depots 2 weeks after virus delivery (FIG. ID), yet in the diet-induced DPN model, only an increase in TH was observed (FIG. 3D). As TH is a marker of nerve activation and not necessarily innervation, there are a few possibilities for interpretation. One potential explanation is that despite 2 weeks post-infection previously revealing robust transduction in adipose, the study may have needed to allow for more time post- AAV -delivery for physiologically measurable changes. However, this proved to not have influence, as diet-induced DPN mice assessed 7 weeks post-AAVRec2-BDNF delivery did not show an increase in nerve or axon outgrowth markers (FIG. 9). Etiology of DPN is most likely different between genetic (leptin-deficient ob / ob) versus diet-induced (leptin-resistant) models and this may explain the differential response to neurotrophic factors that were observed.

[0185] Another possibility is that with the diet-induced DPN model, AAVRec2-BDNF treatment may not be effective at regrowing axons but can still increase sympathetic drive in the remaining axons in adipose tissue (demonstrated by increased TH). However, for mice on a long-term HFND, an increase of TH was not observed (FIG. 4E), and therefore, the ability of BDNF to affect adipose innervation only in the early stages of neuropathy indicates that there is a critical window of efficacy for therapy interventions early in disease progression. The exact temporal ‘point of no return’ when nen es are no longer responsive to plasticity-inducing cues may be different across neurotrophic factors. For example, during peripheral nene injury, there are changes in temporally-expressed growth factors during the axon repair process. While certain growth factors such as ciliary neurotrophic factor (CNTF) are highly expressed in the first few days post-injury and essentially undetectable by 7 days post-injuiy. others such as NGF do not peak until 7 days post-injury.

[0186] There is another likely explanation as to why BDNF gene therapy did not appear as effective as NGF gene therapy in this study, and that is tied to the Rec2 vector used, which targets adipocytes. BDNF, which has been previously implicated in controlling adipose innervation, is secreted from the stromal vascular fraction (SVF) of ing-scWAT (numerous cell types including myeloid-lineage immune cells, and others as revealed by more recent single-cell data; BDNF is also released by the nerve itself), but not from mature adipocytes. On the other hand, both the SVF and adipocyte fractions are sources of NGF in scWAT. Therefore, Rec2-delivery of NGF to adipocytes would be more physiologically relevant than delivery of BDNF to adipocytes. This fits with the working model that the various neurotrophic factors expressed in adipose tissue are released by different cell types in order to ‘wire up’ nerve subsets (marked by different Trk receptors) to their appropriate cellular targets (as modeled in FIG. 13).

[0187] Despite improvements to adipose innervation in AAVRec2-NGF treated mice compared to AAVRec2-Empty vector controls in the HFD group, adipose-specific overexpression of NGF may have a negative impact on glucose control in scWAT of obese mice (FIG. 6C), potentially through NGF actions that are independent of neurite survival / outgrowth. NGF has a non-neuronal role in regulating metabolism, specifically in regulating glucose-mediated insulin secretion in pancreatic islet cells. Elevated glucose leads to NGF secretion (from pancreatic vascular contractile cells), which stimulates TrkA phosphorylation in islet P cells, and this increased TrkA activity triggers insulin secretion from P cells. Similarly. NGF may exert non-neuronal effects on adipocytes that might dysregulate glucose homeostasis. However, further studies would be needed to explain the observed glucose impairment with NGF overexpression in scWAT of HFD mice. While the data correspond with a clinical study that found a strong positive correlation between increased circulating NGF levels and increased glucose levels in patients with gestational diabetes, other studies have found the opposite correlation. Circulating levels of both NGF and BDNF are decreased in Type 2 Diabetes patients, especially those with DPN, yet other studies have reported increased serum levels of NFG with obesity. These studies, along with others, underscore that there is clearly a link between neurotrophic factors and metabolic function, but the mechanisms involved are not fully understood.

[0188] Advancements in gene therapy (such as modified vectors like Rec2 that target one single cell type) can minimize off-target effects and may provide opportunities for combo therapy by targeting different treatments to their appropriate cells of action in the same individual patient. In all likelihood, a combination growth factor therapy that is temporally administered during a critical early window (for example, delivering NGF to adipocytes while delivering BDNF to SVF cell types or the peripheral nerves themselves) would provide more effective outcomes for PN treatment. Peripheral nerve repair, as has been best studied with nerve injuries, relies on highly orchestrated temporal and spatial bioavailability of growth factors, and many growth factors exhibit paracrine functions. For example, BDNF gene expression in the dorsal root ganglia is stimulated by NGF, which may be potentiating the increased innervation observed with AAVRec2-NGF treatment compared to AAVRec2-BDNF treatment by promoting a physiol ogically-relevant release of local nerve-derived BDNF in the tissue. On the other hand. NGF / activated TrkA signaling on oligodendrocytes inhibits and suppresses the cell death signal triggered by the binding of BDNF to p75NTR52, and this mechanism may exist in peripheral nerve NGF signaling, which could also explain the increased innervation observ ed with AAVRec2-NGF treatment compared to AAVRec2- BDNF. Furthermore, it is not yet known how AAV -mediated overexpression of BDNF andNGF in ing-scWAT impacts local glial cells, neuroimmune cells, and the vascular supply, which would be important for promoting and maintaining physiological innervation, as these are also sources of neurotrophic factors in adipose. Whether or not the microneedle array delivery of AAVs could impact the local tissue environment versus systemic injections has also not been assessed yet.

[0189] In summary, this study revealed that AAV -mediated delivery of neurotrophic factors specifically to adipocytes in scWAT may be an effective treatment in alleviating obesity / diabetes- induced adipose neuropathy. This approach may be modified in the future to target growth factors in a combinatorial approach to the cell types that are their physiological source in order to treat PN across multiple tissues impacted by the disease. This study has also shown that the DEN-TEN theragnostic device as a microneedle array is effective to deliver AAV-mediated gene therapies specifically to subdermal scWAT through the skin, presenting a viable approach for delivery' of transdermal treatments to tissues in a targeted manner. Taken together, this approach avoids potential undesirable effects by’ targeting vector delivery to scWAT with the additional specific viral tropism to adipocytes only, making the platform highly relevant for translation to human treatments.

[0190] Materials and Methods

[0191] Mice

[0192] The following mouse strains were obtained from The Jackson Laboratory: PGP9.5-EGFP Strain # 022476; C57BL / 6J Strain # 000664; BTBR.Cg-£e 7WiscJ Strain #:004824 and bred in the lab. BTBRob / obmice were generated from breeding pairs of BTBR11' 'obX BTBR”' 'obanimals. Age matched cohorts were then utilized for experimental studies. For studies involving LFD / HDF, male mice were obtained at 12 weeks old as an age-matched cohort from The Jackson Laboratory: C57BL / 6J DIO Strain #:380050 C57BL / 6J; and DIO Control Strain #:380056. All animals were caged in groups of 2-4 mice and maintained in a temperature and humidity-controlled facility' with a 12-hour light / dark cycle. Mice had ad libitum access to food and water. As the animal became diabetic (8-week-old BTBRob / ob; and C57BL / 6J on high-fat diets), bedding changes were performed frequently to minimize skin lesions due to polyuria. All procedures were performed in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and was approved by an Institutional Animal Care and Use Committee.

[0193] Physiological assessments

[0194] Dietary Interventions and Body Weight

[0195] To achieve a diabetic state known to induce functional neuropathy, adult (at least 12 weeks old) male C57BL / 6J mice were fed a 58% high-fat diet from (Research Diets Cat D12330) for at least 16 weeks. For the HFD vs LFD study, HFD (60 kcal% fat) and LFD (10 kcal% fat) were purchased from Research Diets, Inc. ( D12492 and #D12450B, respectively); animals were placed on diet at 6 weeks of age. Animals had ad libitum access to food and water during dietary interventions. For diets high in fat content (58-60%), food was replaced every 2-3 days; for chow or 10%, the LFD diet was replaced every’ week. Where noted in results section body weight was recorded weekly, terminal body weight and tissue weights were taken at end of study.

[0196] Body Composition

[0197] Body composition of adult male mice was measured using the EchoMRI™ 3-in-l analyzer (EchoMRI, LLC). Statistical analysis was performed in Microsoft Excel and GraphPad Prism. Glucose Tolerance Testing (GTT)

[0198] Mice were fasted overnight for 16 hours, then received an i.p. injection of 20% glucose / dextrose (at 10 uL per gram of body’ weight). A hand-held glucometer (OneTouch UltraMini, LifeScan, Milpitas, CA, Johnson & Johnson, New Brunswick, NJ) was used to measure blood glucose levels (from tail vein blood) at time t=0 and intervals of 15 min, 30 min, 60 min, and 120 min after glucose injection.

[0199] Comprehensive Laboratory Animal Monitoring System (CLAMS) Metabolic Cages

[0200] For physiological assessment, adult mice were placed individually in metabolic cages for respiratory analyses (Comprehensive Laboratory Animal Monitoring System (CLAMS); Columbus Instruments, Columbus, OH). Following the 48hr acclimation period, oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured every 15 minutes, from which both respiratory exchange ratio (RER) and energy expenditure (Heat) were calculated using the following equations: RER = VCO / VC ,' Energy expenditure (heat) = CV*VOj cal / hr, where CV is the “caloric value” as given by CV (3.815 + 1.232) *RER. Throughout the assessment, animals were singly- housed in a bedding-free cage at room temperature on a 12-hour light / dark cycle. Following a 48- hour acclimation period, waveform analysis of CLAMS data was performed by matching every 15- minute measurement across two 24 h-cycles. Two-way analysis of variance (ANOVA) was performed for average VO2. VCO2. RER, and Heat per group. An uncorrected Fisher’s Least Significance Difference test was performed for each time point between dietary groups as a post-hoc test. Interaction P values are reported, representing differences in 48 h data between groups, as well as multiple comparison results for differences that were only day / night phase specific.

[0201] FLIR Thermography

[0202] Skin surface temperature above the inguinal scWAT and BAT was measured using a FLIR T560 thermal camera. Images were acquired by placing mice on the grid top of a cage lid at the same distance away from the thermal camera; the fur was slicked away from the skin with olive oil, and the animal was held in place by its tail. Images of each adipose depot were captured in replicate (x3). Images were analyzed using the FLIR Research Studio R&D Software. Software drawing tools were used to draw a region of interest (ROI, equal size for each depot throughout a cohort) around the adipose depot. The average surface temperature was calculated using that ROI. The surface temperatures of the three replicate images were averaged for each animal, and the statistics w ere analyzed using GraphPad Prism.

[0203] AAV vector and delivery

[0204] A A V Vector Construction and Package

[0205] Virus was constructed by Dr. Lei Cao as previously described. HA-tagged human BDNF cDNA was subcloned into a novel AAV plasmid of dual cassettes that restricts off-target transduction in liver. The r AAV plasmid contains a vector expression cassette consisting of the CMV enhancer and CBA promoter. WPRE, and bovine growth hormone (bGH) poly -A flanked by AAV2 inverted terminal repeats. Engineered hybrid serotype Rec2 vectors were packaged and purified as described previously.

[0206] AAV delivery

[0207] Animals were injected using either a syringe or a hollow microneedle array. For syringe injections, a 28G insulin syringe was used to inject vims directly into the ing-scWAT with IxlO10vg per depot dose of AAVRec2-BDNF, AAVRec2-NGF, AAVRec2-Empty vector control, or Vehicle (1XPBS, 0.001% Pluronic F-68) in a 30uL volume. Three lOuL injections were performed transdermally using an insulin syringe. For device-assisted AAV-dehvery, a hollow needle electrode 3x3 array held together with a printed circuit board (PCB) was used, as previously described15. Tygon™ tubing (1 / 32" I.D. x 3 / 32" O.D. x 1 / 32" Wall) was attached to the shorter ends of the needles that penetrated through a PCB, an Idex F- 182 MicroTight™ Tubing Sleeve, Natural PEEK, 0.009" ID x 0.025" OD sleeve was used to ensure a tight, leak proof fit between the tubing and the needles. Animals were placed under 1-2.5% isofl urane anesthesia and Plane 2 anesthetic effects were confirmed by lack of response to a plantar reflex. The microneedle array was inserted into the animal’s depilated (1 day prior) flank skin above the ing-scWAT, and gently pushed so that the 2mm needle length penetrated into the ing-scWAT. Ties at both ends of the array were used to wrap around the animal and hold the device in place during injections. A multi-syringe pump was used to dispense 30uL of vims at a rate of 0.03mL / min, for a final dose of IxlO10vg per depot (vims diluent was 1XPBS, 0.001% Pluronic F-68). Animals were carefully observ ed and scored for malaise for 48 hours after virus injection, and then observed daily, and showed no adverse reaction to the treatment. After 2, 4, 7, or 10 weeks (depending on study) animals were sacrificed, tissues were harvested and processed for downstream analysis (either snap frozen for molecular analyses or fixed in 10% buffered formalin for histological analyses).

[0208] Western Blotting

[0209] Frozen tissues were lysed in RIPA buffer (Thermo Scientific™, Cat # 89901) with protease (Sigma-Aldrich, Cat. # P8340) and phosphatase (Sigma-Aldrich, Cat. # P0044 and Cat# P5726) inhibitor cocktails and homogenized in Bullet Blender™ (Next Advance, Troy NY, USA). Total protein content of lysates was measured by Bradford Assay, protein was diluted in RIPA Buffer with IX Laemmli buffer to get equal concentration of protein. Protein (30pg / lane) was loaded on SDS- polyacrylamide gel and transferred to PVDF membranes for one-hour blocking with Roche western blocking reagent (Sigma-Aldrich, Cat# 11921681001). Membranes were incubated with primary antibody at 4°C overnight. Primary antibodies used: anti-PGP9.5 (Abeam, Cambridge, U.K. Cat. # abl08986, 1:500), anti-TH (Millipore Sigma-Aldrich, Cat. # AB152, 1: 1000), anti-GAP43 (Novus Biologicals, Centennial CO, USA. Cat. # NB300-143, 1 :3000), anti-VASP (Cell Signaling Technology7, Cat. # 3132, 1:1000), Anti-MPZ (Abeam, Cat. # ab31851, 1:2000), anti-BDNF (Abeam Cat. # abl08319, 1: 1000). Housekeeper antibodies included anti-P-tubulin (Cell Signaling Technology, Cat. # 2146S, 1: 1000). anti-Cyclophilin B (Abeam, Cat. # ab 16045, 1 :40,000). and anti- vinculin (Cell Signaling Technology, Cat. # 4650S, 1 : 1000. Following overnight incubation with primary7antibody blots were rinsed with IX TBS-T, 3x10 mins each time, and incubated in antirabbit HRP secondary antibody (Cell Signaling Cat # 7074, 1 :3000) at room temperature for an hour. Blots were rinsed and visualized with enhanced chemiluminescent substrate (ECL; Pierce). Pictures were taken on Syngene G:BOX Chemi XRQ (Syngene, Frederick MD, USA). Protein expression was normalized to either P-tubulin or cyclophilin B and quantified in ImageJ.

[0210] For NGF ELISA, tissues lysis was performed using Pierce™ IP Lysis Buffer (Cat.# 87787, Thermofisher) with protease (Sigma-Aldrich. Cat. # P8340) and phosphatase (Sigma-Aldrich, Cat. # P0044 and Cat# P5726) inhibitor cocktails and homogenized in Bullet Blender™ (Next Advance, Troy NY, USA). NGF ELISA was performed on undiluted ing-scWAT lysates according to manufacturer’s instructions using the Mouse NGF / NGF Beta ELISA Kit PicoKine™ (Cat. # EK0470, Boster Bio).

[0211] Histology

[0212] Adipose tissue was fixed in 10% buffered formalin and processed for paraffin embedding. Paraffin-embedded tissues were sectioned on a microtome at 7pM, deparaffinized, and stained with Hemalum (Sigma- Aldridge Cat. #75290) per manufacturer’s recommendations to visualize tissue cellularity and cell size. For each animal assessed, 3 tissue sections were evaluated under brightfield microscopy (Nikon) at 10X magnification. Average cell size (area and penmeter) was calculated for each image using Fiji software, and graphed in GraphPad Prism.

[0213] Whole Tissue Processing and Imaging

[0214] Intact scWAT depots were excised from mice, fixed overnight in 2% PFA at 4°C, and processed following the Z-depth reduction method as described previously, with accompanying protocol. To allow obese tissues to fit on glass slides for imaging, tissues were bisected longitudinally to include an intact sub-iliac lymph node, stained, and imaged. Tissues were immunostained sequentially against the sympathetic nerve marker ty rosine hydroxylase (TH, 1 :200, EMD Millipore, AB 152), secondary antibody (goat anti -rabbit IgG Alexa Fluor Plus 594, 1: 1000, ThermoFisher Cat#A32740), and GFP (1:500, Invitrogen, A-21311) to boost PGP9.5-EGFP endogenous fluorescence. Adipocyte autofluorescence was captured by exciting tissue with a diode 405nm laser. Confocal micrographs were captured on a Leica Stellaris 5 laser scanning confocal microscope using LASX software. Fluorescent labels were excited with either a diode 405 nm laser: autofluorescence (405 nm) or a white light laser: Alexa Fluor Plus 594 (590 nm)), GFP (499 nm). Scanning speed was set to 600 Hz. Photons were detected with Power HyD S detectors. Objectives included: HC PL APO 10*70.40 CS2, HC PL APO 40x / 1.30 OIL. PinholeAiry 1.00 AU. Confocal zoom was applied to further increase magnification of NANs. Whole tissue images were generated by tiling Z-maximum intensity projections of 10X objective magnification micrographs captured at 720 x 720 pixel resolution wi th a Z-step size of 10 pm. Image processing performed in Leica Application Suite X and Fiji software.

[0215] Statistical Analyses

[0216] All plots represent mean ± SEM. Statistical calculations were earned out in Excel or GraphPad Prism 9.0, utilizing the ANOVA or Student’s T-test as indications of significance. For all figures, *P<0.05, **P<0.01, *** <0.005, ****P<0.001.

[0217] Lastly, it should be understood that while the present disclosure has been provided in detail with respect to certain illustrative and specific aspects thereof, it should not be considered limited to such, as numerous modifications are possible without departing from the broad spirit and scope of the present disclosure as defined in the appended claims.

[0218] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims

CLAIMSWhat is claimed is:

1. A method of treating peripheral neuropathy or peripheral nerve degeneration or dysfunction in a subject in need thereof, the method comprising delivering a vector to the subject, wherein the vector comprises one or more neurotrophic factor gene(s), wherein said one or more neurotrophic factor gene(s) treats peripheral neuropathy in the subject.

2. The method of claim 1 , wherein peripheral neuropathy is treated by supporting or improving tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nerve function.

3. The method of claim 1, wherein the peripheral neuropathy is adipose neuropathy.

4. The method of any one of claims 1-3, wherein the vector is delivered directly to a dermal tissue, a subdermal tissue or a tissue of the subject.

5. The method of claim 4, wherein the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue.

6. The method of any one of claims 1-5, wherein the vector comprises adipose-tropic, a neurotropic vector, a hepatotropic vector, a myotropic vector, a cardiotropic vector, a pulmotropic vector, a renotropic vector, a hematopoietic tropic vector, a pancreatic tropic vector and an osteotropic vector.

7. The method of any one of claims 1 -6, wherein the vector comprises an adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle.

8. The method of claim 7, wherein the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO and Rec2.

9. The method of any one of claims 1-8, wherein the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s).

10. The method of claims 1 -9, wherein the vector can be combined with at least one additional form of treatment or therapy.

11. The method of claim 10, wherein the additional form of treatment inhibits signals which prevent or block nerve regrowth or regeneration.

12. The method of claim 10, wherein the additional form of treatment comprises tissue nanotransfection.

13. The method of claim 12, wherein said tissue nanotransfection takes place before, or during, delivery of vector.

14. The method of claim 10, wherein the additional form of treatment comprises an additional gene or genes for therapeutic purposes.

15. The method of claim 14, wherein the additional gene or genes are within the same vector.

16. The method of claim 14, wherein the genes are in different vectors or other delivery vehicles.

17. The method of claim 10, wherein the additional form of treatment comprises one or more therapeutic composition(s).

18. The method of claim 10. wherein the additional form of therapy comprises lifestyle modification.

19. The method of claim 18, wherein said lifestyle modification comprises a blood sugar regulation, calorie-restricted diets / exercise, cholesterol-free diet, low sugar diet and / or low- fat diet.

20. The method of any one of claims 1-19, wherein the vector is delivered by syringe injection.

21. The method of any one of claims 1-19, wherein the vector is delivered through an adipose tissue targeting device.

22. The method of claim 21, wherein the adipose tissue targeting device is a DEN-TEN device.

23. The method of any one of claims 1-22, wherein the dosage of the one or more neurotrophic factor gene(s) is about IxlO13vg per fat depot.

24. The method of any one of claims 1-23, wherein the peripheral neuropathy is obesity / diabetes-associated.

25. The method of any one of claims 1-23, wherein the peripheral neuropathy is related to drug or environmental toxicity, aging, idiopathy. or is related to viral or immune issues, or is caused by injury / surgery, or is medically related, or is genetically related.

26. A platform for direct delivery of a vector to adipose tissue, wherein said vector can treat peripheral neuropathy, wherein the vector comprises one or more gene(s) encoding at least one neurotrophic factor, wherein said the at least one neurotrophic factor supports and / or improves tissue innervation in a subject.

27. The platform of claim 26, wherein peripheral neuropathy is treated by improving tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nerve function.

28. The platform of claim 26, wherein the peripheral neuropathy is adipose neuropathy.

29. The platform of any one of claims 26-28, wherein the vector is delivered directly to a dermal tissue, a subdermal tissue or a tissue of the subject.

30. The platform of claim 29, wherein the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue.

31. The platform of any one of claims 26-30, wherein the vector comprises adipose-tropic, a neurotropic vector, a hepatotropic vector, a myotropic vector, a cardiotropic vector, a pulmotropic vector, a renotropic vector, a hematopoietic tropic vector, a pancreatic tropic vector and an osteotropic vector.

32. The platform of any one of claims 26-31, wherein the vector comprises adenovirus- associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virusvector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle.

33. The platform of claim 32, wherein the AAV vector comprises AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrhlO and Rec2.

34. The platform of any one of claims 26-33. wherein the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF) Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s).

35. The platform of any one of claims 26-34, wherein the platform further comprises an additional form of treatment or therapy.

36. The platform of claim 35, wherein the additional form of treatment comprises an additional gene or genes for therapeutic purposes.

37. The platform of claim 36, wherein the additional gene or genes are within the same vector.

38. The platform of claim 36, wherein the genes are in different vectors.

39. The platform of claim 36, w herein the additional form of treatment comprises one or more therapeutic composition(s).

40. The platform of any one of claims 26-39. wherein the platform further comprises a syringe for injection.

41. The platform of any one of claims 26-40, wherein the platform further comprises an adipose tissue targeting device.

42. The platform of claim 41, wherein the device is a DEN-TEN device.

43. The method of claim 17. wherein the one or more therapeutic composition(s) comprise an immune- and / or inflammation-modulatory treatment and / or a growth factor.

44. The method of claim 10, wherein the additional form of treatment is delivered to a different tissue of the subject than the vector (e.g., skin or subdermal tissue), is targeted to a differenttissue of the subject than the vector, and / or has a different treatment timescale (i.e., shortterm vs. long-term) than the vector.

45. The method of claim 10, wherein the additional form of treatment comprises gene deletion / modification / knock-down with a gene overexpression.

46. The method of any one of claims 1-25 or 43-45, wherein a first vector comprising a first neurotrophic factor is delivered to the subject at a first time point, and wherein a second vector comprising a second neurotrophic factor is delivered to the subject at a second time point later than the first time point.

47. The method of claim 46, wherein the first time point and the second time point are from about 1 week to about 1 year apart.

48. A kit for direct deliver}’ of a vector to adipose tissue, wherein the vector can treat peripheral neuropathy in a subject, wherein the kit comprises the vector and an adipose tissue-targeting device, wherein the vector comprises one or more gene(s) encoding at least one neurotrophic factor, wherein said the at least one neurotrophic factor supports and / or improves tissue innervation in the subject.

49. The kit of claim 48, wherein peripheral neuropathy is treated by supporting and / or improving tissue innervation, synaptic contacts, nerve and / or axonal plasticity, or nerve function.

50. The kit of claim 48 or claim 49, wherein the peripheral neuropathy is adipose neuropathy.

51. The kit of any one of claims 48-50, wherein the vector is delivered directly to a dermal tissue, subdermal tissue or a tissue of the subject.

52. The kit of claim 51, wherein the tissue comprises subcutaneous white adipose tissue (scWAT), brain tissue, spinal cord tissue, peripheral nerves, liver tissue, kidney tissue, muscle tissue, heart tissue, lung tissue, pancreatic tissue, bone marrow, and bone tissue..

53. The kit of any one of claims 48-52, wherein the vector is adipose-tropic.

54. The kit of any one of claims 48-53, wherein the vector comprises adenovirus-associated virus (AAV), a lentiviral vector, an adenoviral vector, a herpes simplex virus vector, a retroviral vector, a nanoparticle, an aptamer-conjugated vector, an extracellular vesicle.

55. The kit of claim 54, wherein the AAV vector comprises AAV1, AAV2. AAV5, AAV6, AAV8, AAV9, AAVrhlO and Rec2.

56. The kit of any one of claims 48-55, wherein the neurotrophic factor is Brain-Derived Neurotrophic Factor (BDNF) Nerve Growth Factor (NGF), Bone Morphogenic Proteins (BMPs), or other growth factors alone or in combination with one or more neurotrophic factor(s) or one or more neurotrophic factor gene(s).

57. The kit of any one of claims 48-56, w herein the kit further comprises an additional form of treatment or therapy.

58. The kit of claim 57, wherein the additional form of treatment inhibits signals which prevent or block nerve regrowth or regeneration.

59. The kit of claim 57, wherein the additional form of treatment comprises tissue nanotransfection.

60. The kit of claim 59, wherein said tissue nanotransfection takes place before, or during, delivery' of a vector.

61. The kit of claim 57, wherein the additional form of treatment comprises an additional gene or genes for therapeutic purposes.

62. The kit of claim 61, wherein the additional gene or genes are within the same vector.

63. The kit of claim 61, wherein the genes are in different vectors or other delivery vehicles.

64. The kit of claim 57. wherein the additional form of treatment comprises one or more therapeutic composition(s).

65. The kit of claim 64. wherein the one or more therapeutic composition(s) comprise an immune- and / or inflammation-modulatory treatment and / or a growth factor.

66. The kit of any one of claims 48-65, wherein the kit further comprises a syringe for injection.

67. The kit of any one of claims 48-66, wherein the adipose tissue-targeting device is a DEN- TEN device.

68. The kit of any one of claims 48-67, wherein the dosage of the neurotrophic factor gene is about IxlO13vg per fat depot.

69. The kit of claim 57. wherein the additional form of treatment is delivered to a different tissue of the subj ect than the vector (e g., skin or subdermal tissue), is targeted to a different tissue of the subject than the vector, and / or has a different treatment timescale (i.e., short-term vs. long-term) than the vector.

70. The kit of claim 57, wherein the additional form of treatment comprises gene deletion / modification / knock-down with a gene overexpression.

71. The kit of any one of claims 48-70, wherein a first vector comprising a first neurotrophic factor is delivered to the subj ect at a first time point, and wherein a second vector comprising a second neurotrophic factor is delivered to the subject at a second time point later than the first time point.

72. The kit of claim 71, wherein the first time point and the second time point are from about 1 week to about 1 year apart.

73. The kit of any one of claims 48-72, wherein the peripheral neuropathy is obesity / diabetes- associated.

74. The kit of any one of claims 48-73. w herein the peripheral neuropathy is related to drug or environmental toxicity, aging, idiopathy, or is related to viral or immune issues, or is caused by injury / surgery, or is medically related, or is genetically related.

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