EP4665379A1 - Methods and compositions to treat axonal injury pathologies - Google Patents

Abstract

Methods and compositions for promoting axon growth and/or regeneration in a mammal in need thereof.

Description

METHODS AND COMPOSITIONS TO TREAT AXONAL INJURY PATHOLOGIES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/485,662, filed February 17, 2023, which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R01NS 110681 awarded by the National Institute of Neurological Disorders and Stroke. The Government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on February 19, 2024, as an .XML file entitled “103361-443WOl_ST26.xml” created on February 13, 2024, and having a file size of 9,929 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

[0004] Injury to the adult mammalian spinal cord causes devastating neurological deficits and long-term disability due to axon regeneration failure. In the United States, 17,730 new spinal cord injuries (SCI) occur yearly, and an estimated 1.5 million Americans sustain a traumatic brain injury (TBI). Currently, no therapeutic strategy that restores function is available for these individuals.

[0005] Recovery of neuronal function in adults might be attained by promoting axon sprouting, regeneration, and de-novo formation of neural circuits. In the adult central nervous system (CNS), however, injured neurons have limited capacity to sprout and even less to regenerate, often failing to reestablish functionally meaningful connections. Several strategies have recently challenged this notion by reprogramming adult neurons’ deficient intrinsic growth state and eliminating extracellular growth inhibitors, triggering the regrowth of axonal tracts that project to the spinal cord. A significant challenge is that implementing these complex strategies are not available for clinical interventions.

SUMMARY

[0006] Platelet-derived growth factors (PDGFs) play a crucial role during the development of the vascular system. In the CNS, PDGF-BB is secreted by endothelial cells. PDGF-BB binds to PDGF receptor beta (PDGFrP) expressed on pericytes to stimulate the proliferation and migration of pericytes and their recruitment to growing blood vessels and into the wound area. The method disclosed herein can encourage axon growth and regeneration by administration of PDGF-BB to a spinal cord injury (SCI), which can convert adult pericytes from a growth- inhibitory to a growth-supporting substrate. Administration of PDGF-BB directly into the lesion site 7 days after a SCI (e.g., a clinically relevant paradigm) can promote robust regeneration of ascending sensory axons. There, sensory axons can regenerate along pericytepositive vascular bridges that form into and beyond the lesion site due to PDGF-BB stimulation. As such, pericytes can be programmed to promote axon growth and regeneration after injury to the adult spinal cord. The simplicity of this delivery approach makes this treatment strategy readily translational across different species. The administration of PDGF- BB promotes axon regeneration and the formation of new blood vessels necessary to reestablish vascular function at the lesion site and penumbra region.

[0007] In an aspect, provided is a method to promote axon growth and/or regeneration in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of axon growth and/or regeneration; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal.

[0008] In another aspect, provided is a composition comprising platelet-derived growth factor-BB (PDGF-BB) encapsulated in a nanoparticle.

[0009] In another aspect, provided is a composition comprising platelet-derived growth factor-BB (PDGF-BB) encapsulated in a hydrogel.

[0010] In another aspect, provided is a method to downregulate type I collagen in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of downregulating type I collagen; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal.

[0011] Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF DRAWINGS

[0012] FIGURES 1A-1G depict that SCI leads to profound changes in vasculature architecture and pericyte coverage. FIG. 1A shows a schematic of T12 SCI (WM: white matter; GM: grey matter; D: dorsal and V: ventral). FIG. IB shows representative three-dimensional imaging of the unsectioned spinal cord of adult NG2-eGFP mice depicting changes in vasculature network and pericyte coverage after SCI. A fluorescent gel perfusate was injected via transcardial perfusion to trace the vasculature. The asterisks indicate the lesion site. Scale, 200 pm. FIG. 1C shows a quantification of FIG. IB. Mean and SEM (1-way ANOVA followed by Dunnett’s multiple comparisons test **p<0.01 and ***p<0.001, ns not significant, naive n=4, 3DPI n=4, 7DPI n=4, 14DPI n=3, 28DPI n=6 and 90DPI n=5 biological replicates). FIG. ID shows a three-dimensional reconstruction of the vasculature network at the lesion site and pericyte location to the traced vasculature. Scale, 70 pm. FIG. IE shows a quantification of FIG. ID. Mean and SEM (1-way ANOVA followed by Dunnett’s multiple comparisons test *p<0.05 **p<0.01 and ***p<0.001, ns not significant, naive n=4, 3DPI n=4, 7DPI n=5, 14DPI n=3, 28DPI n=6 and 90DPI n=5). FIG. IF shows representative fluorescent images of adult mouse spinal cords at 3DPI. Scale, 20 pm. FIG. 1G shows a quantification of FIG. IF. Mean and SEM (n=5 biological replicates).

[0013] FIGURES 2A-2E depict that NG2-eGFP mice allow visualization of pericytes and pericyte coverage within the vasculature network in the adult spinal cord. FIGS. 2A-2B show representative fluorescent images of pericytes (FIG. 2A) and oligodendrocyte precursor cells (FIG. 2B) in the spinal cord of adult NG2-eGFP mice. Scale, 25 pm. FIG. 2C shows representative three-dimensional imaging of vasculature networks and pericyte coverage in the adult spinal cord (low thoracic-lumbar region) of adult NG2-eGFP mice (R: rostral, C: caudal). A fluorescent gel perfusate was injected via transcardial perfusion to trace the vasculature. Scale, 200 pm. FIG. 2D shows representative three-dimensional imaging of the endothelial marker CD31 and pericyte coverage in the injured spinal cord of adult NG2-eGFP mice at 3DPI. The yellow and blue arrow heads indicate colocalization and no colocalization between NG2-eGFP and CD31 respectively. Scale, 100 pm. FIG. 2E shows a quantification of FIG. 2D. Mean and SEM (unpaired 2-tailed Student’s t-test *p<0.05, ns not significant, n=6 biological replicates).

[0014] FIGURES 3A-3K shows that cultured adult pericytes expressed both inhibitory and permissive substrates for axon growth. FIG. 3A is an agarose gel electrophoresis of RT- PCR products showing cultured pericytes originating from the adult spinal cord of FVB mice express Ng2, Pdgfrb and Desmin. FIG. 3B is immonoblots showing NG2, PDGFrP and Desmin expression in cultured pericytes isolated from the adult spinal cord of FVB mice. GAPDH is used as a loading control (n=3 biological replicates). FIG. 3C shows a representative fluorescence image of cultured pericyte originating from the spinal cord of adult NG2-eGFP mice. Scale, 50 pm. FIG. 3D shows representative fluorescence images of cultured pericytes originating from the spinal cord of adult FVB mice. Scale, 50 pm. FIG. 3E shows representative fluorescence images of growth cones from adult DRG neurons growing for 24 hours on laminin coated dishes and pericyte monolayer. Scale, 5 pm. FIG. 3F shows a quantification of collapsed growth cones in (E). Mean and SEM (unpaired 2-tailed Student’s t- test ***p<0.001, n=3 independent experiments, 362-417 growth cones per condition). FIG. 3G shows a quantification of neuron survival in FIG. 3E. Mean and SEM (unpaired 2-tailed Student’s t-test, ns not significant, n=3 independent experiments, 2574-2604 neurons per condition). FIG. 3H shows representative fluorescence images of growth cones from adult DRG neurons growing for 24 hours on laminin coated dishes and pericyte monolayer. Scale, 5 pm. FIG. 31 shows a quantification of FIG. 3H. Mean and SEM (unpaired 2-tailed Student’s t-test ***p<0.001, 27-31 growth cones per condition). FIG. 3J shows a quantification of axon growth in adult DRG neurons growing for 24 hours on pericyte monolayers in the presence or absence of intracellular sigma peptide (ISP). Mean and SEM (unpaired 2-tailed Student’ s t-test **p<0.01, n=3 independent experiments, 186-233 neurons per condition). FIG. 3K shows a quantification of axon growth in adult DRG neurons growing for 24 hours on laminin and CSPG-coated dishes in the presence or absence of ISP. Mean and SEM (1-way ANOVA **p<0.01 and ***p<0.001, n=3 independent experiments, 79-95 neurons per condition).

[0015] FIGURES 4A-4H depict that pericytes cause detrimental structural and functional changes in adult DRG neurons. FIG. 4 A shows representative fluorescence images of adult DRG neurons cultured for 24 hours on laminin-coated dishes and pericyte monolayer. Scale, 200 pm. FIG. 4B shows a quantification of FIG. 4A. Mean and SEM (1-way ANOVA followed by Dunnett’s multiple comparisons test *p<0.05, **p<0.01 and ***p<0.001, n=3 independent experiments; 151-195 neurons per condition). FIG. 4C shows representative fluorescence images of cell bodies of adult DRG neurons cultured for 24 hours on laminin- coated dishes and pericyte monolayer. Scale, 20 pm. FIG. 4D shows a patch-clamp recording of adult DRG neurons cultured on laminin-coated dishes and pericyte monolayer. Representative traces show current- and voltage-clamp recordings. Initial inward currents (voltage-clamp) represent conductance mediated via sodium channels. Scale (DIC image), 20 pm. FIG. 4E shows a quantification of passive membrane properties in FIG. 4D. Mean and SEM (unpaired 2-tailed Student’s t-test, ns not significant, laminin n=8 neurons, pericyte monolayer n=15 and n=10 (minimum current to trigger action potential) neurons. FIG. 4F shows a quantification of sodium current in FIG. 4D. I-V curse for sodium current was generated by plotting the maximum inward current amplitudes against the corresponding membrane potentials (Mixed model ***p<0.001, Laminin n=8 and Pericytes n=15 neurons). FIG. 4G shows that calcium traces show spontaneous activity in adult DRG neurons cultured for 24 hours on laminin-coated dishes and pericyte monolayer (laminin n=16 and pericyte monolayer n=21 neurons). FIG. 4H shows a quantification of FIG. 4G.

[0016] FIGURES 5A-5K depict that PDGF-BB converts adult pericytes into a permissive substrate for axon growth. FIG. 5A shows representative fluorescence images of adult DRG neurons cultured for 24 hours on pericyte monolayer with or without PDGF-BB stimulation. Scale, 100 pm. FIG. 5B shows a quantification of FIG. 5A. Mean and SEM (unpaired 2-tailed Student’s t-test **p<0.01 and ***p<0.001, triplicate experiments; 100-103 neurons per condition). FIG. 5C shows representative fluorescence images of adult pericytes cultured for 24 hours with or without PDGF-BB. Scales, 100 (top panels) and 50 (bottom panels) pm. FIG. 5D shows a quantification of fibronectin fiber length in FIG. 5C. Mean and SEM (2-way ANOVA **p<0.01, ns not significant, triplicate experiments; 26-31 cells per condition; 248- 277 fibronectin fibers per condition). FIG. 5E shows representative images of a Cryo-EM grid and adult pericytes cultured on Cryo-EM grids. Scales, 500 (phase image) and 20 (fluorescence image) pm. FIG. 5F shows representative Cryo-EM images of adult pericytes cultured for 24 hours with or without PDGF-BB. FIG. 5G shows a quantification of the angle of deviation along fiber structures in FIG. 5F. Mean and SEM (unpaired 2-tailed Student’s t-test ***p<0.001, vehicle n=6 and PDGF-BB n=10 cells; 46-76 fiber structures per condition). FIG. 5H is an immunoblot showing fibronectin expression in adult pericytes cultured for 24 hours with or without PDGF-BB. GAPDH is used as a loading control (n=3 independent experiments). FIG. 51 shows a quantification of FIG. 5H. Mean and SEM (unpaired 2-tailed Student’s t-test **p<0.001, vehicle n=3 and PDGF-BB n=3 biological replicates). FIG. 5J shows representative fluorescence images of adult DRG neurons cultured for 24 hours on a human pericyte monolayer with or without PDGF-BB stimulation. Scale, 100 pm. FIG. 5K shows a quantification of FIG. 5 J. Mean and SEM (unpaired 2-tailed Student’s t-test ***p<0.01 and ***p<0.001, triplicate experiments; 135-182 neurons per condition).

[0017] FIGURES 6A-6I depict that fibronectin matrix and integrin signaling are required to sponsor axon outgrowth on PDGF-BB stimulated pericyte monolayers. FIG. 6A shows a quantification of axon growth in adult DRG neurons growing for 24 hours on laminin coated dishes in the presence or absence of PDGF-BB. Mean and SEM (unpaired 2-tailed Student’s t- test, ns not significant, n=4 independent experiments, 134-149 neurons per condition). FIG. 6B shows a quantification of axon growth in adult DRG neurons growing for 24 hours on laminin coated dishes in the presence of pericyte/control or pericyte/PDGF-BB conditioned medium. Mean and SEM (unpaired 2-tailed Student’s t-test, ns not significant, n=3 independent experiments, 67-82 neurons per condition). FIG. 6C shows representative differential interference contrast (DIC) images of cultured pericytes. Scale, 50 pm. FIG. 6D shows a quantification of pericyte circularity in FIG. 6C. Mean and SEM (unpaired 2-tailed Student’s t-test **p<0.01, n=3 independent experiments, 222-223 cells per condition). FIG. 6E shows a distribution of laminin expression in cultured pericytes 24 hours after plating (two-sample Kolmogorov-Smirnov test *p<0.05, experiments, 81-117 cells per condition). FIG. 6F is an immunoblot showing NG2 and COL1A1 expression in adult pericytes cultured for 48 hours with and without PDGF-BB. GAPDH is used as a loading control. FIG. 6G shows a quantification of FIG. 6F. Mean and SEM (unpaired 2-tailed Student’s t-test *p<0.05, ***p<0.001, n=3 independent replicates). FIG. 6H is an immunoblot showing fibronectin expression in adult pericytes cultured for 48 hours after transfection with CTR or two different sets of Fnl siRNA. GAPDH is used as a loading control. FIG. 61 shows a quantification of FIG. 6H. Mean and SEM (1-way ANOVA **p<0.01, ***p<0.001, n=3 independent replicates). FIG. 6J shows representative fluorescence images of adult DRG neurons cultured for 24 hours with PDGF-BB stimulation on siRNA-transfected pericyte monolayers. Scale, 200 pm. FIG. 6K shows a quantification of FIG. 6J. Mean and SEM (1-way ANOVA followed by Dunnett’s multiple comparisons test ***p<0.001, triplicate experiments; 67-137 neurons per condition). FIG. 6L shows representative fluorescence images of adult DRG neurons cultured for 24 hours with PDGF-BB stimulation on pericyte monolayers. Specific integrin inhibitors were added at the time neurons were plated. Scale, 200 pm. FIG. 6M shows a quantification of FIG. 6L. Mean and SEM (1-way ANOVA followed by Dunnett’s multiple comparisons test **p<0.01 and ***p<0.001, triplicate experiments; 101-131 neurons per condition).

[0018] FIGURES 7A-7I depict in vivo conversion of adult pericytes via PDGF-BB promotes axon regeneration after SCI. FIG. 7A shows a schematic of T12 SCI and experimental timeline. FIG. 7B shows tepresentative 3D imaging of the unsectioned spinal cords of adult NG2-mEGFP mice generated by automated tile scanning at 4 weeks after SCI. Dorsal column sensory axons were labeled by injecting AAV-tdTomato into the left sciatic nerve. The asterisk indicates the lesion epicenter (R: rostral, C: caudal). Scale, 200 pm. FIG. 7C shows a quantification of regenerating axons in FIG. 7B. Scatter plot and mean (2-way ANOVA ***p<0.001; LS lesion site; vehicle n=6, PDGF-BB n=7 biological replicates). FIG. 7D shows representative 3D imaging of the lesion site of adult NG2-mEGFP mice at 4 weeks after SCI. Asterisk indicates the lesion epicenter (R: rostral, C: caudal). Scale, 200 pm. FIG. 7E shows a quantification of NG2 filament distribution at the lesion epicenter in FIG. 7D. Mean and SEM (unpaired 2-tailed Student’s t-test ***p<0.001, ns not significant, vehicle n=6 and PDGF-BB n=7 biological replicates). FIG. 7F shows a higher magnification image of the unsectioned spinal cord shown in FIG. 7A. The asterisk indicates the lesion site (R: rostral, C: caudal). Arrowheads indicate regenerating axons growing on pericyte-decorated cellular bridges. Scale, 200 pm. FIG. 7G shows a quantification of FIG. 7F. Mean and SEM (unpaired 2-tailed Student’s t-test ***p<0.001, ns not significant, vehicle n=5 and PDGF-BB n=6 biological replicates). FIG. 7H shows representative fluorescence images of astrocyte reactivity 4 weeks after SCI. The asterisk indicates the lesion site (R: rostral, C: caudal). Scale, 200 pm. FIG. 71 shows a quantification of FIG. 7H. Mean and SEM (unpaired 2-tailed Student’s t-test *p<0.05 and **p<0.01, ns not significant, vehicle n=4 and PDGF-BB n=4 biological replicates).

[0019] FIGURES 8A-8H depict that PDGF-BB localized delivery at the lesion site positively affects the physical and chemical nature of the lesion environment. FIG. 8A shows an experimental timeline. FIG. 8B shows representative 3D imaging of the unsectioned spinal cords of adult NG2-CreERT2/Ai9 (RCL-tdTomato) mice generated by automated tile scanning at 4 weeks after SCI. Dorsal column sensory axons were labeled by injecting AAV-eGFP into the left sciatic nerve. The asterisk indicates the lesion epicenter (R: rostral, C: caudal). Scale, 200 pm. FIG. 8C shows a quantification of regenerating axons in FIG. 8B. Scatter plot and mean (2-way ANOVA ***p<0.001; LS lesion site; vehicle n=7, PDGF-BB n=7 biological replicates). FIG. 8D shows a quantification of tdTomato positive pericyte density at the lesion site 4 weeks after SCI. Mean and SEM (unpaired 2-tailed Student’ s t-test *p<0.05, SCI/vehicle n=4, SCI/PDGF-BB n=5 biological replicates). FIG. 8E shows a schematic of flow cytometry. FIG. 8F depicts flow cytometry showing no change in the total number of myeloid cells and the percentage of neutrophils and microglia three days after vehicle and PDGF-BB administration at the lesion site. In contrast, the number of macrophages increased in mice administered PDGF-BB. All samples were collected 10 days after SCI. Mean and SEM (1-way ANOVA **p<0.01, ns not significant, SCI n=5, SCI/Vehicle n=5 and SCI/PDGF-BB n=5 biological replicates). FIG. 8G depicts flow cytometry showing detailed characterization of Arg, MRC and CD 14 positive macrophage and microglia populations three days after vehicle and PDGF-BB administration at the lesion site. All samples were collected 10 days after SCI. Mean and SEM (1-way ANOVA **p<0.01 and ***p<0.001, ns not significant, SCI n=5, SCI/Vehicle n=5 and SCI/PDGF-BB n=5 biological replicates). FIG. 8H depicts multiplex analysis showing expression of inflammatory chemokines and cytokines 3 days after vehicle and PDGF-BB administration at the lesion site. All samples were collected 10 days after SCI. Mean and SEM (unpaired 2-tailed Student’ s t-test *p<0.05, ns not significant, SCI/vehicle n=3- 4 and PDGF-BB n=5 biological replicates).

DETAILED DESCRIPTION

[0020] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

DEFINITIONS

[0021] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0022] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0023] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0024] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can 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. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0025] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0026] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0027] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0028] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0029] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0030] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0031] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0032] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0033] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0034] 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 a neurological disorder or injury. 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 a neurological disorder or injury 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 (subjects 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.

[0035] 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.

[0036] As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0037] As used herein, the phrase “axon growth and/or regeneration” relates to promoting growth, survival, regeneration (e.g. neurogenesis) and/or repair of neural tissue (e.g. axons).

METHODS AND COMPOSITONS

[0038] In an aspect, provided is a method to promote axon growth and/or regeneration in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of axon growth and/or regeneration; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal.

[0039] Platelet-derived growth factor (PDGF) is one among numerous growth factors that regulate cell growth and division. In particular, PDGF plays a significant role in blood vessel formation, the growth of blood vessels from already-existing blood vessel tissue, mitogenesis, i.e., proliferation, of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells and mesenchymal stem cells as well as chemotaxis, the directed migration, of mesenchymal cells. Platelet-derived growth factor is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF- AB).

[0040] PDGF-BB (Platelet-Derived Growth Factor BB) is a homodimeric molecule expressed by hepatocytes and nonresorbing osteoclasts, and it promotes osteoblast development and bone formation. It is also produced by platelets, macrophages, and mast cells. At sites of injury, it promotes neutrophil and macrophage infiltration for debridement, fibroblast secretion of new extracellular matrix, and IGF-I-mediated re-epithelialization. The traditional receptor for PDGF is either a homodimer or heterodimer created from two type I transmembrane RTKs, PDGF R alpha and PDGF R beta. PDGF-BB has been shown to bind the alpha/alpha homodimer, alpha/beta heterodimer, and the beta/beta homodimer in vitro, and act through the beta/beta homodimer in vivo.

[0041] The PDGF-BB composition disclosed herein can be naturally occurring or can be recombinant. Commercially available recombinant human PDGF-BB may be obtained from a variety of sources. In some aspects, PDGF can be obtained from natural sources. In other aspects, PDGF can be produced by recombinant DNA techniques. In some aspects, PDGF or fragments thereof may be produced using peptide synthesis techniques known to one of skill in the art, such as solid phase peptide synthesis.

[0042] When obtained from natural sources, PDGF can be derived from biological fluids. Biological fluids, according to some aspects, can comprise any treated or untreated fluid associated with living organisms including blood. Biological fluids can also comprise blood components including platelet concentrate, apheresed platelets, platelet-rich plasma, plasma, serum, fresh frozen plasma, and buffy coat. Biological fluids can comprise platelets separated from plasma and resuspended in a physiological fluid.

[0043] When produced by recombinant DNA techniques, a DNA sequence encoding a single monomer (e.g., PDGF B-chain) can be inserted into cultured prokaryotic or eukaryotic cells for expression to subsequently produce the homodimer (e.g., PDGF-BB). The homoodimer PDGF-BB produced by recombinant techniques may be used in some aspects. In other aspects, a PDGF-BB homodimer can be generated by inserting DNA sequences encoding for both monomeric units into cultured prokaryotic or eukaryotic cells and allowing the translated monomeric units to be processed by the cells to produce the homodimer. Commercially available recombinant human PDGF-BB may be obtained from a variety of sources. [0044] Examples of recombinant PDGF-BB include, but are not limited to, those available from RND Systems, such as Recombinant Human PDGF-BB Protein, CF (catalog # 220-BB); Recombinant Human PDGF-BB GMP Protein, CF (catalog 220-GMP); Recombinant Rat PDGF-BB Protein (catalog # 520-BB); Recombinant Human PDGF-BB, Biotinylated Protein (catalog # BT220); Recombinant Human PDGF-BB, Animal-Free Protein (catalog # AFL220); and Recombinant Equine PDGF-BB Protein (catalog # 8585-BB). In some aspects, the PDGF- BB can be becaplermin. In some aspects, the PDGF-BB can be administered via localized injection. In some aspects, the PDGF-BB can be formulated for slow release. This is discussed in more detail below.

[0045] In some aspects, the PDGF-BB can be becaplermin. Becaplermin can be produced by recombinant DNA technology by insertion of the gene for the B chain of platelet derived growth factor (PDGF) into the yeast, Saccharomyces cerevisiae for example. Becaplermin has a molecular weight of approximately 25 KD. The sequence of becaplermin is SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNV QCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT (SEQ ID NO: 7). Contemplated herein are fragments or variants of bercaplermin which can also function to promote axon growth or regeneration. For example, contemplated herein is a composition which is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 7, and which is capable of generating axon growth or regeneration.

[0046] In some aspects of the present invention, PDGF-BB can be in a highly purified form. Purified PDGF-BB, as used herein, comprises compositions having greater than about 95% by weight PDGF-BB prior to incorporation in solutions of the present invention. The solution may be prepared using any pharmaceutically acceptable buffer or diluent. In other aspects, the PDGF-BB can be substantially purified. Substantially purified PDGF-BB, as used herein, comprises compositions having about 5% to about 95% by weight PDGF-BB prior to incorporation into solutions of the present invention. In one aspect, substantially purified PDGF-BB can comprise compositions having about 65% to about 95% by weight PDGF-BB prior to incorporation into solutions of the present invention. In other aspects, substantially purified PDGF-BB can comprise compositions having about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, or about 90% to about 95%, by weight PDGF-BB, prior to incorporation into solutions of the present invention. Purified PDGF-BB and substantially purified PDGF-BB may be incorporated into the scaffolding matrix.

[0047] In a further aspect, PDGF-BB can be partially purified. Partially purified PDGF- BB, as used herein, comprises compositions having PDGF-BB in the context of platelet-rich plasma, fresh frozen plasma, or any other blood product that requires collection and separation to produce PDGF-BB. Aspects of the present invention contemplate that any of the PDGF-BB isoforms provided herein can be purified or partially purified. Compositions of the present invention comprising PDGF-BB mixtures may comprise PDGF-BB isoforms or PDGF-BB fragments in partially purified proportions. Partially purified and purified PDGF-BB, in some aspects, can be prepared as described in U.S. Ser. No. 11/159,533 (U.S. Publication 20060084602).

[0048] In some aspects, solutions comprising PDGF-BB can be formed by solubilizing PDGF-BB in one or more buffers. Buffers suitable for use in PDGF-BB solutions of the present invention can include, but are not limited to, carbonates, phosphates (e.g. phosphate-buffered saline), histidine, acetates (e.g. sodium acetate), acidic buffers such as acetic acid and HC1, and organic buffers such as lysine, Tris buffers (e.g. tris(hydroxymethyl)aminoethane), N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be selected based on biocompatibility with PDGF- BB and the buffer's ability to impede undesirable protein modification. Buffers can additionally be selected based on compatibility with host tissues. In one aspect, sodium acetate buffer can be used. The buffers may be employed at different molarities, for example about 0.1 mM to about 100 mM, about 1 mM to about 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM, or any molarity within these ranges. In some aspects, an acetate buffer can be employed at a molarity of about 20 mM.

[0049] In another aspect, solutions comprising PDGF-BB may be formed by solubilizing lyophilized PDGF in water, wherein prior to solubilization the PDGF-BB is lyophilized from an appropriate buffer.

[0050] In some aspects, the mammal can be in need of axon growth and/or regeneration due to a cerebrovascular disease, a neurodegenerative disease, a developmental skeletal malformation, or a traumatic injury. In some aspects, the mammal can have experienced a stroke, aneurysm, moyamoya disease, or hemorrhage. In some aspects, the mammal can have Alzheimer’s disease, Parkinson’s disease, or cerebral small vessel disease. In some aspects, the mammal can have a spinal cord injury, peripheral nerve injury, or a traumatic brain injury. Methods of diagnosing a subject in need of axon regrowth and/or regeneration include, but are not limited to, physical examination/diagnosis by a medical practitioner, including screening for neurophysiological hallmarks; screening for various markers, including protein and/or genetic biomarkers; conducting sensory nerve conduction studies (NCS) such as on-nerve needle (ONN) and surface electrode (SE); and sural nerve biopsy .

[0051] By “promoting axon growth and/or regeneration” is meant that axons can grow, or regenerate, at a rate faster than would be achieved in a control which is not exposed to PDGF- BB. This rate can be about a 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 a 100% improvement in growth or regeneration compared to a control. The rate of growth (or regrowth) compared to a control can also be about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times faster, or more. Specifically, for example, an axon can regenerate when exposed to PDGF-BB, whereas a control would not have ever regenerated or grown.

[0052] The PDGF-BB can be formulated to be released at a rate needed by the subject. In one aspect, this can be a slow release formula. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 90% to about 95%, about 50% to about 85%, about 60% to about 85%, about 70% to about 85%, or about 50% to about 80% of the PDGF-BB can be released within about 24 hours. In some aspects, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 90% to about 95%, about 50% to about 85%, about 60% to about 85%, about 70% to about 85%, or about 50% to about 80% of the PDGF-BB can be released within about 1 hour, about 6 hours, about 8 hours, about 12 hours, or about 48 hours. In some aspects, the PDGF-BB release can be measured in vivo. In some aspects, the PDGF- BB release can be measured in vitro. In some aspects, the PDGF-BB can be released into the surrounding region. In some aspects, the PDGF can be given via localized injection. In some aspects, the PDGF-BB can be released into the surrounding area. In some aspects, the PDGF- BB can be given systemically.

[0053] In some aspects, the slow release PDGF-BB can be encapsulated in a biocompatible and/or biodegradable polymer. In some aspects, the biocompatible and/or biodegradable polymer can include collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof.

[0054] In some aspects, the slow release PDGF-BB can be encapsulated in a nanoparticle comprising the biocompatible and/or biodegradable polymer. In some aspects, the nanoparticle can be targeted to a site of low axon density. In some aspects, the nanoparticle can be targeted to pericytes and/or axons. In some aspects, the nanoparticle can be administered via localized injection.

[0055] In some aspects, the slow release PDGF-BB can be encapsulated in a hydrogel comprising the biocompatible and/or biodegradable polymer. In some aspects, the hydrogel can be targeted to a site of low axon density. In some aspects, the hydrogel can be targeted to pericytes and/or axons. In some aspects, the hydrogel can be administered via localized injection.

[0056] In some aspects, solutions comprising PDGF-BB can further comprise additional components, such as other biologically active agents. In other aspects, solutions comprising PDGF-BB can further comprise cell culture media, other stabilizing proteins such as albumin, antibacterial agents, protease inhibitors (e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethylether)-N,N,N',N' -tetraacetic acid (EGTA), aprotinin, E- aminocaproic acid (EACA), etc.) and/or other growth factors such as fibroblast growth factors (FGFs), epidermal growth factors (EGFs), keratinocyte growth factors (KGFs), insulin-like growth factors (IGEs), bone morphogenetic proteins (BMPs), or other PDGFs including compositions ofPDGF-AA, PDGF-AB, PDGF-CC and/or PDGF-DD.

[0057] Compositions and methods of the present invention, according to some aspects, can further comprise one or more biologically active agents in addition to PDGF-BB. Biologically active agents that can be incorporated into compositions of the present invention, in addition to PDGF-BB, can comprise organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, small-interfering ribonucleic acids (siRNAs), gene regulatory sequences, nuclear transcriptional factors and antisense molecules), nucleoproteins, polysaccharides (e.g., heparin), glycoproteins, and lipoproteins. Non-limiting examples of biologically active compounds that can be incorporated into compositions of the present invention, including, e.g., anti-cancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, hormones, muscle relaxants, prostaglandins, trophic factors, osteoinductive proteins, growth factors, and vaccines, are disclosed in U.S. Ser. No. 11/159,533 (U.S. Publication 20060084602). Biologically active compounds that can be incorporated into compositions of the present invention, in some aspects, include osteoinductive factors such as insulin-like growth factors, fibroblast growth factors, or other PDGFs. In accordance with other aspects, biologically active compounds that can be incorporated into compositions of the present invention preferably include osteoinductive and osteostimulatory factors such as bone morphogenetic proteins (BMPs), BMP mimetics, calcitonin, calcitonin mimetics, statins, statin derivatives, fibroblast growth factors, insulin-like growth factors, growth differentiating factors, and/or parathyroid hormone. Additional factors for incorporation into compositions of the present invention, in some aspects, include protease inhibitors, as well as osteoporotic treatments that decrease bone resorption including bisphosphonates, and antibodies to the NF-kB (RANK) ligand.

[0058] Standard protocols and regimens for delivery of additional biologically active agents are known in the art. Additional biologically active agents can be introduced into compositions of the present invention in amounts that allow delivery of an appropriate dosage of the agent to the damaged tendon and/or the site of tendon attachment. In most cases, dosages are determined using guidelines known to practitioners and applicable to the particular agent in question. The amount of an additional biologically active agent to be included in a composition of the present invention can depend on such variables as the type and extent of the condition, the overall health status of the particular patient, the formulation of the biologically active agent, release kinetics, and the bioresorbability of the biocompatible matrix. Standard clinical trials may be used to optimize the dose and dosing frequency for any particular additional biologically active agent.

[0059] In some aspects, the one or more other treatments for axon growth and/or regeneration can include a PTPc blocker. In some such aspects, the PTPc blocker can be intracellular sigma peptide (ISP). In some aspects, the PTPc blocker can be administered in an amount of at least 200 pg/day (e.g., at least 250 pg/day, at least 300 pg/day, at least 350 pg/day, at least 400 pg/day, at least 450 pg/day, at least 500 pg/day, at least 600 pg/day, at least 700 pg/day, at least 800 pg/day, at least 900 pg/day, at least 1000 pg/day, at least 1200 pg/day, at least 1400 pg/day, at least 1600 pg/day, at least 1800 pg/day, at least 2000 pg/day, at least 2200 pg/day, at least 2400 pg/day, at least 2600 pg/day, at least 2800 pg/day). In some aspects, the PTPG blocker can be administered in an amount of up to about 2800 pg/day (e.g., up to about 2600 pg/day, up to about 2400 pg/day, up to about 2200 pg/day, up to about 2000 pg/day, up to about 1800 pg/day, up to about 1600 pg/day, up to about 1400 pg/day, up to about 1200 pg/day, up to about 1000 pg/day, up to about 900 pg/day, up to about 800 pg/day, up to about 700 pg/day, up to about 600 pg/day, up to about 500 pg/day, up to about 450 pg/day, up to about 400 pg/day, up to about 350 pg/day, up to about 300 pg/day, up to about 250 pg/day, up to about 200 pg/day).

[0060] It is considered that the PTPG blocker can be administered in any amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the PTPG blocker can be administered in an amount of from about 200 pg/day to about 2800 pg/day (e.g., from about 250 pg/day to about 2600 pg/day, from about 300 pg/day to about 2400 pg/day, from about 350 to about 2200 pg/day, from about 400 pg/day to about 2000 pg/day, from about 450 pg/day to about 1800 pg/day, from about 500 pg/day to about 1600 pg/day, from about 600 pg/day to about 1400 pg/day, from about 700 pg/day to about 1200 pg/day, from about 800 pg/day to about 1000 pg/day, from about 200 pg/day to about 1000 pg/day, from about 250 pg/day to about 900 pg/day, from about 300 pg/day to about 800 pg/day, from about 350 pg/day to about 700 pg/day, from about 400 pg/day to about 600 pg/day, from about 450 pg/day to about 500 pg/day, from about 1000 pg/day to about 2800 pg/day, from about 1200 pg/day to about 2600 pg/day, from about 1400 pg/day to about 2400 pg/day, from about 1600 pg/day to about 2200 pg/day, from about 1800 pg/day to about 2000 pg/day).

[0061] In some aspects, the one or more other treatments for axon growth and/or regeneration can include a gabapentinoid. In some such aspects, the gabapentinoid can include gabapentin and/or pregabalin. In some aspects, the gabapentinoid can be administered in a starting amount of about 200 mg. In some such aspects, the dosage of the gabapentinoid can be increased in increments of from about 300 mg to about 400 mg (e.g., from about 320 mg to about 380 mg, from about 340 mg to about 360 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg) until the dosage reaches a final value. In some aspects, the gabapentinoid can be administered in a final dosage of at least about 900 mg/day (e.g., at least about 1000 mg/day, at least about 1100 mg/day, at least about 1200 mg/day, at least about 1300 mg/day, at least about 1400 mg/day, at least about 1500 mg/day, at least about 1600 mg/day, at least about 1700 mg/day, at least about 1800 mg/day). In some aspects, the gabapentinoid can be administered in a final dosage of up to about 1800 mg/day (e.g., up to about 1700 mg/day, up to about 1600 mg/day, up to about 1500 mg/day, up to about 1400 mg/day, up to about 1300 mg/day, up to about 1200 mg/day, up to about 1100 mg/day, up to about 1000 mg/day, up to about 900 mg/day)/

[0062] It is considered that the gabapentinoid can be administered in a final dosage of any amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the gabapentinoid can be administered in a final dosage of from about 900 mg/day to about 1800 mg/day (e.g., from about 1000 mg/day to about 1700 mg/day, from about 1100 mg/day to about 1600 mg/day, from about 1200 mg/day to about 1500 mg/day, from about 1300 mg/day to about 1400 mg/day, from about 900 mg/day to about 1400 mg/day, from about 1000 mg/day to about 1300 mg/day, from about 1100 mg/day to about 1200 mg/day, from about 1300 mg/day to about 1800 mg/day, from about 1400 mg/day to about 1700 mg/day, from about 1500 mg/day to about 1600 mg/day).

[0063] In some aspects, the one or more other treatments for axon growth and/or regeneration can include a PTPc blocker and a gabapentinoid. In some such aspects, the PTPc blocker and the gabapentinoid can be administered in an amount according to any of the dosages described above.

[0064] As discussed above, the PDGF-BB and the one or more other treatments for axon growth and/or regeneration can be formulated for slow release. In some aspects, the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration can be encapsulated in a biocompatible and/or biodegradable polymer. In some aspects, the biocompatible and/or biodegradable polymer can include collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof.

[0065] In some aspects, the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration can be encapsulated a nanoparticle comprising the biocompatible and/or biodegradable polymer. In some aspects, the nanoparticle can be targeted to a site of low axon density or injury. In some aspects, the nanoparticle can be targeted to pericytes and/or axons In some aspects, the nanoparticle can be administered via localized injection. [0066] In some aspects, the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration can be encapsulated in a hydrogel comprising the biocompatible and/or biodegradable polymer. In some aspects, the hydrogel can be targeted to a site of low axon density. In some aspects, the hydrogel can be targeted to pericytes and/or axons. In some aspects, the hydrogel can be administered via localized injection.

[0067] In some aspects, administration can be repeated at least about every 3 weeks (e.g., at least about every 3.5 weeks, at least about every 4 weeks, at least about every 4.5 weeks, at least about every 5 weeks, at least about every 5.5 weeks, at least about every 6 weeks). In some aspects, administration can be repeated up to about every 6 weeks (e.g., up to about every 5.5 weeks, up to about every 5 weeks, up to about every 4.5 weeks, up to about every 4 weeks, up to about every 3.5 weeks, up to about every 3 weeks).

[0068] It is considered that administration can be repeated at any interval ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, administration can be repeated from about every 3 weeks to about every 6 weeks (e.g., from about every 3.5 weeks to about every 5.5 weeks, from about every 4 weeks to about every 5 weeks, from about every 3 weeks to about every 4.5 weeks, from about every 3.5 weeks to about every 4 weeks, from about every 4.5 weeks to about every 6 weeks, from about every 5 weeks to about every 5.5 weeks).

[0069] In some aspects, administration can be repeated for at least about 7 months (e.g., at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months). In some aspects, administration can be repeated for up to about 12 months (e.g., up to about 11.5 months, up to about 11 months, up to about 10.5 months, up to about 10 months, up to about 9.5 months, up to about 9 months, up to about 8.5 months, up to about 8 months, up to about 7.5 months, up to about 7 months).

[0070] It is considered that administration can be repeated for any interval ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, administration can be repeated for from about 7 months to about 12 months (e.g., from about 7.5 months to about 11.5 months, from about 8 months to about 11 months, from about 8.5 months to about 10.5 months, from about 9 months to about 10 months, from about 7 months to about 9.5 months, from about 7.5 months to about 9 months, from about 8 months to about 8.5 months, from about 9.5 months to about 12 months, from about 10 months to about 11.5 months, from about 10.5 months to about 11 months).

[0071] In some aspects, the PDGF-BB can be administered in an amount of at least about 50 pg (e.g., at least about 60 pg, at least about 70 pg, at least about 80 pg, at least about 90 pg, at least about 100 pg, at least about 120 pg, at least about 140 pg, at least about 160 pg, at least about 180 pg, at least about 200 pg, at least about 225 pg, at least about 250 pg, at least about 275 pg, at least about 300 pg, at least about 325 pg, at least about 350 pg, at least about 375 pg, at least about 400 pg, at least about 425 pg, at least about 450 pg, at least about 475 pg, at least about 500 pg). In some aspects, the PDGF-BB can be administered in an amount of up to about 500 pg (e.g., up to about 475 pg, up to about 450 pg, up to about 425 pg, up to about 400 pg, up to about 375 pg, up to about 350 pg, up to about 325 pg, up to about 300 pg, up to about 275 pg, up to about 250 pg, up to about 225 pg, up to about 200 pg, up to about 180 pg, up to about 160 pg, up to about 140 pg, up to about 120 pg, up to about 100 pg, up to about 90 pg, up to about 80 pg, up to about 70 pg, up to about 60 pg, up to about 50 pg).

[0072] It is considered that the PDGF-BB can be administered in any amount ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the PDGF-BB can be administered in an amount of from about 50 pg to about 500 pg (e.g., from about 60 pg to about 475 pg, from about 70 pg to about 450 pg, from about 80 pg to about 425 pg, from about 90 pg to about 400 pg, from about 100 pg to about 375 pg, from about 120 pg to about 350 pg, from about 140 pg to about 325 pg, from about 160 pg to about 300 pg, from about 180 pg to about 275 pg, from about 200 pg to about 250 pg, from about 50 pg to about 225 pg, from about 60 pg to about 200 pg, from about 70 pg to about 180 pg, from about 80 pg to about 160 pg, from about 90 pg to about 140 pg, from about 100 pg to about 120 pg, from about 225 pg to about 500 pg, from about 250 pg to about 475 pg, from about 275 pg to about 450 pg, from about 300 pg to about 425 pg, from about 325 pg to about 400 pg, from about 350 pg to about 375 pg).

[0073] In some aspects, the mammal can be selected from the group comprising: livestock, companion animal, and human.

[0074] In an aspect, provided is a composition comprising platelet-derived growth factor- BB (PDGF-BB) encapsulated in a nanoparticle. In some aspects, the nanoparticle can include a biocompatible and/or biodegradable polymer. In some aspects, the biocompatible and/or biodegradable polymer can include collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof.

[0075] In some aspects, the nanoparticle can further include one or more other treatments for axon growth and/or regeneration. These are discussed in detail above. In some aspects, the nanoparticle can be targeted to a site of low axon density. In some aspects, the nanoparticle can be targeted to pericytes and/or axons.

[0076] In another aspect, provided is a composition comprising platelet-derived growth factor-BB (PDGF-BB) encapsulated in a hydrogel. In some aspects, the hydrogel can include a biocompatible and/or biodegradable polymer. In some aspects, the biocompatible and/or biodegradable polymer can include collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof. The hydrogel can further include one or more other treatments for axon growth and/or regeneration. In some aspects, the hydrogel can be targeted to a site of low axon density. In some aspects, the nanoparticle can be targeted to pericytes and/or axons.

[0077] In another aspect, provided is a method to downregulate type I collagen in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of downregulating type I collagen; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal. PDGF-BB can be beneficial where excessive collagen deposition is found in the skin and internal organs. It is well established that collagen exerts immunogenic properties and that tolerance of collagen is regulated by T-lymphocytes and B-cells. As such, PDGF-BB can be useful for the downregulation of type I collagen due to its immunomodulatory properties causing or contributing to a sustained inflammatory response. In some aspects, the PDGF-BB can be becaplermin. In some aspects, the mammal can have rheumatoid arthritis, dermatomyositis, or scleroderma.

EXAMPLES

Example 1: In Vivo Programming of Adult Pericytes Aids Axon Regeneration by Providing Cellular Bridges for SCI Repair

[0078] In mammals, injury to the adult spinal cord yields devastating neurological deficits and long-term disability due to axon regeneration failure [1], Not only do central nervous system (CNS) neurons lose axon growth ability and regeneration competence during later stages of development [2] [3], but the presence of a hostile cellular environment at the lesion site also actively contributes to regeneration failure after CNS injury [4], The regenerative capacity of neurons profoundly differs between animal lineages [5], In fish and amphibians, a more permissive lesion environment sponsors axon regeneration [6] [7], allowing complete restoration of function after spinal cord injury (SCI). Although described as the absolute barrier to axonal regeneration after SCI in mammals, the fibrotic scar has mainly been understudied. In mammals, current strategies that allow the conversion of the growth-inhibitory lesion environment into a growth-supportive state primarily focus on the elimination of myelin- derived inhibitors [8], digestion of chondroitin sulfate proteoglycans (CSPG) [9] [10] [11] and, more recently, ablation of specific cell populations including pericytes that participate to fibrotic scarring [12] [13] [14],

[0079] Of the cellular sources contributing to fibrotic scarring, pericytes have been the subjects of genetic ablation studies aimed at promoting axon regeneration and functional recovery following SCI in adult mice [13] [14] [15], Pericytes are multifunctional mural cells of the microcirculation where they control blood flow, vascular permeability, and homeostasis [16] [17], Whereas revascularization and vascular normalization ensure the delivery of oxygen and nutrients needed to sustain the high metabolic demands of neural networks, aberrant reorganization of microvasculature networks and perivascular cell function blunts axon regeneration after SCI in adult mice [15], Structured reorganization of vasculature networks at the lesion site is also instrumental for axon regeneration in the peripheral nervous system [18], Since pericytes are highly plastic and motile during vascular development [19], manipulation of pericyte-neuron interaction may be exploited to prime regeneration after SCI by providing vascular bridges for axon elongation across the lesion site.

[0080] Here, a study was conducted which shows that profound changes in vasculature architecture and pericyte coverage fail to normalize vasculature function after SCI in adult mice. Whereas pericytes cause detrimental structural and functional changes in adult dorsal root ganglion (DRG) neurons associated with poor axon outgrowth, PDGF-BB stimulation effectively converts adult pericytes into a permissive substrate for axon growth and regeneration. Mechanistically, reconfiguration of fibronectin matrix and integrin signaling are necessary to sponsor axon elongation on PDGF-BB stimulated pericyte substrates. Finally, PDGF-BB-mediated pericyte conversion at the lesion site enables axon regeneration after SCI and positively affects the physical and chemical nature of the lesion environment. Thus, this study underscores the strong potential for manipulating pericyte behavior as a strategy to create more favorable conditions for SCI repair. Materials and Methods

[0081] Mice: All animal experiments were performed following protocols approved by the Institutional Animal Care and Use Committee at The Ohio State University. Adult (7-9 weeks old) female and male FVB mice (Stock no. 001800, RRID: IMSR_JAX:001800) were used for all experiments except those specifying NG2-mEGFP [20], NG2-CreER™ [82], and Ai9 (RCL-tdTomato) [83], NG2-mEGFP (Stock no. 022735, RRID:IMSR_JAX:022735), NG2- CreER™ (Stock no. 008538, RRID:IMSR_JAX:008538) and Ai9 (RCL-tdTomato) (Stock no. 007909; RRID: IMSR_JAX:007909) were purchased from The Jackson Laboratory. Mice were randomly assigned to experimental groups. Experimenters were blind to group assignment and experimental conditions.

[0082] Spinal cord injury. Adult NG2-mEGFP mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) and a T12 laminectomy was performed. The spinal cord was crushed with modified forceps (no. 5, Cat #11254-20, FST). The forceps were positioned to completely sever ascending dorsal column sensory axons on the left side. For the control surgery, a laminectomy was performed without any direct manipulation of the spinal cord. In another cohort, NG2-mEGFP mice were allowed to recover for seven days before reopening the wound and injecting either vehicle (dH2O) or PDGF-BB (10 ng/pl) into the lesion site (volume: 2 pl, depth: 500 pm, speed: 200 nl/minute). The same experimental procedure was repeated in NG2-CreER™/Ai9 (RCLtdTomato) mice (C57B16/J background) to confirm results in a common genetic background and to determine pericyte’s proliferation following PDGF-BB stimulation. NG2-CreER™/Ai9 (RCL-tdTomato) mice were subjected to T12 SCI. At 7 days after SCI, vehicle and PDGF-BB were injected directly at the lesion site as described above. Tamoxifen (100 mg/kg of body weight) or the corresponding volume of com oil (C0136, Spectrum Chemical) was administered (intraperitoneal injections, 2 times/day for 4 days) beginning 1 hour after SCI. To trace sensory dorsal column axons, 1.5 pl of AVV1 particles expressing either tdTomato (>lxl0¹² vg/ml, Cat. No. 105554-AAV1, Addgene) or eGFP (8xl0¹² vg/ml, Cat. No. 105530-AAV1, Addgene) were injected into the left sciatic nerve 2 weeks after SCI. At 28 days after SCI, mice were transcardially perfused and tissue dissected for further histological analysis or cleared for three- dimensional imaging.

[0083] Vasculature tracing, in toto staining three-dimensional imaging and reconstruction'. The vasculature was traced using a method described earlier [25] [84], Briefly, adult NG2-mEGFP mice were transcardially perfused with 4% PF A in PBS (pH 7.4). Mice were then perfused with 5 ml of 0.05% albumin-tetramethylrhodamine isothiocyanate bovine (A2289, Sigma) in 2% gelatin from porcine skin (G1890, Sigma). At the time of injection, the temperature of the gel solution was maintained at 45°C. After clamping the heart, mice were placed on ice to lower their body temperature and allow for gel formation. In another cohort of adult NG2-mEGFP mice, the spinal cord was fixed, dissected and subjected to CD31 in toto staining 3 days after SCI. Specifically, a 3 mm segment of the spinal cord containing the lesion site was blocked for 12 hours at room temperature with bovine serum albumin (A3059, Sigma- Aldrich) in PBS with 0.5% Triton-XlOO and incubated for 48 hours at room temperature with a rat monoclonal anti-CD31 (1 : 100). After washing 3-4 times (3 hours/each) with PBS, tissues were incubated for 24 hours at room temperature with Alexa Fluor-conjugated secondary antibodies (1 :200, Life Technologies) and finally washed 3-4 times (3 hours/each) with PBS. [0084] The spinal cords were cleared using the advanced CUBIC protocol [67] and imaged in 3D using a confocal microscope (C2 plus, Nikon). Vasculature networks were reconstructed and analyzed using the visualization and analysis software Imaris (version 10.0.0, Oxford Instruments). To exclude any confounding variable originating from OPC in NG2-mEGFP mice, the deposition of mEGFP-positive fibrous structure from pericytes located within the lesion site was calculated using the Imaged plugin ‘SNT’. The Imaris software was then used to determine the density and position of mEGFP- (for NG2-mEGFP mice) or tdTomato- positive (for NG2-CreER™/Ai9 (RCL-tdTomato) mice) pericytes at the lesion site in relation to vasculature structures filled with albumin-TRITC or stained with an antibody directed against CD31.

[0085] Immunohistochemistry. At the end of the study, mice were transcardially perfused with 4% paraformaldehyde (PF A) in PBS (pH 7.4). The spinal cords were dissected, post-fixed at 4°C in 4% PFA for 24 hours and dehydrated in 30% sucrose. Tissues were then embedded in optimum cutting temperature (OCT) compound (Tissue-Tek), frozen, sectioned (20 pm thick, HM525 NX, Thermo Fisher Scientific) and mounted on slides. Slides were warmed at 37°C for 30 minutes and OCT was washed away with PBS. Sections were then blocked at room temperature with 2.5% bovine serum albumin (A3059, Sigma- Aldrich) in PBS with 0.1% Triton-XlOO for 1 hour and incubated overnight at 4°C with the primary antibody. After washing 3 times with PBS, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1 :400, Life Technologies). When necessary, sections were counterstained with DAPI (1 : 10,000, D9542, Sigma-Aldrich). Images were taken using a confocal (C2 plus, Nikon) or epifluorescence microscope (Axio Observer Zl, Zeiss) and linear fluorescence intensity was calculated using Fiji (version 2.0.0-rc-69/1.52p) after background subtraction. The number of biological replicates analyzed for each condition is indicated in the corresponding figure description.

[0086] Primary pericyte culture '. The spinal cords from two adult (6-7 weeks) FVB mice were rapidly dissected and placed in cold HBSS medium (14175103, Gibco). After removing the meninges, the spinal cords were cut into 3-5 mm segments, washed twice with sterile HBSS and dissociated in 0.125% trypsin (25200056, Gibco) for 30 minutes at 37°C. After incubation, fetal bovine serum (FBS) (16000044, Gibco) was added to stop digestion and the spinal cords were dissociated in 30% Percoll (17-0891-01, GE Healthcare) in MEM without Ca²⁺ and Mg²⁺ using a 10 ml sterile pipette. The cell suspension was then layered into 70% Percoll. After centrifugation at 2000 rpm for 30 minutes at 4°C, the 30-70% interface was removed and collected into a sterile tube, washed and spun down at 1000 rpm for 5 minutes. The cell pellet was resuspended in DMEM/F12 (21041025, Gibco) supplemented with 10% FBS and plated at the desired density on poly-D-Lysine (P6407, Sigma) coated dishes. The medium was changed 2 hours after plating to remove any dead cell. Half of the medium was replaced with fresh medium 2 times/week. In a separate set of experiments, the same protocol was used to purify pericytes from NG2-mEGFP mice (Stock number 022735). In another set of experiments, vehicle (e.g., H2O) and PDGF-BB (20 ng/ml) were added to the medium immediately after plating pericytes. The culture was maintained in a humidified atmosphere containing 5% CO2 in air at 36.5°C.

[0087] RNA isolation and RT-PCR Total RNA was extracted from mouse cultured pericyte using the RNeasy kit (Qiagen) and cDNA was synthesized from 0.1-2 pg of RNA using random hexamers from the SuperScript VILO cDNA synthesis kit (11754050, Thermo Fisher Scientific). The subsequent cDNA was used in PCR reactions using Choice Taq Master Mix (cb4070-7, Denville Scientific). PCR products were run on agarose gel. The sequences of the primers used are listed in TABLE 1.

TABLE 1. Key resources table.

[0088] Primary DRG culture'. Murine DRG were dissected and collected in ice-cold Hank’s balanced salt solution (HBSS, GIBCO). The ganglia were transferred into a sterile tube, washed twice with HBSS and incubated in Neurobasal-A medium (GIBCO) containing collagenase type I (3,000 U/mL, Worthington) at 36.5°C for 15 min, followed by 30 min with trypsin (0.25%, GIBCO). Serum was then added to stop trypsin digestion. Ganglia were dissociated by gently pipetting up and down. The cell suspension was filtered using a nylon cell strainer (70 pm) and centrifuged at 900 rpm for 5 min. Dissociated neurons were resuspended in DMEM/F12 supplemented with 10% FBS and plated at low density on laminin (5 pg/mL, Roche), CSPG (10 pg/mL, Millipore) or pericyte-coated coverslips. When needed, RGDS peptide (0.02 mg/ml, Cat. No. 3498, Tocris), cilengitide (2 pM, Cat. No. 5870, Tocris) and K34c (20 pM, Cat. No. 5114, Tocris) were added to the culture medium while plating the neurons on pericyte-coated dishes. The culture was maintained in a humidified atmosphere containing 5% CO2 in air at 36.5°C. For the siRNA gene silencing experiment, dissociated DRG neurons were plated on pericyte-coated dishes 24 hours after pericytes were transfected with pre-designed siRNA oligos (Ambion) using the polyfect transfection reagent (Cat. No. 301105, Qiagen). Neurons were then fixed 24 hours after plating.

[0089] For co-culture experiments, pericytes were plated at 80% confluency on poly-D- Lysine coated 13 mm glass coverslips and dissociated DRG neurons were added 24 hours after plating pericytes.

[0090] Immunocytochemistry. Cells (e.g., pericytes, DRG neurons and pericyte-DRG neurons co-cultures) were fixed with 4% paraformaldehyde in PBS. Coverslips were then blocked at RT for 1 hour with 2.5% BSA and 0.1% Triton-XlOO in PBS and incubated at 4°C overnight with the appropriate primary antibodies. After three rinses in PBS, the coverslips were incubated with Alexa Fluor conjugated secondary antibodies (1:400, Life Technologies) and washed in PBS before mounting them onto microscope slides. Fluorescence images were randomly taken with a confocal microscope (C2 plus, Nikon) or epifluorescence microscope (Axio Observer Zl, Zeiss). This process was repeated for at least three independent experiments. The number of cells quantified for each condition is indicated in the corresponding figure description.

[0091] Morphometric analysis'. DRG neurons were fixed with 4% paraformaldehyde in PBS, and stained for Tuj l (Covance). Images were randomly taken with an epifluorescence microscope (Zeiss) and analyzed using Fiji software (NIH). This was carried out for at least three independent experiments. The number of neurons quantified for each condition is indicated in the corresponding figure description.

[0092] Immunoblotting'. Cultured pericytes were lysed 24 hours after plating on ice in RIP A buffer (0.5 M Tris-HCl pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) containing phosphatase and protease inhibitors (Sigma-Aldrich). The homogenates were then centrifuged, and the supernatant was collected. Using Bradford reagent (Bio-Rad), the protein concentration of the lysate was determined and a portion of the lysate (10 pg total protein) was then fractionated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a 0.2-pm nitrocellulose membrane (Bio-Rad) that was stained to confirm equal loading and transfer of the samples with Ponceau S (P7170, Millipore Sigma). After blocking at room temperature with 5% non-fat milk (1706404, Bio- Rad) in Tris-buffered saline with 0.1% Tween 20 detergent for 1 hour, the membrane was probed with mouse monoclonal anti-NG2 (Millipore), rabbit polyclonal anti-PDGFrP (Sigma) and goat polyclonal anti-Desmin (R&D Systems). For the siRNA experiment, transfected pericytes were lysed 48 hours after plating and the membrane was probed with rabbit polyclonal anti-fibronectin (Sigma). Rabbit monoclonal anti-GAPDH (Cell Signaling Technology) antibody was used as the loading control. Densitometry analysis was performed using ImageJ (NIH). After background subtraction, the intensity of fibronectin bands was measured and normalized to the loading control (e.g., GAPDH). Three biological replicates for each experimental condition were analyzed.

[0093] Patch-clamp recording'. Whole-cell patch-clamp recordings were performed on cultured mouse DRG neurons plated on either laminin or pericyte monolayer. One coverslip was transferred at a time to a submerged recording chamber mounted on a Nikon upright FN1 microscope, and the neurons were visualized through a CMOS camera (ORCA-Flash 4.0LT, Hamamatsu). Neurons were recorded in voltage-clamp mode (Axopatch 200A, Molecular Devices) and hold at -70mV while being continuously perfused with artificial cerebrospinal fluid (ACSF) at room temperature. The ACSF contained (in mM): 124 NaCl, 3 KCL, 1.25 NaH₂PO₄, 2 MgCh, 2 CaCh, 26 NaHCOs, 10 glucose (gassed with 95% O₂/5% CO₂). The perfusion rate was set at 1.5 ml/min. To record the minimum current to trigger an action potential, the recording was switched to the current-clamp mode and the resting potential was maintained at -70 mV by adjusting the baseline current injection. Patch pipettes were pulled using a vertical pipette puller (Model PC-100, Narishige) with a resistance of 3.0 - 4.0 MW. Pipette solution contained (in mM): 125 K-gluconate, 4 Na₂-ATP, 2 MgCh, 10 HEPES, 20 KC1, 3 NaCl, 0.5 EGTA (pH=7.3, 280-290 mOsm). The liquid junction potential was corrected by adjusting the zero-current position to -10 mV before the sealing procedure. The recordings were acquired using WinWCP software (Strathclyde Electrophysiology Software, University of Strathclyde Glasgow). The sampling rate was set at 20 kHz (NI USB-6229, National Instruments) and all recordings were low-pass filtered at 5 kHz. Data analysis was performed using Igor Pro software (WaveMetrics). For voltage-clamp recording, an off-line leak subtraction procedure was performed using a P/-4 protocol. A series of voltage steps from -90 mV to +40 mV with 10 mV increments were applied. The traces show both inward sodium and outward potassium currents. To generate the I-V curve in FIG. 4F, the peak inward current amplitude was used. [0094] To calculate the passive properties of recorded neurons, a negative voltage step pulse (-5 mV, 400 ms duration) was applied 5 times every 5 seconds. The series resistance (Rs), cell input resistance (Rm) and cell capacitance (Cm) were then calculated as previously described [85] using the average response. The average Rs from all recorded neurons was 7.02±0.4 M (n=23 across both experimental groups).

[0095] Calcium imaging'. The membrane-permeable calcium indicator Fluo-4 AM was added to the cell culture medium (final concentration: 5 pM) and kept at 36.5°C in the cell culture incubator for 45-60 minutes. The coverslip was then transferred to a submerged imaging chamber mounted on a Nikon upright NI microscope. Plated neurons were then washed and bathed with gassed (95% 02/5% CO2) ACSF at room temperature. Spontaneous calcium signals were acquired (interval: 3 seconds, duration: 5 minutes) in frame-scan mode using a 60X water-dipping objective (NA 1.0, Nikon) mounted on a laser-scanning confocal system (C2 plus, Nikon) with a 525/40 bandpass filter. The data analysis was performed with Fiji (NIH) and Igor Pro software (Wavemetrics). Calcium profiles for each individual neuron analyzed were then normalized to baseline level and displayed as AF/F.

[0096] Cryo-EM imaging: Pericytes were grown on EM grids coated with PDL (0.1 mg/ml, Sigma). At 24 hours after plating, the grids were flash-frozen using a manual Cryo-plunger. The frozen grids were screened using Thermo Scientific™ Glacios™ Cryo-TEM. Grids with suitable ice thickness were transferred to a Thermo Scientific™ Titan Krios™ for Cryo-ET data collection. The Titan Krios was equipped with an AMETEK Gatan’s K3™ direct detector and AMETEK Gatan™ energy filter. Cryo-ET data were collected at 300 kV, with a pixel size of 0.39 nm/pixel at a magnification of 19,500X. The tilting range was from -60 degrees to 60 degrees. Data were collected at 3- or 4-degree intervals. A dose-symmetric tilt scheme was applied as previously described. The total dose for one data set was 80-100 electrons per square angstrom. The angle of deviation (fibril structures) was calculated using Fiji.

[0097] Quantification of dorsal column regeneration after SCI: For each image of the unsectioned injured spinal cord, the number of regenerating axons at different distances from the lesion epicenter was normalized to the number of labeled axons caudal (200-400 pm) to the lesion. The lesion epicenter was identified based on axon morphology and the presence of infiltrating macrophages containing autofluorescent phagocytic material [86], Samples with poor tracing for each specific experiment or altered by clear experimental flaws (e.g., incomplete lesion) were excluded from the analysis. Blinding strategies were adopted. [0098] Statistical analysis'. Statistical analysis was performed using Prism (version 9.3.1; GraphPad) as follows: 1-way ANOVA (FIG. 1C, FIG. IE, FIG. 3K, FIG. 4B, FIG. 61, FIG. 6K, FIG. 6M, FIGS. 8F-8G), 2-way ANOVA (FIG. 5D, FIG. 7C, FIG. 8C), mixed model (FIG. 4F), unpaired 2-tailed Student’s t-test (FIG. 2E, FIGS. 3F-3G, FIG. 31, FIG. 4E, FIG. 5B, FIG. 5G, FIG. 51, FIG. 5K, FIGS. 6A-6B, FIG. 6D, FIG. 6G, FIG. 7E, FIG. 7G, FIG. 71, FIG. 8D, FIG. 8H), two-sample Kolmogorov-Smirnov test (FIG. 6E). For all analyses performed, significance was defined as p<0.05. The exact values of n and the definition of measures are shown in the corresponding figure descriptions. Randomization and blinding strategies were adopted to eliminate any potential bias in the interpretation of the results.

Results

[0099] SCI leads to profound changes in vasculature architecture and pericyte coverage'. Aberrant reorganization of microvascular structures and perivascular cell function interferes with physiological recovery following central nervous system (CNS) injury and disease. To determine the extent to which pericytes participate in the reorganization of microvasculature structure and vascular normalization at the lesion site, the study assessed changes in vasculature architecture and pericyte coverage after SCI. To this end, adult FVB mice expressing membrane-anchored enhanced GFP (eGFP) under the control of the chondroitin sulfate proteoglycan (Cspg) 4 - also known as neuron-glial antigen 2 (NG2) - promoter on a BAC transgene [20], hereafter called NG2-eGFP mice, were subjected to a thoracic (T)12 SCI that completely severed dorsal column axons on one side (FIG. 1A). Of note, both pericytes and oligodendrocyte precursor cells in the spinal cord expressed eGFP in this transgenic line (FIGS. 2A-2B). Although it is possible to distinguish the two cell types based on their unique morphology without any specific staining procedure, pericyte identity was confirmed by immunostaining using canonical pericyte markers like desmin, platelet-derived growth factor receptor P (PDGFrP) and CD13 (FIG. 2A) [21], Consistent with previous reports [22] [23], NG2-eGFP oligodendrocyte precursor cells express PDGFra and display a highly branched morphology (FIG. 2B). Under normal physiological conditions, it was found that NG2-eGFP pericytes were closely associated with microvasculature structures in the adult mouse spinal cord (FIG. 2C). At each time point after SCI, mice were transcardially perfused and the entire vasculature was labeled by filling the blood vessel lumen with a fluorescent gel perfusate (FIG. 2C). This procedure improves image contrast, allowing vasculature reconstruction at the capillary level [24] [25], After dissecting and clearing the spinal cords, the unsectioned tissues were imaged to visualize three-dimensional changes in vasculature architecture and pericyte coverage at the lesion site over days and months after SCI (FIGS. 1B-1C). Newly formed NG2-eGFP filamentous structures displayed complex and tortuous trajectories (FIG. IB). The data indicate that pericytes already migrated into the lesion site 3 days post-injury (DPI) during capillary sprouting as shown by the colocalization with the endothelial cell marker CD31 (FIG. 2D) [26], However, a mismatch in pericyte coverage and microvasculature filling was found at the lesion epicenter at acute and subacute time points (e.g., 3, 7 and 14 DPI) (FIG. IB). Even at chronic time points (e.g., 28 and 90 DPI), incomplete systemic perfusion of the lesion site was still present (FIG. IB). Yet, pericyte-decorated microvessels continued to fill the injury site (FIGS. 1B-1C). Given that pericytes are embedded in the vascular basement membrane [27], the distance between eGFP pericyte and the closest vasculature structure filled with albumin-TRITC was close to zero in the uninjured spinal cord (FIGS. 1D-1E). In contrast, the distance increased at 3 DPI, suggesting pericytes dissociated from mature microvasculature structures during capillary sprouting after SCI. Previous studies have shown that pericyte deficiency and detachment from the vasculature lead to increased vascular permeability [28] [29] [30], In line with this concept, it was found that claudin 5, a major cell adhesion molecule of tight junctions in endothelial cells maintaining barrier function [31], was only expressed along -30% of the total vasculature length within the lesion site at 3 DPI, underscoring a compromised barrier function early after SCI (FIGS. 1F-1G). Together, the data indicate that profound changes in vasculature architecture and pericyte coverage fail to normalize vasculature function in the injured spinal cord, likely contributing to a lack of functional recovery in adulthood.

[0100] Pericytes cause detrimental structural and functional changes in adult DRG neurons'. Next, the study assessed the contribution of pericytes on axon growth and regeneration failure. Accordingly, adult pericytes from the spinal cord of adult FVB mice were purified and cultured. Isolated pericytes expressed classical pericyte markers including NG2, PDGFrP and desmin [21] (FIGS. 3A-3B). The pericyte isolation protocol was further validated using adult NG2-eGFP mice (FVB background), and it was confirmed that cultured pericytes express eGFP (FIG. 3C). Not only do pericytes express NG2, a potent growth-inhibitory proteoglycan [32], but they also express permissive substrates for axon growth including laminin and fibronectin (FIG. 3D) [33] [34], Thus, it was questioned whether pericyte-neuron interaction inhibits or promotes axon growth. Adult mouse DRG neurons were plated on a confluent pericyte monolayer and, 24 hours later, plated adult DRG neurons. Laminin-coated dishes served as control. DRG neurons grown on a pericyte monolayer showed a drastic reduction in axon length and increased branching compared to the control condition (FIGS. 4A-4B) Of note, axon growth inhibition was comparable to that seen in DRG neurons plated on growth-inhibitory chondroitin sulfate proteoglycans (FIG. 4B). Interestingly, adult DRG neurons grown on pericyte monolayer had disorganized microtubules around the cell body (FIG. 4C) and collapsed growth cones resembling punctate adhesive contacts (FIGS. 3E-3F). After SCI, a disorganized microtubule network is also present in dystrophic end bulbs [35], a hallmark of regeneration failure in the adult mammalian CNS. Next, patch clamp recording and time-lapse calcium imaging were performed to determine the extent to which pericytes impair the physiological properties of adult DRG neurons. Input resistance, capacitance and resting membrane potential were comparable between DRG neurons cultured on laminin- coated dishes and pericyte monolayers (FIGS. 4D-4E). In contrast, the study found a reduced voltage-gated sodium current in DRG neurons growing on pericytes (FIG. 4F), indicating reduced neuronal excitability. Calcium imaging was then performed, and it was found that >40% of control neurons displayed spontaneous calcium events 24 hours after plating (FIGS. 4G-4H) In contrast, <20% of neurons cultured on pericyte monolayer showed spontaneous calcium activity (FigFIGS. 4G-4H). Of note, neuron survival was not affected in these neurons (FIG. 3G)

[0101] Prolonged exposure to CSPG can cause over-adhesion with no axon outgrowth [36], In addition, protein tyrosine phosphatase G (PTPo), a CSPG receptor [37], actively contributes to the conversion of neuronal growth cones into dystrophic bulbs via stabilization with CSPG- rich substrates [36], While searching for strategies to overcome pericyte-mediated inhibition on axon outgrowth, increased PTPG expression was found within the dystrophic growth cones of neurons plated on pericyte monolayer (FIGS. 3H-3I). In turn, the study asked whether incubation with a membrane-permeable peptide that binds to PTPG and blocks CSPG-mediated inhibition of axon growth [36] would be sufficient to rescue axon growth defects in adult DRG neurons plated on a pericyte monolayer. Incubation with a PTPG peptide rescued, at least in part, axon outgrowth in the co-culture system (FIGS. 3J-3K). Taken together, these data support the conclusion that pericytes cause detrimental structural and functional changes in adult DRG neurons, likely due to over-adhesion to CSPG-rich substrates.

[0102] PDGF-BB converts adult pericytes into a permissive substrate for axon growth'. Platelet-derived growth factors (PDGF) play a crucial role during the development of the vascular system. PDGF-BB binds to PDGFrP expressed on pericytes to stimulate the proliferation and migration of pericytes and their recruitment to growing blood vessels and into the wound area [38], Since pericytes are highly plastic and can respond to PDGF-BB exposure by secreting pro-regenerative molecules including numerous neurotrophic factors [39], the study next addressed whether PDGF-BB would be sufficient to convert adult pericytes into a permissive substrate for axon outgrowth. Strikingly, adult DRG neurons cultured for 24 hours on a PDGF-BB stimulated pericyte monolayer extended long and sparsely branched axons (FIGS. 5A-5B). Of note, the extent of axon growth was comparable to that found in DRG neurons grown for 24 hours on permissive laminin-coated dishes (FIGS. 4A-4B). As PDGF- BB alone did not improve axon growth of adult DRG neurons plated on laminin-coated dishes (FIG. 6A), it was tested whether PDGF-BB pericyte-conditioned medium promotes axon growth in adult DRG neurons. Adult mouse DRG neurons were plated on laminin-coated dishes and 2 hours after plating, replaced the medium with either pericyte-control or PDGF- BB pericyte-conditioned media. Surprisingly, exposure to PDGF-BB pericyte-conditioned medium failed to enhance axon growth in adult DRG neurons (FIG. 6B), indicating additional variables may be at play in neurons plated on a PDGF-BB stimulated pericyte monolayer. Adult pericytes exposed to PDGF-BB acquired an elongated phenotype (FIGS. 6C-6D), as reported by others [39], Fibronectin is a multifunctional adhesive glycoprotein and ubiquitous extracellular matrix (ECM) component that plays a critical role in tissue repair, cell attachment and motility [40] [41], Since fiber structure alignment supports axon elongation and regeneration [42] [43], the study questioned whether PDGF-BB stimulation also causes structural changes in fibronectin matrix assembly in cultured pericytes. The immunocytochemistry and Cryo-EM analysis confirmed that pericytes stimulated for 24 hours with PDGF-BB display fibronectin elongation and fibril alignment (FIGS. 5C-5G). The overall expression of fibronectin and laminin, a basal lamina component, decreased in cultured pericytes exposed to PDGF-BB (FIGS. 5H-5I, FIG. 6E). Similarly, expression of NG2 and pro-alphal chain of type I collagen decreased upon exposure to PDGF-BB (FIGS. 6F-6G). This may be due to the fact that fibronectin accelerates collagen nucleation and that the presence of collagen fibrils induces the formation of highly co-localized fibronectin fibrils and subsequent matrix assembly.

[0103] Neurons adhere to ECM components including fibronectin substrates through transmembrane receptor proteins of the integrin family [44] [45], To test the role of fibronectin matrix assembly and integrin signaling during axon outgrowth on PDGF-BB stimulated pericyte monolayers, the study turned to well-established tools that enable acute fibronectin silencing and pharmacological disruption of integrin signaling. Indeed, silencing fibronectin expression in pericyte monolayers abrogated PDGF-BB-dependent axon outgrowth of adult DRG neurons (FIGS. 6H-6K). Similarly, pharmacological blockade of integrin signaling using the RGD peptide (e.g., a broad-spectrum inhibitor of integrin receptor function), Cilengitide (e.g., a_vp3 and a_vps integrins inhibitor) and K34c (e.g., asPi integrin inhibitor) strongly inhibited axon outgrowth of DRG neurons plated on PDGF-BB stimulated pericyte monolayers (FIGS. 6L-6M)

[0104] To test the clinical relevance of PDGF-BB-mediated pericyte conversion, adult DRG neurons were plated on a PDGF-BB-stimulated human pericyte monolayer. Once again, adult mouse DRG neurons cultured for 24 hours on human pericytes exposed to PDGF-BB extended longer and sparsely branched axons compared to the control (FIGS. 5J-5K).

[0105] Together, these results indicate that PDGF-BB effectively converts adult pericytes from an inhibitory to a growth-promoting substrate for axon outgrowth and that reconfiguration of fibronectin matrix and integrin signaling are necessary to sponsor axon outgrowth on PDGF- BB stimulated pericyte monolayers.

[0106] In vivo conversion of adult pericytes via PDGF-BB creates favorable conditions for SCI repair '. Next, the study examined whether in vivo PDGF-BB administration promotes axon regeneration after SCI. Adult (8-10 weeks old) NG2-eGFP mice (FVB background) were subjected to a T12 SCI that completely transected dorsal column sensory axons on one side (FIG. 7A). Seven days later, at a clinically relevant therapeutic window, either vehicle (dH2O) or PDGF-BB was administered at the lesion site. To visualize dorsal column axons in the spinal cord, adeno-associated viral (AAV1) particles expressing tdTomato were injected into the left sciatic nerve to transduce dorsal column axons originating from L3-5 DRG. The spinal cords were dissected and cleared 28 days after SCI. Three-dimensional imaging of the unsectioned spinal cord showed that PBGF-BB localized delivery at the lesion epicenter promoted robust regeneration of dorsal column axons into and beyond the lesion site (FIGS. 7B-7C). As they crossed the lesion site, these regenerating axons exhibited tortuous trajectories, a common feature often seen in experimental models associated with regenerative growth [3] [46], Imaging segmentation using Al-based convolutional reconstruction confirmed that one single administration of PDGF-BB 7 days after SCI led to the formation of pericyte-decorated cellular bridges at the lesion site (FIGS. 7D-7E). Strikingly, numerous regenerating axons were observed growing on these cellular bridges that had formed throughout the lesion site in response to PDGF-BB stimulation (FIGS. 7F-7G). As genetic variations impact axon regeneration and CNS repair [47] [48], the study sought to replicate the regenerative phenotype associated with PDGF-BB-mediated pericyte conversion in a common mouse background strain. Accordingly, adult NG2-CreERT2/Ai9 (RCL-tdTomato) mice (C57B16/J background) were subjected to T12 SCI and, 7 days later, injected vehicle (dH2O) or PDGF-BB at the site of injury (FIG. 8A). To visualize pericytes, tamoxifen was administered for four consecutive days beginning on the same day vehicle and PDGF-BB were injected. Two weeks after SCI, AAV1 particles expressing GFP were injected into the left sciatic nerve to transduce dorsal column axons originating from L3-5 DRG. The spinal cords were then dissected and imaged in 3D 28 days after SCI. The results confirmed that PBGF-BB localized delivery at the lesion epicenter also promoted robust regeneration of dorsal column axons into and beyond the lesion site in a common mouse background strain (e.g., C57BL/6J) (FIGS. 8B-8C). Analysis of tdTomato expressing pericytes confirmed that one single PDGF-BB administration promoted pericyte proliferation at the lesion site (FIG. 8D), as reported by others [49], As a result of SCI, astrocytes surrounding the lesion site become reactive and undergo hypertrophy [50] [51], Reactive astrogliosis is part of the tissue repair process [52], but can lead to the formation of a dense astrocyte border that produces growth inhibitory molecules such as CSPG, which curtail axon regeneration beyond the lesion site following SCI [53] [54], The results indicate that astrocyte limitans in mice receiving PDGF-BB were loosely entwined and had reduced CSPG deposition compared to control cells (FIGS. 7H-7I), thus facilitating axon regeneration into and beyond the lesion site. As microglia coordinate the cellular response at the lesion site in either a pathological or protective fashion [55] [56], the study tested whether localized delivery of PDGF-BB also impacts the behavior of myeloid cells. To test this, NG2-eGFP mice were subjected to a T 12 SCI. On day 7, vehicle and PDGF-BB were injected at the lesion site and, three days later, dissected the lesion site and subjected it to cell sorting (FIG. 8E). The total number of CD45⁺ myeloid cells present at the lesion site was comparable in all experimental conditions (FIG. 8F). Whereas the percentage of neutrophils and microglia was comparable, the study found an increase in the percentage of macrophages in mice administered PDGF-BB (FIG. 8F) This may occur because inflammatory signals and macrophages are essential for pericyte-mediated angiogenesis [57] and blood vessel formation to sponsor nerve regeneration [58] respectively. A more detailed characterization of the different macrophage populations did not identify a specific enrichment other than a non-significant downtrend in the percentage of CD14⁺ macrophages (FIG. 8G). In contrast, the study found a decrease in the percentage of CD14⁺ microglia at the lesion site of mice administered PDGF-BB (FIG. 8G). Given that CD14 acts as a key organizer in shaping microglia response after tissue damage [59], a decrease in CD 14 expression in microglia could be supportive of decreased proinflammatory microglia leading to diminished production of inflammatory chemokines and cytokines. In turn, the expression of classical chemokines and cytokines associated with SCI pathology were measured three days after vehicle and PDGF-BB administration. The multiplex data confirmed that mice administered PDGF-BB had decreased expression of TNF-a, IL1-P, CXCL2 and IL- 6 expression at the lesion site (FIG. 8H).

[0107] Altogether, these results indicate that adult pericytes can be programmed in vivo to sponsor axon regeneration after injury to the adult mammalian spinal cord and that PDGF-BB localized delivery at the lesion site positively affects the physical and chemical nature of the lesion environment thereby creating more favorable conditions for SCI repair.

Discussion

[0108] Evidence suggests that pericytes play a role in the pathophysiology of CNS injury and disease [14] [60] [61] [62] [63], In addition to their participation in fibrotic scarring, pericytes also impair capillary blood flow and functional recovery at chronic stages of SCI. Although experimental data indicate pericyte ablation enables axon regeneration and restoration of function after SCI in adult mice [13], the extent to which pericyte manipulation alone may be sufficient to create more favorable conditions for SCI repair has never been explored. This study provides evidence that pericytes can be converted into a growth- permissive substrate at the lesion site, enabling robust regeneration after SCI. In addition, pericyte conversion positively impacts the physical and chemical nature of the lesion environment. Given the simplicity of the delivery approach, this study identifies a treatment strategy that is readily translational across different species, CNS injury, and neurodegenerative conditions.

[0109] Lack of neurological recovery after SCI is associated with long-term impairment of sensation, cognition, locomotion, bladder and bowel function, contributing to deterioration of quality of life in SCI individuals. Axon sprouting and regeneration can restore connectivity within the injured spinal cord. Moreover, revascularization and normalization of vascular function are necessary to diffuse nutrients and metabolites needed for neuronal growth and function and to eliminate waste material at acute and chronic stages after SCI. In mammals, however, adult neurons have limited capacity to sprout and even less to regenerate [64], often failing to restore connectivity across the lesion site. In addition, detrimental changes in vasculature structure and pericyte function contribute to a lack of physiological recovery after injury and disease [60] [63] [65], The development of genetic tools that specifically label pericytes with durable fluorescent [20] together with vascular tracing methods [24] [25], tissue clearing, three-dimensional microscopy and volumetric analysis [66] [67] allowed the study of changes in vascular architecture and pericyte coverage over the course of the days, weeks and months after SCI. The data indicate that the injured spinal cord fails to restore normal vasculature function at acute and chronic time points, likely contributing to poor functional recovery in adulthood. In contrast, perinatal injuries to the murine spinal cord enable full restoration of vasculature architecture and robust regeneration in the absence of fibrotic scarring [33],

[0110] After tissue injury in the adult, pericytes detach from the mature vasculature and migrate to the lesion epicenter during capillary sprouting, contributing to the deposition of the growth inhibitory ECM [14] [68], The data show pericyte detachment and reduced claudin 5 expression along newly formed microvessels at the lesion epicenter, indicating compromised barrier function early after SCI. An increase in vascular permeability with consequent leakage of larger molecules and cells into the spinal cord results in edema, inflammation and progression of acute SCI pathophysiology [69], Pro- and anti angiogenic signaling are tightly regulated during embryonic vasculature development. An imbalance of such signaling after injury and disease is often associated with uncontrolled vessel growth that results in the formation of immature microvascular networks with profound structural and functional abnormalities [70], Whether normalization of pro- and anti angiogenic signaling may be required to promote the formation of structurally sound and functionally normal vasculature networks after SCI remains to be tested.

[OHl] Both oligodendrocyte precursor cells and pericytes express NG2, a potent axon growth inhibitory proteoglycan upregulated after SCI [71], Whereas oligodendrocyte precursor cells preferentially localize within the glial scar and lesion penumbra [15] [72], pericytes migrate into the fibrotic core after SCI [14] [15], In addition to NG2, the data indicate that adult pericytes also express large quantities of laminin and fibronectin, also known as positive substrates for axon growth and regeneration. Under normal physiological conditions, however, pericytes isolated from the adult mouse spinal cord cause detrimental structural and functional changes in adult DRG neurons associated with diminished axon growth. Filous and colleagues reached a similar conclusion after testing axon outgrowth of DRG neurons plated on NG2 expressing cells [72], Indeed, exposure to CSPG can cause overadhesion leading to no neurite outgrowth [36], The data indicate that incubation with a membrane permeable peptide that binds to a CSPG receptor rescues, at least in part, axon growth defects of adult DRG neurons growing on a pericyte monolayer.

[0112] PDGF-BB plays a key role during the development of the vasculature system. Upon binding to PDGFrP expressed on pericytes, PDGF-BB stimulates the proliferation and migration of pericytes and their recruitment to growing vessels. Pericytes are highly plastic and can respond to PDGF-BB exposure by secreting trophic factors and regenerative molecules needed to repair and restore neuron structure and function after injury and disease [39], Accordingly, the study tested whether PDGF-BB administration would be sufficient to convert adult pericytes into a permissive substrate for axon growth and regeneration. Surprisingly, PDGF-BB stimulation fully rescues axon growth defects of adult DRG neurons plated on a pericyte monolayer originating from the adult mouse spinal cord as well as the adult human brain. Similarly, the in vivo data provide evidence that one single administration of PDGF-BB at the lesion site at a clinically relevant time point (e.g., seven days after SCI) promotes robust regeneration of sensory ascending dorsal column axons in adult mice. The three-dimensional imaging clearly indicates axons regenerate into and beyond the lesion site by ‘riding’ pericyte decorated vessel structures that formed in response to PDGF-BB. Independent studies provide further evidence that vascular bridging across the lesion site is crucial for axon regeneration in the central and peripheral nervous systems [18] [73],

[0113] The structural and functional characterization using gain- and loss-off-function experiments indicates that PDGF-BB supports the reorganization of fibronectin matrix and the depletion of NG2 and collagen I expression in adult pericytes and that integrin signaling is necessary for axonal elongation on their associated fibronectin matrix. Along this line, a large body of work underscores the importance of integrin signaling for axon growth and regeneration [74], The fact that fibronectin acts as a permissive molecular substrate for axon growth is well-established in the SCI field. In the absence of collagen I deposition, injury to the spinal cord in neonatal mice leads to the formation of fibronectin bridges by microglia that promote the growth of long projecting axons across the lesion site. Furthermore, another recent study showed that SCI in zebrafish leads to the recruitment of PDGFrb myoseptal and perivascular cells to the site of injury, where they contribute to the deposition of a permissive ECM that promotes axon regeneration and functional recovery [75],

[0114] PDGF-BB has been shown to stimulate the conversion of pericytes to fibroblasts in the tumor microenvironment [76], Several studies suggest that perivascular cells, including fibroblasts and pericytes, give rise to stromal fibroblasts [77] [78], the main cellular source of fibrotic scarring. However, a comprehensive transcriptional analysis of virtually all cells of the brain vasculature indicates that perivascular fibroblast and pericyte show a different gene signature [79], As such, it is unlikely that the two cell types may be interchangeable in terms of contribution to fibrotic scarring [80], Controversy also exists about the actual number of pericyte subtypes in the adult CNS. Betsholtz and colleagues identified only one type of pericyte in the brain [79], Determining the trajectory of PDGF-BB stimulated pericytes by conducting fate mapping studies combined with the analysis of transcriptional signatures at predetermined time intervals after SCI will be an important direction for future investigation. [0115] Intrinsic neuronal and extrinsic non-neuronal mechanisms must be targeted simultaneously to promote successful regeneration and SCI repair [81], Not only does one single administration of PDGF-BB promote axon regeneration across the lesion site, but it also dampens astrogliosis and microglia reactivity, leading to reduced expression of classical chemokines and cytokines associated with SCI pathology. Therefore, quantitative inference and analysis of cell-cell communication at the lesion site will be an important direction for future investigations.

[0116] In summary, these observations highlight the strong potential for pericyte programming as a promising treatment strategy for SCI repair and other neurodegenerative conditions.

[0117] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

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CAGGCCGGTCGGGTGACCTA (SEQ ID NO: 1)

GGGCCACGTGGAAGACACGG (SEQ ID NO: 2)

ACCTGCAGAGACCTCAAAAGTAGGT (SEQ ID NO: 3)

ACCACGGTGACCTCCTGCGA (SEQ ID NO: 4)

AGCCAGCGCGTGTCCTCCTA (SEQ ID NO: 5)

AGCGTCGGCCAGGGAGAAGT (SEQ ID NO: 6)

SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNV QCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVT (SEQ ID NO: 7; bercaplerimin)

Claims

What is claimed is:

1. A method to promote axon growth and/or regeneration in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of axon growth and/or regeneration; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal.

2. The method of claim 1, wherein the mammal is in need of axon growth and/or regeneration due to a cerebrovascular disease, a neurodegenerative disease, a developmental skeletal malformation, or a traumatic injury.

3. The method of claim 2, wherein the mammal has experienced a stroke, aneurysm, moyamoya disease, or hemorrhage.

4. The method of claim 2, wherein the mammal has Alzheimer’s disease, Parkinson’s disease, or cerebral small vessel disease.

5. The method of claim 2, wherein mammal has a spinal cord injury, peripheral nerve injury, or a traumatic brain injury.

6. The method of any one of claims 1-5, wherein the PDGF-BB is becaplermin.

7. The method of any one of claims 1-6, wherein the PDGF-BB is administered via localized injection.

8. The method of any one of claims 1-7, wherein the PDGF-BB is formulated for slow release.

9. The method of claim 8, wherein the slow release PDGF-BB is encapsulated in a biocompatible and/or biodegradable polymer.

10. The method of claim 9, wherein the slow release PDGF-BB is encapsulated in a nanoparticle comprising the biocompatible and/or biodegradable polymer.

11. The method of claim 10, wherein the nanoparticle is targeted to a site of low axon density.

12. The method of any one of claims 10-11, wherein the nanoparticle is targeted to pericytes and/or axons.

13. The method of any one of claims 10-12, wherein the nanoparticle is administered via localized injection.

14. The method of claim 9, wherein the slow release PDGF-BB is encapsulated in a hydrogel comprising the biocompatible and/or biodegradable polymer.

15. The method of claim 14, wherein the hydrogel is targeted to a site of low axon density.

16. The method of any one of claims 14-15, wherein the hydrogel is targeted to pericytes and/or axons.

17. The method of any one of claims 14-16, wherein the hydrogel is administered via localized injection.

18. The method of any one of claims 1-17, wherein the PDGF-BB is given in combination with one or more other treatments for axon growth and/or regeneration.

19. The method of claim 18, wherein the one or more other treatments for axon growth and/or regeneration comprises a PTPc blocker and/or a gabapentinoid.

20. The method of claim 19, wherein the PTPc blocker is intracellular sigma peptide (ISP).

21. The method of claim 19, wherein the gabapentinoid comprises gabapentin and/or pregabalin.

22. The method of any one of claims 18-21, wherein the PDGF-BB and the one or more other treatments for axon growth and/or regeneration are formulated for slow release.

23. The method of claim 22, wherein the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration are encapsulated in a biocompatible and/or biodegradable polymer.

24. The method of claim 23, wherein the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration are encapsulated a nanoparticle comprising the biocompatible and/or biodegradable polymer.

25. The method of claim 24, wherein the nanoparticle is targeted to a site of low axon density or injury.

26. The method of any one of claims 24-25, wherein the nanoparticle is targeted to pericytes and/or axons.

27. The method of any one of claims 24-25, wherein the nanoparticle is administered via localized injection.

28. The method of claim 23, wherein the slow release PDGF-BB and one or more other treatments for axon growth and/or regeneration are encapsulated in a hydrogel comprising the biocompatible and/or biodegradable polymer.

29. The method of claim 28, wherein the hydrogel is targeted to a site of low axon density.

30. The method of any one of claims 28-29, wherein the hydrogel is targeted to pericytes and/or axons.

31. The method of any one of claims 28-30, wherein the hydrogel is administered via localized injection.

32. The method of any one of claims 1-31, wherein administration is repeated from about every 3 weeks to about every 6 weeks, for from about 7 months to about 12 months.

33. The method of any one of claims 1-32, wherein the PDGF-BB is administered in an amount of from about 50 pg to about 500 pg.

34. The method any one of claims 1-33, wherein the mammal is selected from the group comprising: livestock, companion animal, and human.

35. A composition comprising platelet-derived growth factor-BB (PDGF-BB) encapsulated in a nanoparticle.

36. The composition of claim 35, wherein the nanoparticle comprises a biocompatible and/or biodegradable polymer.

37. The composition of claim 36, wherein the biocompatible and/or biodegradable polymer comprises collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof.

38. The composition of any one of claims 35-37, wherein the PDGF-BB is becaplermin.

39. The composition of any one of claims 35-38, wherein the PDGF-BB is formulated for slow release.

40. The composition of any one of claims 35-39, wherein the nanoparticle further comprises one or more other treatments for axon growth and/or regeneration.

41. The composition of claim 40, wherein the one or more other treatments for axon growth and/or regeneration comprises a PTPc blocker and/or a gabapentinoid.

42. The composition of claim 41, wherein the PTPc blocker is intracellular sigma peptide

(ISP).

43. The composition of claim 41, wherein the gabapentinoid comprises gabapentin and/or pregabalin.

44. The composition of any one of claims 40-43, wherein the PDGF-BB and the one or more other treatments for axon growth and/or regeneration are formulated for slow release.

45. The composition of any one of claims 35-44, wherein the nanoparticle is targeted to a site of low axon density.

46. The composition of any one of claims 35-45, wherein the nanoparticle is targeted to pericytes and/or axons.

47. A composition comprising platelet-derived growth factor-BB (PDGF-BB) encapsulated in a hydrogel.

48. The composition of claim 47, wherein the hydrogel comprises a biocompatible and/or biodegradable polymer.

49. The composition of claim 48, wherein the biocompatible and/or biodegradable polymer comprises collagen, gelatin, hyaluronic acid, chitosan, polyethylene glycol, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, polypropylene fumarate, polyvinyl alcohol, or any combination thereof.

50. The composition of any one of claims 47-49, wherein the PDGF-BB is becaplermin.

51. The composition of any one of claims 47-50, wherein the PDGF-BB is formulated for slow release.

52. The composition of any one of claims 47-51, wherein the hydrogel further comprises one or more other treatments for axon growth and/or regeneration.

53. The composition of claim 52, wherein the one or more other treatments for axon growth and/or regeneration comprises a PTPc blocker and/or a gabapentinoid.

54. The composition of claim 53, wherein the PTPc blocker is intracellular sigma peptide (ISP).

55. The composition of claim 53, wherein the gabapentinoid comprises gabapentin and/or pregabalin.

56. The composition of any one of claims 52-55, wherein the PDGF-BB and the one or more other treatments for axon growth and/or regeneration are formulated for slow release.

57. The composition of any one of claims 47-56, wherein the hydrogel is targeted to a site of low axon density.

58. The composition of any one of claims 47-57, wherein the nanoparticle is targeted to pericytes and/or axons.

59. A method to downregulate type I collagen in a mammal in need thereof, the method comprising: a) diagnosing a mammal as being in need of downregulating type I collagen; and b) administering platelet-derived growth factor-BB (PDGF-BB) to the mammal.

60. The method of claim 59, wherein the PDGF-BB is becaplermin.

61. The method of any one of claims 59-60, wherein the mammal has rheumatoid arthritis, dermatomyositis, or scleroderma.