WO2024096877A1

WO2024096877A1 - Beta-glucan treatment to promote axon regeneration

Info

Publication number: WO2024096877A1
Application number: PCT/US2022/048719
Authority: WO
Inventors: Steven KAREL; Roman Giger; Ryan PASSINO; Stephen Smith
Original assignee: Biothera Inc dba Immuno Research Inc
Current assignee: Biothera Inc dba Immuno Research Inc
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2024-05-10
Anticipated expiration: 2025-05-02

Abstract

Intra-ocular injection of polymeric β(1,3; 1,6)-glucan (β-glucan) triggers a strong immune response in the eye. When combined with retro-orbital optic nerve crush injury (ONC), β-glucan promotes lengthy regeneration of severed retinal ganglion cell (RGC) axons.

Description

BETA-GLUCAN TREATMENT TO PROMOTE AXON REGENERATION

BACKGROUND

Broadly speaking, the vertebrate nervous system includes two major divisions: the central nervous system (CNS), and the peripheral nervous system (PNS). The major structures of the CNS are the brain and spinal cord. The retina, optic nerve, olfactory nerves, and olfactory epithelium are sometimes also considered parts of the CNS in vertebrates. At the cellular level, the nervous system is characterized primarily by various types of neurons. Neurons include projections called axons through which neurons can communicate with other cells. When a neuron sends a signal, an electrochemical action potential progresses along the axon, and the signal is transmitted to a neighboring cell at a synapse. The nervous system also includes neuroglial cells, or glial cells, which provide supporting functions to the nervous system. In the CNS, glial cells include astrocytes, microglia, oligodendrocytes, radial glial cells, and ependymal cells.

Injury or damage to neurons of the adult mammalian CNS typically results in permanent functional deficits. In general, severed neuronal axons — for example, as may be caused by traumatic injury to the neuron — fail to undergo spontaneous regeneration. The lack of significant or sustained regeneration by damaged CNS axons is at least partially responsible for the poor clinical outcomes that are observed following brain or spinal cord injuries. Currently, there are no treatments available to improve these outcomes in humans or animal models. Both neuronal intrinsic and extrinsic mechanisms pose barriers to axon regeneration and efficient CNS repair.

SUMMARY

Intra-ocular injection of polymeric P(l,3; l,6)-glucan (P-glucan) triggers a strong immune response in the eye. When combined with retro-orbital optic nerve crush injury (ONC), P-glucan promotes lengthy regeneration of severed retinal ganglion cell (RGC) axons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an eye.

FIG. 2 is a schematic view of a retina of the eye shown in FIG. 1.

FIG. 3A is an image of a cholera toxin subunit B (CTB-555) tracing of retinal ganglion cell (RGC) axons in a naive optic nerve of a wild-type mouse.

FIG. 3B is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury (ONC). FIG. 3C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 4 is a high magnification image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury and intraocular injection of P-glucan.

FIG. 5A is a plot of a single-cell RNA sequencing of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury (ONC).

FIG. 5B is a plot of a single-cell RNA sequencing of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intraocular injection of P-glucan.

FIG. 6A is a pie chart of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury.

FIG. 6B is a pie chart of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 7A is a gene expression heatmap of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury.

FIG. 7B is a gene expression heatmap of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 8A is a plot of a single-cell RNA sequencing of immune cells that express integrin aM/CDl lb in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury.

FIG. 8B is a plot of a single-cell RNA sequencing of immune cells that express integrin aM/CDl lb in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 9 A is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type (WT) mouse 14 days following optic nerve crush injury (ONC) and intraocular injection of P-glucan.

FIG. 9B is an image of a CTB-555 tracing of RGC axons in an optic nerve of a CD 11b knock out (KO) mouse 14 days following optic nerve crush injury and intraocular injection of P-glucan. FIG. 9C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a CD1 lb KO mouse 14 days following optic nerve crush injury, intra-ocular injection of P-glucan, and treatment with PLX5622.

FIG. 9D is an image of a CTB-555 tracing of RGC axons in an optic nerve of a CDllb KO mouse 14 days following optic nerve crush injury.

FIG. 10 is a graph showing RGC axon density per optic nerve based on a distance from an optic nerve crush lesion.

FIG. 11 A is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type (WT) mouse 14 days following optic nerve crush injury (ONC) and intraocular injection of P-glucan.

FIG. 1 IB is an image of a CTB-555 tracing of RGC axons in an optic nerve of a C3 knock out (KO) mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 11C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a Clq KO mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan.

FIG. 12 is a graph showing RGC axon density per optic nerve based on a distance from an optic nerve crush lesion.

FIG. 13A is a dot plot showing results of flow cytometry analysis of a retina and vitreous of a wild-type (WT) mouse 3 days following intra-ocular injection of P-glucan.

FIG. 13B is a dot plot showing results of flow cytometry analysis of a retina and vitreous of a CDllb knock out (KO) mouse 3 days following intra-ocular injection of P-glucan.

FIG. 14A is a graph showing myeloid cells per retina for a wild- type (WT) mouse and a CDllb knock out (KO) mouse 3 days following intra-ocular injection of P- glucan.

FIG. 14B is a graph showing microglia cells per retina for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan.

FIG. 14C is a graph showing neutrophils cells per retina for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan.

FIG. 14D is a graph showing monocyte/macrophage cells per retina for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P- glucan. FIG. 15 A is a graph showing myeloid cells per optic nerve for a wild-type (WT) mouse and a CD 11b knock out (KO) mouse 3 days following intra-ocular injection of P-glucan.

FIG. 15B is a graph showing microglia cells per optic nerve for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan.

FIG. 15C is a graph showing neutrophils cells per optic nerve for a wildtype mouse and a CD1 lb KO mouse 3 days following intra-ocular injection of P-glucan.

FIG. 15D is a graph showing monocyte/macrophage cells per optic nerve for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan.

DETAILED DESCRIPTION

The immune system and the nervous system are in constant dialogue, and this interaction is particularly intense following nervous system injury. Accordingly, the immune system provides a potential portal for altering CNS repair. However, it is unknown whether communication between the immune system and the nervous system occurs directly between immune components and neurons or whether intermediaries are involved, such as astrocytes, for example. Identifying and mitigating cellular and/or molecular mechanisms that limit axonal regeneration and nervous system repair, or, on the other hand, identifying and exploiting mechanisms that promote axonal regeneration and nervous system repair could have significance for improving the outcome of nervous system injuries and disease. The beta-glucan (P-glucan) treatment described herein is a method for promoting axonal regeneration in an injured optic nerve.

As described in K.T. Baldwin et al., Neuroinflammation triggered by fi- glucan/dectin-1 signaling enables CNS axon regeneration, 112 PNAS 2581 (2015), there is accumulating evidence that, under certain circumstances, endogenous CNS repair mechanisms, including those that facilitate neuronal regeneration, can be unleashed by inducing a local innate immune response. However, the underlying cellular and molecular mechanisms that are involved in promoting CNS repair are not well understood.

Retro-orbital optic nerve crush (ONC) is a widely used paradigm for investigating factors that can influence axonal growth (i.e., axonal regeneration) in the injured CNS. Consistent with outcomes from CNS injuries described above, retinal ganglion cells (RGCs) (the retinal neurons that give rise to the optic nerve) do not normally regenerate lengthy axons beyond the injury site after ONC. However, robust RGC axonal growth has been observed in ONC models after induction of intraocular inflammation. Intraocular inflammation can be induced in models, for example, via lens trauma, intraocular injection of lipopolysaccharide (LPS) (a bacterial cell wall component), intraocular injection of zymosan (a yeast cell wall extract) or constituents of zymosan that are classified as pathogen-associated molecular patterns (PAMPs), or other methods.

PAMPs are small molecular motifs that are highly conserved within a class of microbes and serve as ligands for pattern recognition receptors (PRRs). PRRs are widely expressed on cells of the innate immune system, such as monocytes, macrophages, neutrophils, and myeloid dendritic cells. PRRs for zymosan include Toll-like receptors (TLRs) 1 and 2, complement receptor 3 (CR3), and the C-type lectin family members CLEC7A (dectin-1) and CLEC6A (dectin-2). Activation of PRRs by PAMP-PRR interactions induces phagocytosis, oxidative burst, and cytokine and chemokine production.

Beta-glucans (P-glucans) are a class of PAMPs that are known to interact with the PRRs CR3 and dectin-1, among others. More generally, P-glucans comprise a group of P-D-glucose polysaccharides that occur naturally in the cell walls of cereals (e.g., oats), bacteria, and fungi. Beta-glucans can have significantly different physicochemical properties and biological activity, dependent on source. For example, some P-glucans are soluble, and others are insoluble. Typically, P-glucans have a linear backbone consisting of P(1 ,3) glycosidic bonds, though some P-glucans also have P(l,6) side chains or P(l,4) bonds. The P-glucans from bacterial cell walls are typically P(l,3)-glucans. Zymosan can consist of about 40-50% P( 1,3; l,6)-glucans.

Baldwin et al. (2015) showed that zymosan can elicit axonal regeneration through dectin-1. The dectin-1 ligand curdlan (a particulate form of P(l,3)-glucan) was similarly found to promote axonal regeneration in a dectin-1 -dependent manner. Baldwin et al. (2015) also showed that intra-ocular LPS triggers an immune response that does not support RGC axon regeneration. According to techniques of this disclosure, intra-ocular injection of polymeric P( 1 ,3 ; l,6)-glucan triggers a strong immune response in the eye and promotes lengthy regeneration of severed RGC axons following retro-orbital optic nerve crush injury.

The P-glucan treatment described herein includes the intra-ocular injection of P-glucan into a posterior chamber of the eye following optic nerve crush injury to elicit RGC axon regeneration. It should be understood that in other examples, the P-glucan treatment can include exposing the immune system and/or the nervous system (e.g., the central nervous system) to P-glucan via some other suitable route. For example, the P- glucan could be injected into a biological component, such as a tissue, organ, or structure that is part of a tissue or organ, that is in proximity to the immune system and/or the nervous system. Proximity can be defined either directly based on physical location or indirectly based on communication between the biological component and the immune system and/or the nervous system. In other examples, the P-glucan may not be injected and may instead by provided or administered in a different form suitable for allowing the P-glucan to interact with the immune system and/or the nervous system.

The P-glucan composition used in the testing described herein with reference to FIGS. 1-15D below is a purified insoluble particulate P(l,3; l,6)-glucan from Saccharomyces cerevisiae (yeast) in a suspension at a given concentration. For purposes of clarity and simplicity, the P-glucan composition used in the testing described herein will be referred to generally as “P-glucan” or “purified P-glucan” in the following description. The P-glucan can be isolated from any suitable source, including from yeast, bacteria, or another organism, or can be derivatized P-glucan. In some examples, the P-glucan can be an insoluble particulate. In other examples, the P-glucan can be soluble. The purified P- glucan can have a purity of 75% or greater (i.e., can consist of 75% or greater P-glucan). In some examples, the P-glucan can have a purity of 80% or greater (i.e., can consist of 80% or greater P-glucan). In some examples, the P-glucan can have a purity of 90% or greater (i.e., can consist of 90% or greater P-glucan). Additionally, the purified P-glucan can be in a suspension (for insoluble particulates) or solution (for soluble particulates) with a concentration that is greater than 10 mg/ml. At relatively higher concentrations, the P- glucan composition becomes more viscous, or gel-like. For example, the concentration of the P-glucan can be 25 mg/ml. Specifically, the intra-ocular injection of P-glucan in the testing described herein included 1-2 pl of the purified P-glucan at 25 mg/ml in phosphate- buffered saline (PBS) that was injected into the posterior chamber of an eye following optic nerve crush injury.

Axonal regeneration elicited by the P-glucan used in the testing described herein is generally more reproduceable and more robust compared to previous work with zymosan. That is, there can be a greater density of regenerated axons and/or regenerated axons can extend a greater distance beyond the site of injury following the P-glucan treatment described herein compared to treatment with zymosan. The phenomenon of improved RGC axon regeneration following treatment with the P-glucan compared to zymosan is not fully understood. However, zymosan is a crude yeast cell wall extract with constituents that may bind to several different receptors. There can also be tremendous batch-to-batch variation in zymosan. The P-glucan may elicit a stronger response in the absence of conflicting or interfering signals from the different components of zymosan. For example, there are likely many ligands present in zymosan that have no influence on axonal regeneration. To some extent, there may also be a dosing phenomenon that causes the immune response to activate repair mechanisms when a certain amount of P-glucan is present. For example, a larger quantity of P-glucan may saturate some receptors and/or permit some P-glucan to bind other receptors. Alternatively, the insoluble particulates in the concentrated, gel-like P-glucan suspension may stay in one location longer, or may not be cleared away, such that some other mechanism of action is triggered that may differ from the response to treatment with a soluble P-glucan. Any one or more of these factors, or other factors not listed here, may participate in producing the improved RGC axon regeneration seen following the P-glucan treatment described herein.

For an in-depth analysis of the P-glucan-triggered immune response, mouse retinas and optic nerves were micro-dissected 3 days after optic nerve crush injury and intra-ocular injection of P-glucan or phosphate-buffered saline (PBS). Innate immune cells were isolated by immunopanning and subjected to single cell RNA-sequencing (scRNA- seq). A comparative analysis revealed that the retinal/intra-ocular immune milieu in P- glucan treated mice is dominated by neutrophils, monocytes/macrophages, and few microglia. In the injured optic nerve, microglia greatly outnumber macrophages, and seven different subpopulations were identified. Pharmacological ablation of microglia with the CSF1R inhibitor PLX56622 attenuated P-glucan elicited optic nerve regeneration, suggesting that microglia are necessary for immune-mediated axon regeneration.

Differential gene expression and pathway analysis of scRNA-seq data under regenerating and non-regenerating conditions identified complement activation as one of the top hits. The complement receptor 3 (CR3) is composed of two subunits CDllb (integrin aM) and CD 18 (integrin P2). Because integrin aM directly binds to P-glucan, It gam-/- mice (genetically modified mice with a global, i.e., germline, knock out (KO) of the integrin aM gene such that Itgam is not expressed) were subjected to intro-ocular P- glucan injection and optic nerve crush injury. Itgam-/- mice model inhibition (i.e., inactivation) or disruption of CR3. It should be noted that Itgam-/- mice are also referred to herein as CDllb KO and CR3 KO mice. Surprisingly, optic nerves from Itgam-/- mice showed significant increased axon regeneration when compared to parallel processed wildtype (WT) nerves. Increased RGC regeneration in Itgam-/- mice was blocked when combined with PLX5622 treatment. The complement component C3 is strongly upregulated in the retina of P-glucan injected mice. However, the increased RGC regeneration observed in Itgam-/- mice is not due to disruption of the CR3 receptor, since C3-/- mice failed to mimic the RGC regeneration phenotype observed in Itgam-/- mice.

FIG. 1 is a schematic view of an eye. FIG. 2 is a schematic view of a retina of the eye shown in FIG. 1. FIGS. 1-2 will be discussed together.

The eye (e.g., a mouse eye, as shown in FIG. 1, or a human eye) includes a lens, a vitreous chamber, a retina, and an optic nerve. As shown in FIG. 2, the retina includes multiple layers, including photoreceptors (rods and cones), bipolar cells, and retinal ganglion cells (RGCs). The retina is a light-sensitive layer of the eye. The retina extends over an interior surface of a globe portion of the eye that defines the vitreous chamber therein. The vitreous chamber is located behind the lens and so is a relatively posterior chamber of the eye. With respect to the direction light enters the eye, the retina is located at a back or rear portion of the eye. The optic nerve extends posteriorly from the globe of the eye and is formed of RGC axons. Though not shown in FIG. 1 , the optic nerve connects the eye to the brain.

Light entering the eye passes through the lens and is focused on a portion of the retina. The retinal photoreceptors sense light, and the RGCs receive sensory input from the photoreceptors. RGC axons in turn relay visual input to the brain via the optic nerve. Damage to the RGCs or optic nerve can cause permanent vision loss (e.g., vision loss due to glaucoma, etc.).

FIG. 3A is an image of a cholera toxin subunit B (CTB-555) tracing of retinal ganglion cell (RGC) axons in a naive optic nerve of a wild-type mouse. FIG. 3B is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury (ONC). FIG. 3C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild- type mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIG. 4 is a high magnification image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury and intra-ocular injection of -glucan. FIGS. 3A-4 will be discussed together.

FIGS. 3A-4 are tracings of RGC axons in optic nerves of wild- type mice. The tracings were completed using cholera toxin subunit B conjugated to a chromophore with emission wavelengths of 555 nm (CTB-555). FIGS. 3A-3C each show a longitudinal section through an optic nerve. The white portions of FIGS. 3A-4 represent the RGC axons. FIG. 3A shows RGC axons in a naive optic nerve (a nerve that has not been injured). FIG. 3A shows the high density of RGC axons extending through the naive optic nerve.

FIG. 3B shows RGC axons in an optic nerve 14 days following optic nerve crush injury. Optic nerve crush injury was performed in vivo by surgically crushing the optic nerve just behind the eye. As shown in FIG. 3B, there are very few, if any, RGC axons extending beyond the site of the optic nerve crush injury lesion, showing that the RGC axons do not spontaneously regenerate following optic nerve crush injury.

FIGS. 3C-4 show RGC axons in an optic nerve 14 days following optic nerve crush injury and the intra-ocular injection of P-glucan. The intra-ocular injection of P-glucan referred to herein included 1-2 pl of P-glucan at 25 mg/ml in phosphate-buffered saline (PBS) that was injected into the posterior chamber of the eye following optic nerve crush injury. As shown in FIGS. 3C-4, the P-glucan promotes regrowth of the RGC axons in the optic nerve. FIG. 4 is a high magnification image showing more detail of the regenerated RGC axons. The RGC axons shown in FIG. 4 have characteristic wavy or curved paths that are indicative of regeneration as opposed to imaging artifacts. Comparing FIG. 3C to FIG. 3B, there is significant regrowth of the RGC axons in the optic nerve following optic nerve crush injury due to the intra-ocular injection of P-glucan. This shows that the intra-ocular injection of P-glucan promotes optic nerve axon regeneration. Optic nerve crush injury without intra-ocular P-glucan does not promote axonal regeneration.

FIG. 5A is a plot of a single-cell RNA sequencing of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury (ONC). FIG. 5B is a plot of a single-cell RNA sequencing of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIGS. 5A-5B will be discussed together.

Together, FIGS. 5A and 5B represent a comparative analysis of the intraocular immune response following optic nerve crush injury and injection of PBS (saline) (i.e., non-regenerative conditions) versus optic nerve crush injury and injection of P-glucan (i.e., regenerative conditions). In FIGS. 5A and 5B, each plotted point can represent a single retinal cell or infiltrating immune cell that has been sequenced by single-cell RNA sequencing. Plotted points are distributed in clusters based on the single-cell RNA sequencing data. The clusters can correspond to different cell types and subpopulations. More specifically, raw data from sequenced cells is aligned with a mouse reference genome and principal components that represent alike cells are identified. A dimension reduction is performed on the top principal components to generate a UMAP plot. The UMAP plot is a three-dimensional construct of the principal components projected onto a two- dimensional format. The UMAP plot is manually overlaid with feature plots of marker genes for known cell types to identify cell types corresponding to each cluster in the UMAP plot.

For example, FIGS. 5A and 5B show clusters of immune cell types, including monocytes, macrophages, microglia, neutrophils, granulocytes, T cells, natural killer (NK) cells, and oligodendrocyte progenitor cells. The clusters in FIG. 5A can represent immune cell types that are present in the retina and/or the posterior chamber of the eye 3 days following optic nerve crush injury. The clusters in FIG. 5B can represent immune cell types that are present in the retina and/or the posterior chamber of the eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan.

Cluster sizes corresponding to neutrophils and granulocytes are increased following intra-ocular injection of P-glucan (FIG. 5B) compared to optic nerve crush injury alone (FIG. 5A). A cluster size corresponding to microglia is decreased following intraocular injection of P-glucan (FIG. 5B) compared to optic nerve crush injury alone (FIG. 5 A). Thus, there is a difference in the intra-ocular inflammatory response of mice that were subjected to optic nerve crush injury and received an injection of P-glucan (i.e., regenerative conditions) compared to mice that underwent optic nerve crush injury alone (i.e., non-regenerative conditions), though the significance of this difference is not yet fully understood. The most apparent differences are the increase in granulocytes and the decrease in microglia following intra-ocular P-glucan injection. It is possible that all the changes in the inflammatory response that are observed following intra-ocular P-glucan injection (e.g., comparing FIG. 5A to FIG. 5B) are necessary to drive axonal regeneration, though any one or more of the changes could be sufficient. Characterizing the change in the intra-ocular inflammatory response under regenerative conditions could lead to identification of cell types or signaling pathways that may be involved in promoting P- glucan-mediated axonal regeneration.

FIG. 6A is a pie chart of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury. FIG. 6B is a pie chart of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIGS. 6A-6B will be discussed together.

As shown in FIG. 6 A, a total of 3,916 cells from the retina and/or posterior chamber of an eye were characterized following optic nerve crush injury. About 1% of the characterized cells were classified as monocytes, about 26% were classified as macrophages, about 64% were classified as microglia, about 4% were classified as neutrophils, and about 5% were classified as granulocytes. As shown in FIG. 6B, a total of 4,274 cells from the retina and/or the posterior chamber of an eye were characterized following optic nerve crush injury and intra-ocular injection of P-glucan. About 2% of the characterized cells were classified as monocytes, about 28% were classified as macrophages, about 13% were classified as microglia, about 23% were classified as neutrophils, and about 34% were classified as granulocytes.

Compared to the intra-ocular immune cell profile after optic nerve crush injury alone (FIG. 6A), the percentages of neutrophils and granulocytes were increased, and the percentage of microglia was decreased following optic nerve crush injury and intraocular injection of P-glucan (FIG. 6B). The percentages of monocytes and macrophages were similar after optic nerve crush injury and after optic nerve crush injury and intraocular injection of P-glucan. The data displayed in FIGS. 6A and 6B further quantifies the difference in the intra-ocular immune cell profile of mice that were subjected to optic nerve crush injury and received an injection of P-glucan (i.e., regenerative conditions) compared to mice that underwent optic nerve crush injury alone (i.e., non-regenerative conditions). Regenerative conditions are associated with increased neutrophils and granulocytes and decreased microglia compared to non-regenerative conditions.

FIG. 7 A is a gene expression heatmap of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury. FIG. 7B is a gene expression heatmap of immune cells in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIGS. 7A-7B will be discussed together.

In FIGS. 7 A and 7B, the rows (Itgam to C3arl) correspond to differentially expressed genes, and the columns represent individual cell samples that have been organized, or clustered, based on different cell types (e.g., immune cell types, such as macrophages, microglia, and neutrophils). The genes Itgam, Itgax, and Itgb2 each code for protein subunits of complement receptor 3 (CR3). The genes Clqa, Clqb, Clqbp, Clqc, C3, and C3arl each code for complement components or protein subunits of complement components.

Levels of gene expression are color-coded according to whether expression is relatively increased or decreased over the averaged gene expression in all the cells that were analyzed. Yellow indicates increased expression, black indicates no or minimal change in expression, and pink indicates decreased expression. The color of each tile that corresponds to an individual cell and a gene indicates the relative level of expression of the gene in the individual cell sample. The level of expression of the genes Itgam, Itgax, Itgb2, Clqa, Clqb, Clqbp, Clqc, C3, and C3arl was compared between samples following optic nerve crush injury (FIG. 7A) and samples following optic nerve crush injury and intraocular injection of P-glucan (FIG. 7B). The samples were organized/clustered into groups corresponding to macrophages, microglia, and neutrophils.

The expression profile for Itgam, Itgax, Itgb2, Clqa, Clqb, Clqbp, Clqc, C3, and C3arl is different after optic nerve crush injury alone (FIG. 7A) compared to after optic nerve crush injury and intra-ocular injection of P-glucan (FIG. 7B). The different level of expression of the various genes that can be seen by comparing FIGS. 7 A and 7B indicates that integrin aM/CDllb and complement components are regulated by intraocular P-glucan injection in an optic nerve crush model. The complement cascade has been previously shown to be involved in neurodegenerative diseases, such as Alzheimer’s disease, so it is possible that complement components might also negatively influence neuronal regeneration.

FIG. 8A is a plot of a single-cell RNA sequencing of immune cells that express integrin aM/CDl lb in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury. FIG. 8B is a plot of a single-cell RNA sequencing of immune cells that express integrin aM/CDl lb in a retina and/or posterior chamber of an eye 3 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIGS. 8A-8B will be discussed together.

In FIGS. 8 A and 8B, each plotted point can represent a single sequenced cell (e.g., by single-cell RNA sequencing) that expresses the Itgam gene (integrin aM/CDllb). The UMAP plots in FIGS. 8A and 8B were generated similarly to the plots in FIGS. 5A and 5B and are based on the same single-cell RNA sequencing data. The UMAP plots in FIGS. 8 A and 8B were overlaid with a feature plot showing expression of the Itgam gene.

As can be seen by comparing FIGS. 8 A and 8B, the number of cells that express Itgam increases after intra-ocular injection of P-glucan compared to optic nerve crush injury alone. Identifying Itgam as a top differentially expressed gene under the regenerative conditions described herein is interesting because integrin aM is known to bind P-glucan. Increased Itgam expression under regenerative conditions, therefore, suggests that integrin aM could be involved in a signaling pathway or other mechanism that is associated with P-glucan-mediated axonal regeneration.

FIG. 9 A is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type mouse 14 days following optic nerve crush injury (ONC) and intra-ocular injection of P-glucan. FIG. 9B is an image of a CTB-555 tracing of RGC axons in an optic nerve of a CDllb knock out (KO) mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIG. 9C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a CDllb KO mouse 14 days following optic nerve crush injury, intraocular injection of P-glucan, and treatment with PLX5622. FIG. 9D is an image of a CTB- 555 tracing of RGC axons in an optic nerve of a CD1 lb KO mouse 14 days following optic nerve crush injury. FIGS. 9A-9D will be discussed together.

FIGS. 9A-9D are all tracings of RGC axons in optic nerves following optic nerve crush injury. The tracings were completed using cholera toxin subunit B (CTB-555). FIGS. 9A-9D each show a longitudinal section through an optic nerve. The white portions of FIGS. 9A-9D represent the RGC axons.

FIG. 9 A shows RGC axons in an optic nerve of a wild-type (WT) mouse 14 days following optic nerve crush injury and the intra-ocular injection of P-glucan. As shown in FIG. 9A, there has been regrowth of the RGC axons following the intra-ocular injection of P-glucan.

FIG. 9B shows RGC axons in an optic nerve of a CD1 lb KO mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan. As shown in FIG. 9B, there has been significant regrowth of the RGC axons in the CDllb KO mouse following the intra-ocular injection of P-glucan. Comparing FIG. 9B to FIG. 9A, there is better regrowth of RGC axons in the CDllb KO mouse (FIG. 9B) compared to the wildtype mouse (FIG. 9A) following intra-ocular injection of P-glucan.

FIG. 9C shows RGC axons in an optic nerve of a CD1 lb KO mouse 14 days following optic nerve crush injury, the intra-ocular injection of P-glucan, and treatment with PLX5622. PLX5622 is a pharmacological inhibitor of CSF1R that can cross the blood-retina barrier. Microglia need CSF1R to survive, so treatment with PLX5266 (e.g., PLX5266 can be provided in chow) can ablate retinal microglia. Comparing FIG. 9C to FIG. 9B, there is less regrowth of the RGC axons following treatment with PLX5622 (FIG. 9C). In other words, the enhanced effect on axonal regeneration that is seen in the CDllb KO mouse is attenuated when the mice are treated with PLX5622. This suggests that P- glucan-mediated axonal regeneration is at least partially due to CSF1R activation or involvement of retinal microglia. Comparing FIG. 9C to FIG. 9A, there is also less regrowth of the RGC axons in a CD 11b KO mouse following intra-ocular injection of P- glucan and treatment with PLX5622 (FIG. 9C) than is seen in a wild-type mouse following intra-ocular injection of -glucan (FIG. 9A).

FIG. 9D shows RGC axons in an optic nerve of a CD1 lb KO mouse 14 days following optic nerve crush injury. Comparing FIG. 9D to FIGS. 9B-9C, there is the least amount of RGC axon regrowth following optic nerve crush injury with no further intervention (i.e., intra-ocular injection of -glucan and/or treatment with PLX5622).

The improved axonal regeneration seen in CD 11b KO mice (FIG. 9B) compared to parallel processed wild-type mice (FIG. 9A) suggests that the interaction between CDllb and P-glucan or signaling downstream of CDllb may have some type of negative, or inhibitory, effect on P-glucan-mediated axonal regeneration. This result also suggests that there may be a P-glucan that could preferentially bind and act to promote axonal regeneration through a receptor other than CR3/CDllb/integrin aM, such as, e.g., dectin-1. Increased axonal regeneration in the CDllb KO mouse compared to the wildtype mouse is unexpected because, as discussed above, P-glucan is known to bind CR3/CDllb/integrin aM.

The vertical axis represents a number of RGC axons per optic nerve. The horizontal axis represents a distance the RGC axons extended from the optic nerve crush lesion site as measured in micrometers (pm). The number of RGC axons was quantified at 500 pm, 1000 pm, 1500 pm, and 2000 pm for each sample group. RGC axons were measured from wild-type (WT) mice 14 days following optic nerve crush injury and intraocular injection of P-glucan; CR3 knock-out (KO) mice 14 days following optic nerve crush injury and intra-ocular injection of P-glucan; and CR3 KO mice 14 days following optic nerve crush injury, intra-ocular injection of P-glucan, and treatment with PLX5622. Statistical significance was determined with a one-way ANOVA test.

At the 500 pm length, there were significantly more RGC axons measured from CR3 KO mice following optic nerve crush injury and intra-ocular injection of P- glucan than from WT mice following optic nerve crush injury and intra-ocular injection of P-glucan. At the 1000 pm and 1500 pm lengths, there were significantly more RGC axons measured from CR3 KO mice following optic nerve crush injury and intra-ocular injection of P-glucan than from WT mice following optic nerve crush injury and intra-ocular injection of P-glucan and CR3 KO mice following optic nerve crush injury, intra-ocular injection of P-glucan, and treatment with PLX5622. At the 2000 pm length, there were significantly more RGC axons measured from CR3 KO mice following optic nerve crush injury and intra-ocular injection of P-glucan than from CR3 KO mice following optic nerve crush injury, intra-ocular injection of P-glucan, and treatment with PLX5622.

The graph in FIG. 10 further quantifies the difference in axonal regeneration shown in FIGS. 9A-9C. For almost all the combinations of samples and lengths measured, there were significantly more axons in the optic nerves from CR3 KO mice that underwent optic nerve crush injury and injection of P-glucan compared to the other two sample types. This shows that, when CR3 is knocked out, P-glucan-mediated axonal regeneration is improved over a baseline level of regeneration that is seen in wild-type mouse optic nerves under the regenerative conditions described herein.

FIG. 11 A is an image of a CTB-555 tracing of RGC axons in an optic nerve of a wild-type (WT) mouse 14 days following optic nerve crush injury (ONC) and intraocular injection of P-glucan. FIG. 1 IB is an image of a CTB-555 tracing of RGC axons in an optic nerve of a C3 knock out (KO) mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIG. 11C is an image of a CTB-555 tracing of RGC axons in an optic nerve of a Clq KO mouse 14 days following optic nerve crush injury and intra-ocular injection of P-glucan. FIGS. 11A-11C will be discussed together.

FIGS. 11A-11C are all tracings of RGC axons in optic nerves 14 days following optic nerve crush injury and the intra-ocular injection of P-glucan. The tracings were completed using cholera toxin subunit B (CTB-555). FIGS. 11A-11C each show a longitudinal section through an optic nerve. The white portions of FIGS. 11A-11C represent the RGC axons.

FIG. 11A shows RGC axons in an optic nerve of a wild-type (WT) mouse. FIG. 1 IB shows RGC axons in an optic nerve of a C3 knock out (KO) mouse (genetically modified mice with a global, i.e., germline, knock out of the C3 gene such that C3 is not expressed). FIG. 11C shows RGC axons in an optic nerve of a Clq KO mouse (genetically modified mice with a global, i.e., germline, knock out of the Clq gene such that Clq is not expressed). C3 and Clq are complement components. The complement cascade is a series of enzymatic reactions that occurs during an inflammatory response, and complement components can bind or tag entities to induce phagocytosis. C3 and Clq are also ligands for CD 11b. FIGS . 11 A- 11 C show the difference in RGC axon regrowth between a wildtype mouse, a C3 KO mouse, and a Clq KO mouse. Comparing FIGS. 11A-11C, there was no significant difference in the RGC axon regrowth in the wild-type mouse, the C3 KO mouse, and the Clq KO mouse. Thus, deletion of C3 or deletion of Clq does not mimic the pro-regenerative effects observed in CD 11b KO mice. This shows that P-glucan- mediated axonal regeneration is independent of C3 and Clq (i.e., Clq and C3 are not required for RGC axon regeneration elicited by P-glucan). This also indicates that the enhanced axonal regeneration seen in CD 11b KO mice is not due to a change in the downstream signaling via C3 or Clq.

The vertical axis represents a number of RGC axons per optic nerve. The horizontal axis represents a distance the RGC axons extended from the optic nerve crush lesion site as measured in micrometers (pm). The number of RGC axons was quantified at 500 pm, 1000 pm, 1500 pm, and 2000 pm for each sample group. Statistical significance was determined with a one-way ANOVA test. RGC axons were measured from wild-type (WT) mice 14 days following optic nerve crush injury and intra-ocular injection of P- glucan; C3 knock-out (KO) mice 14 days following optic nerve crush injury and intraocular injection of P-glucan; and Clq KO mice 14 days following optic nerve crush injury, intra-ocular injection of P-glucan.

The graph in FIG. 12 further quantifies the axonal regeneration shown in FIGS. 11A-11C. There was no significant difference in the number of axons between any of the sample groups at any of the lengths. This shows that Clq and C3 are not required for P-glucan-elicited RGC axon regeneration.

FIG. 13A is a dot plot showing results of flow cytometry analysis of a retina and vitreous of a wild-type (WT) mouse 3 days following intra-ocular injection of P-glucan. FIG. 13B is a dot plot showing results of flow cytometry analysis of a retina and vitreous of a CDllb knock out (KO) mouse 3 days following intra-ocular injection of P-glucan. FIGS. 13A-13B will be discussed together.

The y-axis of the plots in FIGS. 13A and 13B represents CDllb antibody labeled cells. The x-axis of the plots in FIGS. 13 A and 13B represents CD45 antibody labeled cells. Cells that are double-positive (i.e., positive for CDllb and CD45) are seen in the upper right-hand portion of the plots. Double-positive cells are innate immune cells. Cells that are double-negative (i.e., negative for CDllb and CD45) are seen in the lower left-hand portion of the plots. Double-negative cells are non-immune cells. In FIG. 13B, a negligible number of cells express CD 11b and CD45. Taken together, the plots in FIGS. 13A and 13B show that the CD1 lb KO mice do not express Itgam (integrin aM/DCl lb).

FIG. 14A is a graph showing myeloid cells per retina for a wild- type (WT) mouse and a CDllb knock out (KO) mouse 3 days following intra-ocular injection of P- glucan. FIG. 14B is a graph showing microglia cells per retina for a wild- type mouse and a CDllb KO mouse 3 days following intra-ocular injection of -glucan. FIG. 14C is a graph showing neutrophils cells per retina for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan. FIG. 14D is a graph showing monocyte/macrophage cells per retina for a wild-type mouse and a CDllb KO mouse 3 days following intra-ocular injection of P-glucan. FIGS. 14A-14D will be discussed together.

FIGS. 14A-14D each show the number of cells per retina for a wild-type (WT) mouse and a CDllb knock out (KO) mouse 3 days following intra-ocular injection of P-glucan. The vertical axis of each graph in FIGS. 14A-14D represents a number of cells (myeloid, microglia, neutrophil, and monocyte/macrophage, respectively) per retina. FIG. 14A shows myeloid cells per retina. The vertical axis representing the number of myeloid cells per retina has a scale from zero to 40,000. As shown in FIG. 14A, there is a significant difference in the number of myeloid cells per retina between the wild-type mouse and the CDllb KO mouse, with the wild-type mouse having more myeloid cells. FIG. 14B shows microglia cells per retina. The vertical axis representing the number of microglia cells per retina has a scale from zero to 200. As shown in FIG. 14B, there is a significant difference in the number of microglia cells per retina between the wild-type mouse and the CDllb KO mouse, with the wild-type mouse having more microglia cells. FIG. 14C shows neutrophil cells per retina. The vertical axis representing the number of neutrophil cells per retina has a scale from zero to 40,000. As shown in FIG. 14C, there is a significant difference in the number of neutrophils cells per retina between the wild-type mouse and the CDllb KO mouse, with the wild-type mouse having more neutrophil cells. FIG. 14D shows monocyte/macrophage cells per retina. The vertical axis representing the number of monocyte/macrophage cells per retina has a scale from zero to 3,000. As shown in FIG. 14D, there was no significant difference in the number of monocyte/macrophage cells per retina between the wild-type mouse and the CDllb KO mouse.

Comparing FIGS. 14A-14D, the CDllb KO mouse affects myeloid cell trafficking to the retina. However, it is unclear if the statistical significance of the decrease in retinal microglia and neutrophils seen in the CD 11b KO mice (FIGS. 14B and 14C, respectively) indicates any biological significance.

FIG. 15 A is a graph showing myeloid cells per optic nerve for a wild-type (WT) mouse and a CD 11b knock out (KO) mouse 3 days following intra-ocular injection of P-glucan. FIG. 15B is a graph showing microglia cells per optic nerve for a wild- type mouse and a CD 11b KO mouse 3 days following intra-ocular injection of P-glucan. FIG. 15C is a graph showing neutrophils cells per optic nerve for a wild- type mouse and a CD1 lb KO mouse 3 days following intra-ocular injection of P-glucan. FIG. 15D is a graph showing monocyte/macrophage cells per optic nerve for a wild-type mouse and a CD 11b KO mouse 3 days following intra-ocular injection of P-glucan. FIGS. 15A-15D will be discussed together.

FIGS. 15A-15D each show the number of cells per optic nerve for a wildtype (WT) mouse and a CD 11b knock out (KO) mouse 3 days following intra-ocular injection of P-glucan. The vertical axis of each graph in FIGS. 15A-15D represents a number of cells (myeloid, microglia, neutrophil, and monocyte/macrophage, respectively) per optic nerve, and the vertical axis of each graph in FIGS. 15A-15D has a scale from zero to 300. FIG. 15A shows myeloid cells per optic nerve; FIG. 15B shows microglia cells per optic nerve; FIG. 15C shows neutrophil cells per optic nerve; and FIG. 15D shows monocyte/macrophage cells per optic nerve. There is not a significant difference in the number of myeloid cells, microglia cells, neutrophil cells, or monocyte/macrophage cells per optic nerve between the wild-type mouse and the CD 11b KO mouse.

Comparing FIGS. 15A-15D, the CDllb KO mouse does not affect myeloid cell trafficking to the optic nerve.

Comparing FIGS. 14A-14D to FIGS. 15A-15D, the overall number of myeloid cells in the retina (FIG. 14A) is around two orders of magnitude greater than the number of myeloid cells in the optic nerve (FIG. 15 A) for both the wild-type and CDllb KO mice. This is largely due to the number of neutrophil cells in the retina (FIG. 14C) being around two orders of magnitude greater than the number of neutrophil cells in the optic nerve (FIG. 15C) for both the wild-type and CD1 lb KO mice, though the number of monocyte/macrophage cells in the retina (FIG. 14D) is also about one order of magnitude greater than the number of monocyte/macrophage cells in the optic nerves (FIG. 15D) for both the wild-type and CD1 lb KO mice. Though not as large of a difference numerically, the number of microglia cells in the wild-type retina (FIG. 14B) is in the hundreds (the number of microglia cells in the CD1 lb KO retina is close to zero), whereas the number of microglia cells in the wild-type and CDllb KO optic nerves (FIG. 15B) is less than 100. The relative difference in the number of myeloid cells present the retina and the optic nerve may have some connection to the observed effect on myeloid cell trafficking, though any connection is not yet understood.

The testing described above with respect to FIGS. 3A-15D demonstrates that intra-ocular injection of the purified P-glucan triggers a strong immune response in the eye and promotes lengthy regeneration of severed RGC axons following retro-orbital optic nerve crush injury. The level of P-glucan-elicited RGC axon regeneration is generally improved compared to zymosan-elicited regeneration. Moreover, the P-glucan-elicited RGC axon regeneration is increased when CR3 is inhibited, as demonstrated by the testing with CR3 KO mice (FIGS .9 A- 10). Though the experiments described herein were conducted on mouse retinas and optic nerves, these experiments can serve as a model for P-glucan-elicited axon regeneration that could be seen in other mammalian central nervous system axons, e.g., spinal cord axons. Thus, the P-glucan-elicited axon regeneration described herein may have various clinical benefits. For example, the P-glucan-elicited axon regeneration described herein could be applied to treat head and spinal cord trauma; neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis, and Parkinson's disease; ocular diseases such as glaucoma, and/or other diseases or conditions for which axon regeneration has an effect.

While the invention has been described with reference to an exemplary embodiment(s), it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

CLAIMS:

1. A method of treating damaged axons, the method comprising: exposing an immune system and/or a nervous system to a P-glucan composition having a purity of P-glucan that is 75% or greater to induce axon regeneration.

2. The method of claim 1, and further comprising: inhibiting complement receptor 3 (CR3).

3. The method of claim 1, wherein exposing an immune system and/or a nervous system to the P-glucan composition having the purity of P-glucan that is 75% or greater further comprises: injecting the P-glucan composition into a biological component in proximity to the immune system and/or the nervous system.

4. The method of claim 1, wherein the purity of P-glucan is 80% or greater.

5. The method of claim 4, wherein the purity of P-glucan is 90% or greater.

6. The method of claim 1, wherein a P-glucan of the P-glucan composition is a P(l,3; l,6)-glucan isolated from a yeast.

7. The method of claim 6, wherein the yeast is Saccharomyces cerevisiae.

8. The method of claim 1, wherein the P-glucan composition is an insoluble particulate P-glucan in a suspension.

9. The method of claim 8, wherein the suspension has a concentration greater than 10 mg/ml.

10. The method of claim 9, wherein the suspension has a concentration of 25 mg/ml.