AU2015252036A1

AU2015252036A1 - Methods and compositions for treating CNS injury

Info

Publication number: AU2015252036A1
Application number: AU2015252036A
Authority: AU
Inventors: James BOURNE; Leon TEO
Original assignee: Monash University
Current assignee: Monash University
Priority date: 2015-11-03
Filing date: 2015-11-03
Publication date: 2017-05-18
Also published as: WO2017075661A1

Abstract

The present invention relates to methods and compositions for treating a condition associated with, or arising from, astrocyte dysfunction or glial scar formation or progression in an individual. The present invention is useful for, but not limited to, the 5 treatment of central nervous system (CNS) injuries. The present invention provides a method for promoting the recovery of central nervous system function in an individual having CNS injury or damage, the method comprising administering ephrin-A1 to the individual, thereby enhancing recovery of central nervous system function. The present invention also provides a method for minimising glial scarring in an individual having 10 CNS injury or damage, the method comprising administering ephrin-A1 to the individual, thereby minimising glial scarring. The invention also provides a method reducing reactive astrogliosis in an individual having CNS injury or damage, the method comprising administering ephrin-A1 to the individual, thereby reducing reactive astrogliosis.

Description

Methods and compositions for treating CNS injury

Field of the invention 2015252036 03 Nov 2015

The present invention relates to methods and compositions for treating a condition associated with, or arising from, astrocyte dysfunction or glial scar formation or 5 progression in an individual. The present invention is useful for, but not limited to, the treatment of central nervous system (CNS) injuries.

Background of the invention

Ischemic strokes are the second leading cause of brain injuries in adult humans, often leading to long-term neurological impairments. Spontaneous functional recovery after 10 adulthood strokes is rarely observed in the clinic. Even so, the options for clinical interventions remain limited and none of the >1,000 therapeutic strategies developed and trialled in rodents for the treatment of stroke have been successfully translated to the clinic. Currently, the administration of thrombolytic compounds, such as tissue plasminogen activator (tPA), remains the only treatment option available. However, the 15 use of thrombolytics imposes strict physiological criterions on patient selection in addition to the hyper-acute time limit for effective treatment. These limitations preclude the majority of stroke patients. Therefore, the development of novel pharmacotherapeutic strategies that extends the therapeutic time window and promotes functional recovery in the sub-acute period after stroke is of tremendous clinical value. 20 The formation of regenerative growth cones by neurones is evidence that the adult central nervous system (CNS) possess the intrinsic capacity for regeneration after injury. However, regenerative outgrowth fails on contact with the growth-inhibitory glial scar and ultimately degenerates resulting in retrograde degeneration of upstream neurones. The glial scar is predominantly composed of reactive astrocytes that are 25 activated from their resting state by the onset of neuropathologies. The exaggerated astrogliotic response, including recruitment of distal population and local proliferation triggered by the injury results in an increased astrocyte density juxtaposing the injury core. This ultimately leads to a fine meshwork of entangled astrocytic processes and secretion of chondroitin sulfate proteoglycans (CSPG), a potent neurite outgrowth 30 inhibitor that is a major component of the glial scar. 1 1001272724

There exists a need for new and/or improved methods and compositions for reducing glial scar formation and/or enhancing recovery after CNS injury or damage. 2015252036 03 Nov 2015

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that 5 this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

The present invention provides a method for minimising glial scarring in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant I0 or analog thereof to the individual, thereby minimising glial scarring.

The invention provides a method for reducing reactive astrogliosis in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby reducing reactive astrogliosis.

The invention provides a method of minimising glial scarring in an individual, the method 15 comprising - identifying an individual with a condition associated with, or arising from, glial scarring, - administering ephrin-A1, variant or analog thereof to the individual, thereby minimising glial scarring in the individual.

The invention also provides a method of reducing astrocyte reactivity in an individual 20 having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby reducing astrocyte reactivity in the individual.

The present invention provides a method for promoting the recovery of central nervous system function in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby enhancing 25 recovery of central nervous system function.

The invention also provides a method for treating or preventing a disorder associated with, or arising from, glial scarring in an individual, the method comprising administering 2 1001272724 ephrin-A1, variant or analog thereof to the individual, thereby treating or preventing a disorder associated with, or arising from, glial scarring in the individual. 2015252036 03 Nov 2015

The present invention provides a method minimising glial scarring in an individual having CNS injury or damage, the method comprising the step of administering a 5 composition to the subject, wherein the composition comprises, consists essentially of or consists of ephrin-A1, variant or analog thereof and a pharmaceutically acceptable diluent, excipient or carrier.

In any method or use of the invention described herein, ephrin-A1, variant or analog thereof may be administered systemically or directly at the site of injury or damage. I0 Ephrin-A1, variant or analog thereof may be formulated for systemic or topical administration. Most preferably, ephrin-A1, variant or analog thereof is applied topically. An example of topical application is delivery by osmotic or peristaltic pump locally to the site of injury. Ephrin-A1, variant or analog thereof may be administered at the injury core, area surrounding and / or juxtaposing the injury core (for example the penumbra). 15 The invention provides a method of minimising glial scar formation and/or progression at an ischemic site in brain or spinal tissue, the method comprising administering ephrin-A1, variant or analog thereof to the site or margins thereof in an amount and for a time to minimise glial scar formation and/or progression. Preferably, the ischemic site arises from occlusion of a blood vessel or brain-supplying artery such as one of the >0 major cerebral arteries (for example the middle or posterior cerebral artery). Preferably, the ischemic site arises from occlusion of a vessel or artery.

The invention provides a pharmaceutical composition for minimising glial scarring in an individual comprising ephrin-A1, variant or analog thereof, and a pharmaceutically acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient 25 present in the composition is ephrin-A1, variant or analog thereof.

The invention provides a pharmaceutical composition for minimising glial scarring in an individual comprising as an active ingredient ephrin-A1 and a pharmaceutically acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient present in the composition is ephrin-A1, variant or analog thereof. 3 1001272724

The invention provides a pharmaceutical composition for minimising glial scarring in an individual comprising as a main ingredient ephrin-A1 and a pharmaceutically acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient present in the composition is ephrin-A1, variant or analog thereof. 2015252036 03 Nov 2015 5 The invention also provides ephrin-A1, variant or analog thereof for use in the treatment of an individual with a CNS injury.

The invention also provides a pharmaceutical composition comprising ephrin-A1, variant or analog thereof and a pharmaceutically acceptable diluent, excipient or carrier for use in minimising glial scarring, or reducing astrocyte reactivity, in an individual. I0 The present invention also provides use of ephrin-A1, variant or analog thereof in the manufacture of a medicament for minimising glial scarring, or reducing astrocyte reactivity, in an individual in need thereof.

The present invention also provides a composition comprising ephrin-A1, variant or analog thereof for use in the treatment of individuals with any other condition or disease 15 described herein.

The present invention also provides a composition comprising ephrin-A1, variant or analog thereof and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention also provides a composition comprising as a main ingredient or active ingredient ephrin-A1, variant or analog thereof. Preferably, the only active 20 ingredient in the composition is ephrin-A1, variant or analog thereof.

The present invention also provides a surgical or implantable device for use in surgery of the CNS coated or impregnated with ephrin-A1, variant or analog thereof. Preferably, the surgical or implantable device is a neuroprosthetic or implantable microelectrode. Any type of in-dwelling brain microelectrodes are contemplated such as Deep Brain 25 Stimulating micro electrodes or microelectrode arrays.

The present invention also provides a composition for use in coating a surgical or implantable device, the composition comprising ephrin-A1, variant or analog thereof.

The composition may be a hydrogel, preferably slow-dissolving, having ephrin-A1, variant or analog thereof incorporated into it. Also, the composition may be a foam or 4 1001272724 sponge where ephrin-A1, variant or analog thereof is incorporated directly into the structure of the foam or sponge. 2015252036 03 Nov 2015

In any aspect of the present invention, the individual may be one that has been identified as having a CNS injury or damage. 5 In any aspect of the present invention, the individual may be one that has been identified as at risk of developing glial scarring.

In any aspect of the present invention, ephrin-A1, variant or analog thereof or a composition comprising ephrin-A1, variant or analog thereof may be administered after an event that causes glial scarring. Preferably, administration of ephrin-A1, variant or I0 analog thereof or a composition comprising ephrin-A1, variant or analog thereof is commence between 3 to 7 days post-injury.

In any aspect of the present invention, ephrin-A1, variant or analog thereof or a composition comprising ephrin-A1, variant or analog thereof is administered for a period of 7, 8, 9, 10, 11, 12, 13 or 14 days. Typically, ephrin-A1, variant or analog thereof or a 15 composition comprising ephrin-A1, variant or analog thereof is be administered as a continuous infusion via an osmotic / peristaltic pump locally to the site of injury over the dosing period.

In any aspect of the present invention, the individual has a condition associated with, or arising from, astrocyte dysfunction (for example reactive astrogliosis) or glial scar 20 formation or progression. Typically, the individual has a central nervous system (CNS) injury such as a brain or spinal cord injury. The CNS injury may have arisen from exogenous factors or endogenous cause. Exogenous factors include physical force trauma such as spinal cord injury, chemical injury or irradiation. Endogenous causes include haemorrhagic strokes, ischemia (including global ischemia and focal ischemia), 25 hypoxia, seizure, epilepsy, status epilepticus, CNS vascular disease and neuroocular disease. Also, an individual may be one undergoing, or who has undergone, a neurosurgical intervention including resection of tissue in situations such as epilepsy or biopsies.

In any aspect of the present invention, the individual may be one that has been 30 identified as having a CNS injury or damage. 5 1001272724

In any aspect of the present invention, the individual may be one that has been identified as at risk of developing glial scarring. 2015252036 03 Nov 2015

Preferably, the individual is an adult, typically, at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, at least 60 years, at least 70 years, 5 at least 80 years or at least 90 years old.

In any aspect of the present invention, ephrin-A1 may be human ephrin-A1. Preferably, human ephrin-A1 has an amino acid sequence shown in SEQ ID NO: 1.

In aspect of the present invention, ephrin-A1, variant or analog thereof may be isolated, recombinant, synthetic, purified or substantially purified. I0 In any statement of the invention above, reference to ephrin-A1 may also include reference to a biologically active variant or analog of ephrin-A1. A biologically active variant or analog of ephrin-A1, preferably human ephrin-A1, is a polypeptide or peptidomimetic that may have, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 15 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1, and also retains the biological activity or ability to minimise glial scarring. The ability to minimise glial scarring may be measured by any method as described herein or known in the art.

The invention also provides a kit or article of manufacture comprising ephrin-A1, variant 20 or analog thereof as described herein, and/or pharmaceutical composition as described above.

In other embodiments there is provided a kit for use, or when used, in mentioned of the invention described herein, the kit including: - a container holding ephrin-A1, variant or analog thereof as described herein, or a 25 pharmaceutical composition comprising ephrin-A1, variant or analog thereof as described herein; - a label or package insert with instructions for use. Preferably, the instructions are for use in a method of the invention as described herein. 6 1001272724

The methods and compositions of the invention described herein, could be used in combination with gelfoams, such as those with hemostatic properties or in combination with rehabilitative training after brain and spinal cord injuries. 2015252036 03 Nov 2015

As used herein, except where the context requires otherwise, the term "comprise" and 5 variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. As used herein “including” and “comprising” are intended to have the same meaning and are used interchangeably.

Further aspects of the present invention and further embodiments of the aspects 10 described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Chronic glial scarring differs after infant and adulthood focal ischemic stroke. (A) Parasagittal Nissl substance stained section of control Adult V1. Bounding 15 box indicates area enlarged in (B). (B) Immunofluorescent photomicrograph revealing the normal distribution of GFAP+ astrocytes within the laminar structure of V1 as revealed by DAPI nuclei stain. (C, E) Parasagittal Nissl substance stained section of adult V1 following infant (C) and adulthood (E) focal stroke. Bounding boxed indicates ischemic core enlarged in (D) and (F), respectively. (D, F) GFAP+ immunofluorescent 20 photomicrograph demonstrating the differences in pattern of chronic glial scarring >365 days following infant and adulthood stroke. (D) Discrete distribution of GFAP+ astrocytes, comprising the chronic glial scar proximal to the ischemic core after infant stroke. (F) In contrast, a more prolific, dense and widespread distribution of GFAP+ astrocyte was observed in the chronic glial scar following adulthood stroke. (G-V) 25 EphA4 is increased on reactive astrocytes in the sub-acute period following infant and adulthood stroke. (G, K) Low levels of EphA4 expression were detected on GFAP+ astrocytes in the control infant (G) and adult (K) V1. (H, L) Parasagittal Nissl substance labelled V1 section of infant (H) and adult (L) V1 at 21 days post ischemia (DPI). Bounding boxed indicates areas proximal to the ischemic core, enlarged in (I) and (M), 30 respectively. EphA4 expression was increased on GFAP+ reactive astrocytes proximal to the ischemic core after infant (I) and (M) adulthood focal stroke. Bounding boxes in (I) 7 1001272724 and (M) indicates enlarged areas in (J) and (N), respectively. (O-R) High magnification immunofluorescent photomicrographs demonstrating the increase in EphA4 expression, correlated to the change in astrocyte morphology between resting (O, Q) and reactive (P, R) astrocytes as revealed by GFAP immunolabelling in both infants (O-P) and adults 5 (Q-R). Morphological changes observed included an increase in EphA4 expression, 2015252036 03 Nov 2015 hypertrophy of cell bodies and the shortening and thickening of processes, which are hallmarks of reactive astrocytes. (S-V) Immunoblot analysis revealed the statistically significant upregulation of EphA4 expression in post-ischemic tissue lysates of infant (S, U) and adult (T, V) V1 (p <0.05). Y-axis in (U, V) represents fluorescent intensity; I0 (arbitrary values). Scale bars: A, C, E, H, L: 1 mm; B, G, I, J, K, Μ, N: 100 pm; F: 500 pm; O-R: 10 pm.

Figure 2. Different ephrins are expressed proximal to the ischemic core after infant and. adulthood ischemic stroke. (A-B) Western blot analyses were performed on tissue lysates obtained from the infant and adult post-ischemic V1 region. Full results 15 of the ephrin screen is summarised in table 3. Histograms describes the mean fluorescent intensity of protein bands (fluorescent intensity; arbitrary values) for control and 2 post-ischemic time points ± SEM. T-test analysis revealed a statistically significant increased of ephrin-A1 at 1 DPI, which was sustained at 21 DPI (p < 0.05) in the post-ischemic infant but not adult V1. In the post-ischemic adult V1, a significant >0 increase of ephrin-A2 (C, D; p < 0.05) and ephrin-A5 (E, F; p < 0.05) was detected at 1 DPI and sustained at 21 DPI. No significant change in ephrin-A2 or -A5 expression was detected in infants apart from an initial decrease at 1 DPI, returning to normal levels by 21 DPI. (H-J) ephrin-A1 in infants and ephrin-A2 and -A5 in adults interact with EphA4 in the normal post-ischemic V1. To confirm the potential interactions between the 25 candidate ephrins above and EphA4, a Co-immunoprecipitation (co-IP) experiment was performed targeted at EphA4 and screened for candidate ephrins. (H) Ephrin-A1 was co-immunoprecipitated with EphA4 from the post-ischemic infant but not adult tissue lysates. In adults, ephrin-A2 (I) and ephrin-A5 (J) was detected after co-IP against EphA4 in the post-ischemic adult tissue lysates. 30 Figure 3. Distribution of ephrin-expressing cells proximal to the ischemic core after infant and adulthood stroke. (A-C) Distribution of ephrin-A1 immunopositive cells in the normal and post-ischemic infant V1. (A) Photomicrograph of control infant 8 1001272724 V1 immunolabelled for ephrin-A1 demonstrating the weak ephrin-A1 labelling predominantly distributed in the subgranular layers of V1. (B) Parasagittal Nissl substance labelled section of the infant V1 at 21 DPI with bounding box indicating area enlarged in (C). (C) Photomicrograph of ephrin-A1 DAB-immunolabelled V1 5 demonstrating the increased expression and distribution of ephrin-A1 proximal to the ischemic core (iv), diminishing distally to control levels (i). Bounding boxes in (C) indicates enlarged photomicrographs in (i-iv). In control adult V1, cellular expression of ephrin-A2 (D) and ephrin-A5 (E) was detected, dispersed in cortical V1. Further analysis revealed that ephrin-A2 (F) and ephrin-A5 (G) is expressed exclusively by interneurones I0 in the normal adult V1, revealed by combination of calbindin, parvalbumin and calretinin labelling. (H) Parasagittal Nissl substance labelled section of the adult V1 at 21 DPI with bounding box indicating area enlarged in (I, J). Ephrin-A2 (I) and ephrin-A5 (J) expression was increased after adulthood stroke. DAB-immunolabelling revealed a similar distribution of immunopositive cells similar to that of ephrin-A1 in infants. 2015252036 03 Nov 2015 15 Specifically, ephrin-A2 (v-viii) and ephrin-A5 (ix-xii) immunopositive cells in the postischemic adult V1 was distributed more densely proximal to the ischemic core and decreasing distally. Bounding boxes in (I) and (J) indicates regions enlarged in (v-viii) and (ix-xii), respectively. Cells with increased expression of ephrin-A2 and ephrin-A5 in the post-ischemic adult V1 appear to be distributed predominantly in the white matter ?0 compared to the cortical distribution of ephrin-A1 immunopositive cells in infants. Scale bars: A, C, D, E, I, J: 500pm; B, H: 1mm; F, G: 10pm; i-xii: 100pm.

Figure 4. Reactive astrocytes are responsible for the increased expression of ephrin-A1 and ephrin-A2/ -A5 after focal stroke in infant and adult V1, respectively. (A) High magnification immunofluorescent photomicropraphs revealed 25 ephrin-A1 expression on GFAP+ astrocytes in control infant V1, consistent with DAB-immunolabelled sections. Ephrin-A1 expression was increased at 21 DPI (B) on morphologically distinct reactive astrocytes proximal to the ischemic core as revealed by GFAP co-immunolabelling. In the post-ischemic adult V1, the expression of ephrin-A2 (C) and ephrin-A5 (D) was on GFAP+ reactive astrocytes were detected proximal to the 30 adult ischemic core at 21 DPI. (E-G) The co-expression of EphA4 receptor and ephrin ligands was detected on reactive astrocytes proximal to the ischemic core in the infant and adult V1. High magnification confocal microscopy and colocalisation analysis revealed that ephrin-A1 (E; infants) and ephrin-A2/ -A5 (F, G; adults) were co- 9 1001272724 expressed with EphA4 on reactive astrocytes but fluorescent puncta were not colocalised in both cases. The Pearson’s coefficient (Rr) calculated described little to no correlation between the EphA4 (red) puncta and ephrin-A1 (E; Rr= -0.61), ephrin-A2 (F; Rr= -0.38) or ephrin-A5 (G; Rr= -0.42) puncta (green). Scale bar: A, B, E, F, G: 10pm; 2015252036 03 Nov 2015 5 C, D: 20pm.

Figure 5. Wound-closure assay reveals opposing astrocyte behaviours in response to ephrin treatment. (A) Temporal analysis of marmoset astrocyte wound-closure capacity over 48 hrs revealed statistically significant differences in the degrees of astrocyte wound-closure capacities elicited by different ephrin treatments (KW; p I0 <0.0001). Graph describes the mean percentage of astrocyte wound closure over 48 hrs (n=3) ± SEM. Specifically, ephrin-A1 (A, C) treatment did not induce astrocyte wound closure relative to untreated controls (A, B). In contrast, treatment of both ephrin-A2 (A, D) and ephrin-A5 (A, E) promoted the rate and final extent of astrocyte wound closure the degree similar to that observed in EphA4 (A) and LIF-treated positive 15 controls (A, F).

Figure 6. Quantitative analysis of in vitro assay reveals that ephrin-A1 and ephrin-A2/ -A5 forward signalling plays opposing roles in regulating astrocyte reactivity after infant and adulthood stroke. (A) Extent of astrocyte wound closure at 48 hrs. Histogram describes the median % wound closure at 48 hrs ± interquartile ranges. Non-20 parametric Kruskal-Wallis test revealed statistically significant (p <0.0001) differences in the extent astrocyte wound closure elicited by differential ephrins treatment. Dunn's post-hoc analysis revealed that ephrin-A1 treatment only achieved a 35.6% wound closure, which was not significant to the 24.9% closure observed in untreated controls.

In contrast, treatment with ephrin-A2 (76.3%; p <0.01) and ephrin-A5 (100%; p <0.001) 25 promoted astrocyte wound closure to achieve a statistically significant increase in % closure compared to untreated controls. The extent of wound closure elicited by ephrin-A5 treatment was most pronounced, which was significantly greater than that elicited by ephrin-A1 (p <0.01) as well as untreated controls. Furthermore, % wound-closure at 48 hrs elicited by ephrin-A5 treatment was not significantly different to that of EphA4 (p 30 >0.05) or LIF-treated positive controls (p >0.05). (B, F) Differences in astrocyte guidance in response to ephrin treatment were also evident in stroke assays. Histogram (F) describes the median percentage of GFAP+ nuclei distributed on control or ephrin 10 1001272724 stripes ± interquartile ranges. Quantitative analysis revealed statistically significant differences in astrocyte guidance response elicited by different ephrin stripes (KW; p < 0.0001). A random, non-preferential distribution of astrocytes was detected on negative control stripes (48.97% astrocytes on stripes). (C, F) Astrocytes were preferentially 5 distributed in the spaces between stripes, with only 38.85% of astrocytes distributed on ephrin-A1 stripes (p <0.05), suggesting ephrin-A1 induced astrocyte-repulsion. In contrast, astrocytes were preferentially distributed on ephrin-A2 (D, F) and ephrin-A5 (E, F) stripes, suggesting ephrin-A2 and -A5 induced astrocyte attraction. Specifically, a significantly large proportion of 79.83% (p <0.05) and 68.18% (p <0.05) of astrocytes I0 were distributed on ephrin-A2 and ephrin-A5 stripes, respectively. (G) Proliferation of marmoset astrocytes are differentially regulated by ephrin-A1 and ephrin-A2/ -A5 treatments in vitro. Histogram describes the median percentage of BrdU nuclei over total nuclei counted ± interquartile ranges. Non-parametric KW test revealed a statistically significant difference in astrocyte proliferation in response to different 15 ephrins treatment, Dunn’s post-hoc test revealed that ephrin-A1 treatment significantly reduced the percentage of BrdU+ nuclei over total nuclei population (39.0%; p >0.05) compared to untreated controls (57.8%). In contrast, a significant increase in % BrdU+ nuclei was achieved following ephrin-A2 (67.5%; p <0.05) and ephrin-A5 (71.4%; p <0.05) treatment, compared to untreated controls. The degree to which astrocyte >0 proliferation was increased by ephrin-A5 treatment was not significantly different to that of EphA4 (71.5; p <0.05) and LIF-treated positive controls (74.1%). Scale bar: B-E: 50pm. 2015252036 03 Nov 2015

Figure 7. Ephrin-A1 treatment inhibits astrocyte reactivity induced by ephrin-A2 and ephrin-A5 in wound closure assay. Administration of ephrin-A1 in addition to 25 ephrin-A2 or ephrin-A5 significantly reduced astrocyte wound closure capacity (KW; p <0.0001). Graphs (A, C) describes the percentage of astrocyte wound closure over 48 hrs ± SEM. Histograms (E, F) describes the median percentage of astrocyte wound closure at the 48 hrs time point ± interquartile ranges. (A, B, E) Treatment of ephrin-A1 in addition to ephrin-A2 reduced the rate wound closure, achieving a final closure of 30 44.5% at 48 hrs, which was significantly reduced (MW; p <0.01) compared to that of ephrin-A2 treatment alone. (C, D, F) Addition of ephrin-A1 similarly reduced the rate of wound closure induced by ephrin-A5, achieving a final closure of 62.7% at 48 hrs, which was also significantly reduced (MW; p <0.01) compared to that of ephrin-A5 treatment 11 1001272724 alone. This result suggests that ephrin-A1 treatment is able to inhibit astrocyte reactivity that is induced by ephrin-A2 and ephrni-A5 forward signalling. 2015252036 03 Nov 2015

Figure 8. MRI-compatible Plastics One Brain Infusion Kit, Model 3280PM.

Figure 9. iPrecio Micro-infusion Pump, Model SMP-300, routinely used in mice 5 experiments.

Figure 10. Administration of ephrin-A1 after brain injury successfully reduces reactive astrogliosis and glial scarring in vivo. (A) Coronal section of the adult rat brain immunostained for glial fibriliary acidic protein, a marker for reactive astrocytes in rodents. Bounding box indicates area enlarged in (B-D), which is also the site of needle I0 stick injury. (B) Normal, uninjured rat brain with little to no GFAP immunoreactivity. (C) GFAP immunoreactivity is markedly increased around the injury core (red arrow), which is consistent with the accumulation of reactive astrocytes and glial scarring. (D) Administration of ephrin-A1 following needle stick injury resulted in a marked reduction in GFAP immunoreactivity at the injury core, consistent with a reduction in reactive 15 gliosis and a less severe glial scar. (E) Quantitative analysis of the glial scar at the macroscopic level demonstrated a statistically significant reduction in glial scar volume in ephrin-A1 treated rats compared to untreated controls (*; p <0.05). (F) Sagittal section of adult rat brain after brain injury immunostained for GFAP+ reactive astrocytes. Reactive astrocytes were densely distributed proximal to the injury core (red 20 arrow; H), initially decreasing distally (H; 600pm-1100pm from core) followed by a secondary increase (H-l; 1600pm-2100pm from core). This high density of GFAP+ reactive astrocytes distal to the core is consistent with on-going recruitment and migration of reactive astrocytes to the core in the sub-acute period after brain injury. (G) Administration of ephrin-A1 after brain injury resulted in a statistically significant 25 reduction in GFAP+ reactive astrocyte density at the injury core, which is progressively decreased distally (H-l). The absence of a secondary increase in astrocyte density distally suggest that ephrin-A1 treatment successfully supressed the secondary migration of reactive astrocytes to the core, contributing to the reduction in astrocyte density and glial scar volume. (J) Brain injury results in widespread apoptotic cell death 30 proximal to the injury core (red arrow) as detected by activated caspase-3 (aCas3) immunolabelling. (K) ephrin-A1 treatment significantly reduced the overall density of 12 1001272724 aCas3+ apoptotic cells at the injury core compared to untreated controls (J; L; p <0.05). This reduction in apoptotic cells indicates after ephrin-A1 treatment indicates a reduction in the severity of secondary injury and may play neuroprotective roles after brain injury. 2015252036 03 Nov 2015 5 Figure 11. Amino acid and nucleotide sequence of human ephrin-A1

Detailed description of the embodiments

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations 10 constitute various alternative aspects of the invention.

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may 15 be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to 20 all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

All of the patents and publications referred to herein are incorporated by reference in their entirety. 25 For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

An advantage of the present invention is that minimising glial scarring would extend the current therapeutic time window for ischemic stroke treatment into the sub-acute period (days-weeks), which would be of tremendous clinical value. Further, a reduction in glial 13 1001272724 scarring would be permissive towards regeneration. Most treatment strategies targeting astrocyte reactivity and glial scarring currently being trialled are administered soon after the injury, which may impede the neuroprotective function of the astrocyte in the acute period post-injury. The advantage of ephrin-A1 administration in the adult after CNS 5 injury, such as ischemic stroke, may induce ‘infant-like’ astrogliotic responses without compromising neuroprotection, leading to a more discrete ‘infant-like’ scar that is more permissible towards functional recovery. 2015252036 03 Nov 2015

While the present invention finds particular application in the treatment of glial scarring arising from ischemia, the invention targets the process of glial scarring that occurs as a 10 result of any type of injury or damage to the CNS. As such, this strategy could be applied to minimise glial scarring and promoting recovery after other CNS injuries, such as traumatic brain and spinal cord injuries. Further, this approach would be beneficial to neuroprosthetics and implantable microelectrodes. The reactive astrocyte response and scarring that encapsulate these implants over time significantly limit the longevity of 15 these implants. This issue could be solved through the integration of reducing astrocyte reactivity and glial scarring around the implant by a method or composition of the invention described herein. CNS injuries can occur as a result exogenous factors such as physical force trauma, or endogenous causes (e.g. Ischemic/ haemorrhagic strokes). Ischemic strokes and ?0 traumatic brain injuries (TBI) are the most common type of CNS injury. Examples of these are those that afflict the visual neocortex. Injury of the visual cortex often results in cortical blindness, which refers to blindness produced by pathologies affecting V1 and the primary visual pathway, not the eye. The severity of blindness depends on the extent of damage to V1 or its immediate afferents, resulting in the loss of vision in the 25 contralateral visual hemifield.

Within minutes of a CNS injury, the first wave of cell death (necrosis) occurs as a direct consequence of the pathology. The necrotic wave is characterised by cell swelling and mitochondria/ endoplasmic reticulum degradation followed by rupturing of the plasma, organelle and nuclear membranes. Injuries that compromises the blood brain barrier 30 (BBB) can lead to intracerebral haemorrhage, initiating a localised neuroinflammatory response from chemotactic recruitment of both neural and peripheral immune cells. 14 1001272724

Depending on the severity, a second wave for cell death (necrosis and apoptosis), known as the secondary injury can occur in the lesion penumbra in the sub-acute period after injury (hours-days). The lesion penumbra refers to an area of tissue surrounding, or adjacent to, the necrotic core, that is less severely affected by the lesion, and retains 5 some level of metabolic stability. The causes of secondary injuries include prolonged hypoxia/ ischemia, excitotoxicity, reperfusion injuries and accumulation of free radicals. Cerebral oedema induced homeostatic and osmotic stress also contributes to secondary injury. In addition, the debris from necrotic cells and degenerating neural processes as a result of axonal shearing can also lead to degeneration of neurones in 10 the penumbra. The result of the secondary injury leads to a compounded effect on cell death and glial recruitment to the lesion core and penumbra and the subsequent severity of glial scarring. As a result of molecular and chemical inhibition the capacity for neuronal regeneration in the penumbra is significantly reduced. 2015252036 03 Nov 2015 A glial scar is predominantly composed of reactive astrocytes that are activated from 15 their resting state by the onset of neuropathologies. The exaggerated astrogliotic response, including local proliferation triggered by the injury results in an increased astrocyte density juxtaposing the injury core. This ultimately leads to a fine meshwork of entangled astrocytic processes and secretion of chondroitin sulfate proteoglycans (CSPG). Formation of the glial scar is an integral component of wound healing in the >0 CNS and is composed primarily of reactive astrocytes, inflammatory cells and a secreted extracellular matrix component, comprising mainly CSPGs. The glial scar functions as a structural barrier, limiting necrotic and inflammatory events and stabilising the injury site. One key element that triggers glial scar formation is the extravasation of blood-born cells or molecules into the CNS parenchyma, for example, cytokines and 25 fibrinogens. The presence of these factors initiates the conversion of resting astrocytes into a reactive state. Reactive astrocytes are characterised by hypertrophic transformation, shortening and thickening of cell processes, changes in gene and protein expression as well as their overall function. Reactive astrocytes perform crucial roles in maintaining homeostatic balance at the injury site by scavenging excessive 30 glutamate or ions that exacerbates secondary injury. Reactive astrocytes have also been known to play neuroprotective roles and provide trophic support to surviving neurones and after injury. However, the exaggerated response of reactive astrocytes over time, especially after adulthood injuries eventually contributes to the inhibition of 15 1001272724 repair in later periods. Local proliferation of reactive astrocytes proximal to the site of injury results in an increase in reactive astrocyte density. The accumulation of reactive astrocytes proximal to the site of injury eventually results in a fine meshwork of entangled processes and secreted extracellular matrix proteins, especially CSPGs, a 5 potent growth inhibitory factor that is a major component of the glial scar. 2015252036 03 Nov 2015

In addition to its physiological role in limiting CNS injuries, the glial scar is also a potent inhibitor of axonal regeneration. Following injury, the inherent capacity for axonal regeneration is manifested in the formation of regenerative growth cones . Growth cones are the motile tip of a growing axon or dendrites that guides extending neurites to 10 their appropriate targets by responding to guidance cues in the extracellular environment. The guidance of growth cones is crucial for the proper establishment of neural pathways and circuitry throughout development and maturation of the CNS. However, regenerative growth is halted on contact with the CSPG-rich matrix of the glial scar and prevented from further extension. Contact with these inhibitory cues causes 15 regenerative growth cones to take on a dystrophic morphology. These dystrophic growth cones are described as “sterile clubs” with bulbous morphology and lack of forward movement. Axons with dystrophic end bulbs survive for a short period, eventually succumbing to anterograde degeneration resulting in neuronal death. The growth-inhibitory environment of the glial scar is viewed as the major hindrance to any ?0 potential regenerative processes. Therefore, the present invention has been devised to limit, if not remove, the growth-inhibitory properties of the glial scar in order to promote regenerative processes.

As used herein, “minimising glial scarring” includes reducing the progression of an existing glial scar or inhibiting or slowing the formation of a glial scar. This may be 25 measured by clinical or biochemical means to determine the density, size and/or volume of the glial scar at a given time. Further, the amount and distribution of reactive astrocytes and secreted extracellular matrix proteins, including chondroitin sulfate proteoglycans (CSPG) proximal to the injury core may be measured using methods know in the art and as described herein. It is possible to measure the extent of scarring 30 indirectly using an MRI technique known as FLAIR-T1 (T1 -weighted fluid-attenuated inversion recovery (FLAIR)). Fluid attenuated inversion recovery cancels out the effect of fluid from the resulting image to reveal the extent of injury. The ischemic core and 16 1001272724 surrounding area which will be the site of glial scarring will be visible as hyper-intense signal. Repeated, longitudinal FLAIR-T1 imaging can be performed on patients to monitor the volume and size of the ischemic core to look for signs of improvement, i.e reduction in FLAIR-T1 hyperintensity. This can be done in combination with T2 weighted 5 imaging (another MRI technique) which will monitor the overall progression of the ischemic core over time. 2015252036 03 Nov 2015

As used herein "promoting the recovery of central nervous system function" should be taken to mean that the treatment promotes, or reduced an inhibitory effect on, neuron regeneration compared to untreated neurons. Typically, the neuron regeneration is I0 axonal regeneration. Typically, this would result in a clinically relevant improvement in at least one of speech, motor coordination function (i.e. posture, balance, grasp or gait), sensory perception (i.e vision, touch, taste, hearing and olfaction), cognition or memory.

As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., 15 causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by clincians.

The terms "treatment" or "treating" of a subject includes the application or administration 20 of a compound of the invention to a subject (or application or administration of a compound of the invention to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or 25 condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration 30 or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. 17 1001272724

The existence of, improvement in, treatment of or prevention of a condition described herein may be by any clinically or biochemically relevant method of the subject or a biopsy therefrom. For example, in addition to minimising glial scarring, treatment of an injury by a method or composition of the invention described herein may result in any 5 one or more of: reduction in overall GFAP+ immunoreactivity proximal to the injury core, reduction in reactivity and density of reactive astrocyte response, reduction in overall glial scar volume, a change in the distribution and density of reactive astrocyte population in both proximal and distal portions, reduction in astrocyte density in proximal and distal portion, reduction in the secondary migration / recruitment of reactive 10 astrocytes from distal portion into the injury core (a major hallmark of glial scarring), and / or reduction in apoptotic (dying) cells at the ischemic core. Further, there may be improved functional recovery of central nervous system function as described herein. 2015252036 03 Nov 2015

In the context of the present invention, the term "individual in need thereof shall be taken to mean an individual who has damaged or injured central nervous system tissue 15 as a result of suffering CNS damage or injury, such CNS damage or injury being caused by ischemia, stroke, or any other cause of CNS damage or injury as described herein (i.e. damage or injury to CNS tissue caused by chemical or physical means, or by irradiation).

As used herein, the term “Traumatic Brain Injury” is art recognized and is intended to 20 include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF). 25 The term "isolated" in relation to a protein or polypeptide means that by virtue of its origin or source of derivation is not associated with naturally-associated components that accompany it in its native state; is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques 30 known in the art. By “substantially purified” is meant the protein is substantially free of 18 1001272724 contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents. 2015252036 03 Nov 2015

The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising or 5 consisting of ephrin-A1, this term does not encompass an ephrin-A1 naturally-occurring within a subject’s body. However, if such a protein is isolated, it is to be considered an isolated protein comprising or consisting of ephrin-A1. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising or consisting of ephrin-A1. A recombinant I0 protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

As used herein unless stated otherwise ephrin-A1, includes to all isoforms, orthologs, paralogs or homologs of human ephrin-A1. Preferably, the ephrin-A1 as used herein is human ephrin-A1. Human ephrin-A1 may comprise, consist essentially of or consist of 15 an amino acid sequence shown in SEQ ID NO: 1 or encoded by the nucleotide sequence shown in SEQ ID NO: 2. The amino acid and nucleotide sequence of human ephrin-A1 is also accessible in the NCBI database using the accession numbers AAH95432.1 and NM_004428.2. An example of a splice variant of human ephrin-A1 is isoform b accessible by the accession number NP_872626.1. The amino acid sequence >0 of Ephrin-A1 from dog and cat can be accessed by accession numbers XP_852071.1 and XP_011289478.1.

Also contemplated for use in the invention is a biologically active variant or analog of ephrin-A1, preferably human, that is a polypeptide or peptidomimetic that may have, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 25 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1, which also retains the biological activity or ability to minimise glial scarring. The ability to minimise glial scarring may be measured by any method as described herein or known in the art.

Also contemplated for use in the invention is a fusion protein comprising an amino acid 30 sequence of ephrin-A1, variant or analog thereof. Preferably, the fusion protein comprises a first amino acid sequence of ephrin-A1 and a second amino acid sequence 19 1001272724 of ephrin-A1, variant or analog thereof. The fusion protein may comprise an Fc portion of an antibody and ephrin-A1, variant or analog thereof (referred to herein as ephrin-A1 -Fc). The Fc portion may be derived from, for example, a human IgG antibody, such as an lgG1 or lgG2 antibody. The ephrin-A1-Fc fusion may comprise, consist essentially of 5 or consist of Fluman Ephrin-A1 (Met1-Ser182) - linker (such as IEGRMD) - Human lgG1 (Pro100-Lys330). 2015252036 03 Nov 2015 “Percent (%) amino acid sequence identity” or “percent (%) identical” with respect to a polypeptide sequence, i.e. a polypeptide, protein or fusion protein of the invention defined herein, is defined as the percentage of amino acid residues in a candidate I0 sequence that are identical with the amino acid residues in the specific polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Those skilled in the art can determine appropriate parameters for measuring alignment, 15 including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a ?0 certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X/Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal 25 to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison 30 of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 20 1001272724 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be 5 used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of I0 sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector 15 NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A nonlimiting examples of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http://www.jalview.org/). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non->0 limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid 25 sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. 2015252036 03 Nov 2015

The polypeptide desirably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide 30 comprised of D-amino acids is expected to have a greater retention of its biological activity in vivo. 21 1001272724

The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source. Recombinant production is preferred. For instance, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be 5 subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available I0 kits also can be employed (e.g., such as manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; InVitrogen, Carlsbad, Calif., and the like). The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids. 2015252036 03 Nov 2015

The term "conservative substitution" as used herein, refers to the replacement of an 15 amino acid present in the native sequence in the peptide or polypeptide with a naturally or non- naturally occurring amino acid or a peptidomimetic having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non- naturally occurring amino acid or with a peptidomimetic moiety which is 20 also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that may be considered to be conservative substitutions for one another: 25 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 22 1001272724 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). 2015252036 03 Nov 2015

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be determined bearing in mind the fact that replacement of charged amino acids by sterically similar 5 non-charged amino acids are considered as conservative substitutions. For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled person and non-natural or unnatural amino acids are described further below. 10 When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

Alterations of the native amino acid sequence to produce mutant polypeptides, such as by insertion, deletion and/or substitution, can be done by a variety of means known to those skilled in the art. For instance, site-specific mutations can be introduced by 15 ligating into an expression vector a synthesized oligonucleotide comprising the modified site. Alternately, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as disclosed in Walder et al., Gene 42: 133(1986); Bauer etal., Gene 37: 73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462. A preferred means for introducing mutations is the QuikChange Site-20 Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

Any appropriate expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, N.Y.: 1985)) and corresponding suitable host can be employed for production of recombinant polypeptides of ephrin-A1, biologically active variants or analogs thereof. Expression hosts include, but are not limited to, 25 bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology 6: 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CFIO, FleLa, and BHK cell lines, and the like. The skilled person is aware that the choice of expression host has ramifications for the type 30 of polypeptide produced. For instance, the glycosylation of polypeptides produced in 23 1001272724 yeast or mammalian cells (e.g., COS-7 cells) will differ from that of polypeptides produced in bacterial cells, such as Escherichia coli. 2015252036 03 Nov 2015

Alternately, a polypeptide of the invention, i.e. ephrin-A1, biologically active variants or analogs thereof, can be synthesized using standard peptide synthesizing techniques 5 well-known to those of ordinary skill in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). If desired, this can be done I0 using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The polypeptide-containing mixture can then be extracted, for instance, with dimethyl ether, to remove non-peptidic organic compounds, and the 15 synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) optionally can be done in order to eliminate any incomplete polypeptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For 20 other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, such as by the methods described herein or other genetic means, or as part of a larger conjugate, such as through physical or chemical conjugation, as known to those of ordinary skill in the art and described herein. A “peptidomimetic” is a synthetic chemical compound that has substantially the same 25 structure and/or functional characteristics of a polypeptide of the invention, the latter being described further herein. Typically, a peptidomimetic has the same or similar structure as a polypeptide of the invention, for example the same or similar sequence of SEQ ID NO: 1 that has the ability to minimise glial scarring. A peptidomimetic generally contains at least one residue that is not naturally synthesised. Non-natural components 30 of peptidomimetic compounds may be according to one or more of: a) residue linkage groups other than the natural amide bond ('peptide bond') linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce 24 1001272724 secondary structural mimicry, i.e , to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. 2015252036 03 Nov 2015

Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literatures, e.g., Organic Syntheses Collective 5 Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot.267:220-234

As used herein, the term "immunoglobulin heavy chain constant region" is used interchangeably with the terms "Fc", "Fc region" and "Fc domain" and is understood to I0 mean the carboxyl-terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding an Fc receptor. As is known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CH1-hinge-CFI2-CFI3(-CFI4). CFI4 is present in IgM, which has no hinge region. The immunoglobulin heavy chain constant 15 region useful in the fusion proteins of the invention may comprise an immunoglobulin hinge region, a CH2 domain and a CH3 domain. As used herein, the term immunoglobulin "hinge region" is understood to mean an entire immunoglobulin hinge region or at least a portion of the immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region. 20 It is contemplated that suitable immunoglobulin heavy chain constant regions may be derived from antibodies belonging to each of the immunoglobulin classes referred to as IgA, IgD, IgE, IgG, and IgM, however, immunoglobulin heavy chain constant regions from the IgG class are preferred. Furthermore, it is contemplated that immunoglobulin heavy chain constant regions may be derived from any of the IgG antibody subclasses 25 referred to in the art as lgG1, lgG2, lgG3, and lgG4. In one embodiment, an Fc region is derived from lgG1. In another embodiment, an Fc region is derived from lgG2.

Immunoglobulin heavy chain constant region domains have cross-homology among the immunoglobulin classes. For example, the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE. Preferred 30 immunoglobulin heavy chain constant regions include protein domains corresponding to a CH2 region and a CH3 region of IgG, or functional portions or derivatives thereof. The 25 1001272724 choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. The Fc regions of the present invention may include the constant region such as, for example, an IgG-Fc, IgG- CH, an 5 Fc or CH domain from another Ig class, i.e., IgM, IgA, IgE, IgD or a light chain constant domain. Truncations and amino acid variants or substitutions of these domains may also be included. 2015252036 03 Nov 2015 A variety of nucleic acid sequences encoding Fc fusion proteins may also be used to make the ephrin-A1-Fc fusion proteins of the invention. For example, the nucleic acid I0 sequences may encode in a 5' to 3' direction, either the immunoglobulin heavy chain constant region and the ephrin-A1 polypeptide, or the ephrin-A1 polypeptide and the immunoglobulin heavy chain constant region. Furthermore, the nucleic acid sequences optionally may also include a "leader" or "signal" sequence based upon, for example, an immunoglobulin light chain sequence fused directly to a hinge region of the 15 immunoglobulin heavy chain constant region. In a particular embodiment, when the Fc region is based upon IgG sequences, the Fc region encodes in a 5' to 3' direction, at least an immunoglobulin hinge region (i.e., a hinge region containing at least one cysteine amino acid capable of forming a disulfide bond with a second immunoglobulin hinge region sequence), an immunoglobulin CH2 domain and a CH3 domain. >0 Furthermore, a nucleic acid sequence encoding the ephrin-A1 -Fc fusion proteins may also be integrated within a replicable expression vector that may express the Fc fusion protein in, for example, a host cell.

In one embodiment, the immunoglobulin heavy chain constant region component of the ephrin-A1-Fc fusion proteins is non-immunogenic or is weakly immunogenic in the 25 subject. The Fc region is considered non- or weakly immunogenic if the immunoglobulin heavy chain constant region fails to generate a detectable antibody response directed against the immunoglobulin heavy chain constant region. Accordingly, the immunoglobulin heavy chain constant region should be derived from immunoglobulins present, or based on amino acid sequences corresponding to immunoglobulins present 30 in the same species as the intended recipient of the fusion protein. In some embodiments, human immunoglobulin constant heavy region sequences are used for the ephrin-A1-Fc fusion protein, which is to be administered to a human. Nucleotide and 26 1001272724 amino acid sequences of human Fc IgG are known in the art and are disclosed, for example, in Ellison et al., Nucleic Acids Res. 10:4071 -4079 (1982). 2015252036 03 Nov 2015

The ephrin-A1-Fc fusion proteins of the invention may be made using conventional methodologies known in the art. For example, ephrin-A1-Fc fusion constructs may be 5 generated at the DNA level using recombinant DNA techniques, and the resulting DNAs integrated into expression vectors, and expressed to produce the ephrin-A1 -Fc fusion proteins of the invention. As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated into the host cell genome, or to replicate I0 autonomously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an adeno-associated virus. As used herein, the term "gene expression" or "expression" of a ephrin-A1-Fc fusion protein, is understood to mean the transcription of a DNA sequence, translation of the mRNA 15 transcript, and secretion of an Fc fusion protein product. As an alternative to fusion of proteins by genetic engineering techniques, chemical conjugation using conventional chemical cross-linkers may be used to fuse protein moieties.

Sequences of constant regions useful for producing the proteins of the present invention, particularly ephrin-A1-Fc, may be obtained from a number of different 20 sources. In some examples, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including lgG1, lgG2, lgG3 and lgG4. In one example, the constant region is human isotype lgG4 or a stabilized lgG4 constant region. 25 In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human lgG1 or lgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector 30 function of an Fc region containing protein are known in the art and/or described herein. 27 1001272724

In one example, the Fc region is an lgG4 Fc region (i.e., from an lgG4 constant region), e.g., a human lgG4 Fc region. Sequences of suitable lgG4 Fc regions will be apparent to the skilled person and/or available in publically available databases (e.g., available from National Center for Biotechnology Information). 2015252036 03 Nov 2015 5 In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an lgG1 Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331. In another example, the Fc region is an lgG1 Fc region comprising one or more of the following changes E233P, L234V, L235A 10 and deletion of G236 and/or one or more of the following changes A327G, A330S and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields etal., J Biol Chem. 276(9):6591-604, 2001). Additional examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177 : 1129-1138 2006; and/or Hezareh J Virol ;75: 12161-12168, 2001). 15 In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CFI2 domain from an lgG4 antibody and at least one CFI3 domain from an lgG1 antibody, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering) (e.g., as described in WO2010/085682). >0 Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

Although the invention finds application in humans, the invention is also useful for therapeutic veterinary purposes. The invention is useful for domestic or farm animals such as cattle, sheep, horses and poultry; for companion animals such as cats and 25 dogs; and for zoo animals.

Ephrin-A1, variant or analog thereof or a composition of the invention may be administered into an individual systemically, orally or directly at the site of injury or damage. Any route of administration which provides ephrin-A1, variant or analog thereof to astrocytes at or near a site of injury or damage is contemplated. Ephrin-A1, variant or 30 analog thereof may be administered at the injury core, area surrounding and / or juxtaposing the injury core (for example the penumbra). Ephrin-A1 may be formulated 28 1001272724 for oral, systemic or topical administration. A particular administration route contemplated is intrathecally. As used herein, the term “intrathecal administration” is intended to include delivering ephrin-A1 or a composition including ephrin-A1 directly into the cerebrospinal fluid of a subject, by techniques including lateral 5 cerebroventricular injection through a burrhole or cisternal or lumbar puncture or the like. The term “lumbar region” is intended to include the area between the third and fourth lumbar (lower back) vertebrae. The term “cistema magna” is intended to include the area where the skull ends and the spinal cord begins at the back of the head. The term “cerebral ventricle” is intended to include the cavities in the brain that are I0 continuous with the central canal of the spinal cord. Administration of ephrin-A1 or a composition including ephrin-A1 to any of the above mentioned sites can be achieved by direct injection or by the use of infusion pumps. For injection, the ephrin-A1 or a composition including ephrin-A1 of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's 15 solution. In addition, the ephrin-A1 or a composition including ephrin-A1 may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included. The injection can be, for example, in the form of a bolus injection or continuous infusion (e.g., using infusion pumps) of ephrin-A1 or a composition including ephrin-A1. 2015252036 03 Nov 2015 20 Ephrin-A1, variant or analog thereof or a composition comprising ephrin-A1, variant or analog thereof may be delivered directly to the site of injury via any means, for example, through an intracerebral cannula thereby providing a minimally invasive method of localised application. While direct / topical administration of ephrin-A1 would be preferred a systemic route is also contemplated. Additionally, implantable infusion 25 pumps, like those currently being used in humans would enhance translatability and ensure accurate timing and dosage of ephrin-A1 delivery to the site of injury to promote functional recovery. In context of ischemia, ephrin-A1 may be administered to, or proximal to, the ischemic core.

Topical administration of ephrin-A1 or a composition including ephrin-A1 may be to an 30 area at risk for the development of a glial scar, such as the exposed meninges immediately after a laminectomy operation. Topical administration may be in form of, but not limited to, a biodegradable polymer, hydrogel or emulsion. 29 1001272724

For any of the conditions described herein, when ephrin-A1, variant or analog thereof is topically or locally administered to a human, the therapeutically effective amount of corresponds to preferably between about 0.1 - 5mg/kg/day. 2015252036 03 Nov 2015

In any aspect of the present invention, ephrin-A1, variant or analog thereof or a 5 composition comprising ephrin-A1, variant or analog thereof may be administered after an event that causes glial scarring. Preferably, administration of ephrin-A1, variant or analog thereof or a composition comprising ephrin-A1, variant or analog thereof is commence between 3 to 7 days post-injury.

In any aspect of the present invention, administration of ephrin-A1, variant or analog I0 thereof or a composition comprising ephrin-A1, variant or analog thereof is administered for a period of 7, 8, 9, 10, 11, 12, 13 or 14 days. Typically, ephrin-A1, variant or analog thereof or a composition comprising ephrin-A1, variant or analog thereof is be administered as a continuous infusion via an osmotic / peristaltic pump locally to the site of injury over the dosing period. 15 Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavouring agents, colouring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as 20 calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binding agents such as starch, gelatine or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the 25 gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Liquid preparations for perioperative CNS surgery including brain, spinal cord and ophthalmic procedures may take the form of, for example, solutions or suspensions, or 30 may be presented as a dry product for its direct application (e.g. powder, gel or impregnated on a solid support) or reconstituted with water or other suitable vehicle (eg 30 1001272724 sterile pyrogen-free water) before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as emulsifying agents (eg lecithin or acacia), non-aqueous vehicles (eg almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-5 hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts and optionally, multiple active agents (e.g. antibiotics) in a suitable carrier such as physiological saline or Ringer's solution. The solution is applied by continuous irrigation of the wound during surgical procedures and diagnostic promote neuroprotection of the CNS. 2015252036 03 Nov 2015 10 Formulations for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active ingredient(s) in admixture with excipients 15 suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (for example, lecithin), condensation products of an alkylene oxide with fatty acids such 20 as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol mono-oleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene 25 sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a 30 vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as 31 1001272724 beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid. 2015252036 03 Nov 2015 5 Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavouring and colouring agents, may 10 also be present.

Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as 15 soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as polyoxyethylene sorbitan monoleate. An emulsion may also comprise one or more sweetening and/or flavouring agents. >0 Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.

Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and 25 mode of delivery. Topical vehicles include organic solvents such as alcohols (for example, ethanol, iso-propyl alcohol or glycerine), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerine, lipid-based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of 30 natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, protein-based materials such as collagen and gelatine, silicone-based materials (both nonvolatile and 32 1001272724 volatile), and hydrocarbon-based materials such as microsponges and polymer matrices. 2015252036 03 Nov 2015 A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, 5 suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, I0 such as hydroxymethylcellulose or gelatine-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules. A topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches. The physical appearance and viscosity of such 15 forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form. Solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or >0 enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity. Both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. 25 Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels, and often do not contain emulsifiers. 30 Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. 33 1001272724

Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not 5 limited to, protective colloids or nonionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer I0 or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within 15 topical formulations. 2015252036 03 Nov 2015

Preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other >0 hydroxy acids and their salts, glycerine, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colours include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, 25 abrasives, absorbents, anticaking agents, antifoaming agents, antistatic agents, astringents (such as witch hazel), alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.

Typical modes of delivery for topical compositions include application using the fingers, 30 application using a physical applicator such as a cloth, tissue, swab, stick or brush, spraying including mist, aerosol or foam spraying, dropper application, sprinkling, 34 1001272724 soaking, and rinsing. Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration (for example, as a transdermal patch). 2015252036 03 Nov 2015

Pharmaceutical compositions may be formulated as sustained release formulations such as a capsule that creates a slow release of modulator following administration. 5 Such formulations may generally be prepared using well-known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable. Preferably, the formulation provides a relatively constant level of modulator release. The amount of modulator contained within 10 a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

In another embodiment there is provided a kit or article of manufacture including ephrin-A1 as described herein, and/or pharmaceutical composition as described above. 15 In other embodiments there is provided a kit for use in a therapeutic or prophylactic application mentioned above, the kit including: - a container holding a therapeutic composition in the form of ephrin-A1 as described herein, or a pharmaceutical composition comprising ephrin-A1 as described herein; - a label or package insert with instructions for use. 20 In certain embodiments the kit may contain one or more further active principles or ingredients for treatment of a condition described herein, such as a CNS injury.

The kit or “article of manufacture” may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of 25 materials such as glass or plastic. The container holds a therapeutic composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used for treating the condition of choice. In one embodiment, the label or 35 1001272724 package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat a condition described herein, such as a CNS injury. 2015252036 03 Nov 2015

The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second 5 container with a second active principle or ingredient contained therein. The kit in this embodiment of the invention may further comprise a package insert indicating the composition and other active principle can be used to treat a disorder or prevent a complication stemming from a condition described herein. Alternatively, or additionally, the kit may further comprise a second (or third) container comprising a 10 pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In certain embodiments the therapeutic composition may be provided in the form of a 15 device, disposable or reusable, including a receptacle for holding the therapeutic, prophylactic or pharmaceutical composition. In one embodiment, the device is a syringe. The device may hold 1-2 mL of the therapeutic composition. The therapeutic or prophylactic composition may be provided in the device in a state that is ready for use or in a state requiring mixing or addition of further components. >0 It will be understood, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy. 25 The present invention also provides a surgical or implantable device for use in surgery of the CNS coated or impregnated with ephrin-A1. Preferably, the surgical or implantable device is a neuroprosthetic or implantable microelectrode. Any type of indwelling brain microelectrodes are contemplated such as Deep Brain Stimulating micro electrodes currently in use for Parkinson’s disease and chronic pain treatment or 30 microelectrode arrays for use in bionic eyes. 36 1001272724

Ephrin-A1, variant or analog thereof may be impregnated, incorporated into, or coated onto any surgical or implantable device described herein and may also be impregnated or incorporated into gel foams and hydrogels that are in used in neurosurgical applications. 2015252036 03 Nov 2015 5 Slow-dissolving hydrogel (e.g. Duraseal) may have ephrin-A1 incorporated into it. In the case of foams or sponges, such as Gelfoam, it can be incorporated directly into the structure of the foam or sponge.

For implantable devices, ephrin-A1 may be coated on to the surface of, or manufactured into the exterior of the device, thereby allowing ephrin-A1 to be released over time. For I0 example, coating the device with a slow-dissolving hydrogel will allow or permit release of ephrin-A1 in situ. For foams and sponges, such as Gelfoam, it will be released into brain tissue as the foam dissolves and is absorbed over time.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features 15 mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

It will be understood that these examples are intended to demonstrate these and other aspects of the invention and although the examples describe certain embodiments of the invention, it will be understood that the examples do not limit these embodiments to 20 these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and/or principles of the invention mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein.

Examples 25 Example 1

The non-human primate model described herein offers high reproducibility of ischemic lesion size and locality throughout life as well as near-identical pathophysiological responses to that of the post-ischemic human brain. The marmoset monkey (Callithrix jacchus) is a New World primate with a visual system that has been well characterised 37 1001272724 throughout development and adult life. Additionally, this model provides the opportunity for accurate downstream behavioural and functional assessments and testing of novel therapeutic strategies, which is of great clinical significance. 2015252036 03 Nov 2015

Animals 5 18 marmoset monkeys (Callithrix jacchus) aged postnatal day (PD) fourteen (n=9), and adults (>18 months; n=9) were utilised in this study, of which n=2 from each cohort were designated uninjured, non-sham controls. Seven animals from each age cohort were subjected to V1 focal ischemia, with post-injury survival of 1 day(s) post injury (DPI; n=2), 21 DPI (n=4) or >1 year (n=1). Gender was not a criterion in the selection of I0 animals, and no siblings were used. Animals were housed in family groups (12:12 hrs light/dark cycle, temperature 31 °C, humidity 65%). Experiments were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Monash University Animal Ethics Committee. 15 Endothelin-1 induced focal ischemia

Induction of focal ischemic injury to the infant and adult marmoset monkey V1 were performed by vasoconstrictor-mediated vascular occlusion of the calcarine artery (PCAca), as detailed previously (Teo and Bourne, Brain Pathology. 2014;24(5):459-74). Anaesthesia was induced and maintained using inspired isoflurane (0.5-4%). Following 20 craniotomy and dural thinning, intracortical injections of endothelin-1 (ET-1) proximal to the calcarine artery (distal P4 branch of the posterior cerebral artery), which supplies operculum V1, were performed. ET-1 doses of 0.1 pi / 30s pulse at 30s intervals, totalling ~0.5 μΙ_ for per site for infants (4 sites) and ~0.7 μΙ_ for adults (7 sites) were administered. Upon completion, the craniotomy was replaced and secured with tissue 25 adhesive (Vetbond; 3M) and the skin sutured closed. Endothelin-1 is a vasoconstrictor, which causes the constriction of the vascular smooth muscles, resulting in the occlusion of a brain-supplying artery. This results in the ischemic stroke. The focal characteristics of the stroke is achieved by targeting the distal P4 branch of the posterior cerebral artery (PCA) also known as the calcarine artery (PCAca) which supplies V1. 30 Fresh tissue collection 38 1001272724

At the end of the designated recovery periods, (1 DPI, 21 DPI, n=2; n=1 control) marmosets were administered an overdose of pentobarbitone sodium (100 mg.kg'1; intraperitoneal). Following apnea, cerebral tissues were removed and dissected under aseptic conditions in sterile ice-cold phosphate buffered saline (PBS; 0.1M; pH 7.2). 2015252036 03 Nov 2015 5 Hemispheres were separated and the occipital lobe was dissected at the level of the diencephalon and bisected coronally. Caudal portions, encompassing V1 and the ischemic-necrotic core were collected and snap-frozen at -40°C in liquid nitrogen.

Perfusion and fixed tissue processing

Animals were euthanased as above (21 DPI: n=2 +1 control). Following apnea, infants I0 were transcardially perfused with warm 0.1M heparinised phosphate buffered saline (PBS, pH 7.2) containing 0.1% sodium nitrite and adults with heparinised PBS, both followed by 4% paraformaldehyde (PFA). Brains were dissected, post-fixed and cryoprotected, as outlined in Warner etal., J Neurosci. 2012;32(48): 17073-85.

Following separation of the hemispheres, each hemisphere was bisected coronally at 15 the start of the caudal pole of the diencephalon and frozen in liquid nitrogen at -40°C.

Western blot

Proteins for western blotting were purified using the TRIzol-LS reagent (Life). Snap-frozen tissue were homogenised in 750pL per 50-100pg-tissue weight and processed according to the established protocols (Chomczynski, 1993; 15(3):532-4, 6-7). Purified 20 proteins were re-suspended in 1% sodium dodecyl sulfate (SDS; Sigma). Protein concentrations were determined through a modified Bradford protein assay (Bio-Rad). Following protein separation on 4-12% bis-tris polyacrylamide gel electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (PVDF; Millipore) and blocked in blocking buffer (1:1; Li-Cor) in 0.1 M PBS for 25 mins. Next, incubation with 25 primary antibodies (Table 1) in blocking buffer diluted 1:1 with 0.1% PBS-Tween overnight at 4°C was undertaken. Following washes, membranes were incubated with secondary antibodies (Table 2) in 0.1% PBS-Tween containing 0.02% SDS for 1 hr at room temperature. Protein bands were visualised using the Odyssey CLx infrared imaging system (Li-Cor). 30 Co-immunoprecipitation 39 1001272724

Tissue for immunoprecipitation was homogenised at 4°C in NP-40 lysis buffer (Life; optimised for membrane-bound proteins) with 1mM phenylmethanesulfonyl fluoride (PMSF; Sigma) and 1X protease inhibitor cocktail (Sigma). Following a 2 hr incubation with agitation at 4°C, insoluble debris was pelleted by centrifugation at 12,000 X g for 15 5 mins at 4°C. Supernatant was collected, aliquoted and stored at -20°C. Protein concentration was determined as above. Immunoprecipitation was performed using magnetised Dynabeads protein-A kit (Life). EphA4 antibody (5pg; SCBT) was preincubated with Dynabeads-protein A for 1 hr at room temperature with agitation. Tissue lysates (500pg.ml'1) were added and incubated overnight at 4°C with agitation. 2015252036 03 Nov 2015 I0 Dynabeads-Ab-Ag complexes were magnetically separated from the supernatant and washed in PBS before elution in the supplied elution buffer. Resulting eluate was pH adjusted with 1M Tris-HCL and separated on 4-12% bis-tris polyacrylamide gel electrophoresis and immunoblotted as outlined above.

Immunohistochemistrv and immunofluorescence 15 For immunohistochemistry, free-floating sections were washed in PBS before treatment with 0.3% hydrogen peroxide and 50% methanol in PBS for 20 min to inactivate endogenous peroxidases. Sections were pre-blocked in a solution of 10% normal goat serum in PBS + 0.3% Triton X-100 (TX; Sigma) before incubation with primary antibodies overnight at 4°C (Table 1). Sections were rinsed in 0.1% PBS-Tween prior to 20 incubation with biotinylated secondary antibodies (Table 2) for 1 hr at room temperature. Sections were then treated with streptavidin-horseradish peroxidase conjugate (GE Healthcare; 1:200) and visualised via a metal-enhanced chromogen, 3,3’-diaminobenzidine (DAB; Sigma).

Table 1: Primary antibodies used

Category Host Clonality Target Source Cat# Application Ms M Calbindin D28K Swant 300 Cell Ms M Calretinin Swant 6B3 IF marker Ms M Glial Fibrillary Millipore MAB 360 40 1001272724 2015252036 03 Nov 2015

Rb P Acidic Protein AB 5804 Ms M Parvalbumin Swant 235 Ms M ephrin-A1 SCBT SC377165 Rb P ephrin-A1 Abeam AB97527 IHC / IF/ WB Ms M ephrin-A2 Abeam AB123877 Rb P ephrin-A2 SCBT SC912 Rb P ephrin-A3 Abeam AB64814 WB ephrins Rb P ephrin-A5 SCBT SC20722 IHC / IF/ WB Gt P ephrin-A5 SCBT SC6075 Gt P ephrin-B1 R&D AF473 WB Rb P ephrin-B2 SCBT SC15397 Rb P ephrin-B3 Abeam AB53063 Rb P EphA4 SCBT SC921 Ephs Ms M EphA4 Invitrogen 37-1600 IP / IF / WB Loading control Ms M b-actin Abeam AB8224 WB

Legend: Ms- mouse, Rb- rabbit, Gt- goat, IF- immunofluorescence, IHC- Immunohistochemistry, WB-western blotting, IP- Immunoprecipitation, M- Monoclonal, P- Polyclonal

Table 2: Secondary antibodies used in this study

Category Host Target Target isotype Conjugate Source Cat# IHC Gt Ms igG Biotin DAKO E0433 41 1001272724 2015252036 03 Nov 2015

Gt Rb IgG Biotin E0432 Gt Ms IgG AF 488 A-11001 Gt Ms lgG1 AF 647 LIFE A-21240 Gt Rb IgG AF 594 A-11037 Gt Ms IgG IRDye 680 926-68020 Gt Rb IgG IRDye 680 Li-Cor 926-68021 Gt Rb IgG IRDye 800 926-32211 D Gt IgG IRDye 800 926-32214

Legend: IHC- Immunohistochemistry, IF- Immunofluorescence, WB- Western blot, D- donkey, Gt- goat, Ms- mouse, Rb- Rabbit, AF- Alexa fluor

Immunofluorescent antigen detection was conducted with similar procedures to those above, except steps including and after secondary antibody incubation were performed in the dark. Following overnight incubation at 4°C with a primary antibodies in blocking 5 solution (Table 1). Sections were then rinsed in 0.1% PBS-Tween before incubation with secondary antibodies (Table 2). The rabbit anti-EphA4 antibody used in IF experiments was raised against a peptide corresponding to amino acids 938-953 of the intracellular Sterile Alpha Motif domain of EphA4. Sections were also treated with either Floechst 333258 or 4',6-diamidino-2-phenylindole (DAPI) nuclei stains. 10 Fluorescent Immunocytochemistry (ICC) experiments were performed similarly.

Incubation of coverslips with primary antibodies was performed for 2 hrs and secondary antibodies for 30 mins, at room temperature.

Flistology

Nissl-substance staining was performed to aid in the anatomical and laminar 15 demarcation of V1 in the infant and adult marmoset brain based on methods previously described (Teo and Bourne, 2014, supra). 42 1001272724

Generation of neurospheres and astrocytes 2015252036 03 Nov 2015

Neurospheres were generated from tissue excised from marmoset V1 at PD14. Excised tissue was minced with a scalpel blade and dissociated in 0.25 mg ml_'1 trypsin, lOpg.mL"1 DNase I, 10 mM HEPES and 0.2 mg.mL'1 EDTA in Ca2+/Mg2+-free Hanks 5 buffered salt solution at 37°C for 15 min. Dissociated cells were pelleted, resuspended and plated (3x104 cells.cm'2) on low-adhesion 100mm diameter culture dishes in NeuroCult NSA medium with proliferation supplement (lOng.mL'1 FGF-2, 20ng.ml_'1 rhEGF) and incubated at 37°C, 5% CO2. Neurospheres formed after 1-week incubation and were large enough to be passaged after 3 weeks. Multipotency of neurospheres I0 was maintained up to 3 passages beyond which only GFAP+ astrocytes were generated (Homman-Ludiye etal., Cereb Cortex. 2014;24(11):2884-98).

Ephrin ligand clustering

Recombinant human (rh) ephrin-A1-Fc (6417-A1-050), -A2-Fc (7856-A2-050) and -A5-Fc (374-EA-200) ligands, as well as rh-EphA4-Fc (6827-A4-050), all obtained from R&D 15 systems, were used in the subsequent in vitro assays. EphA4-Fc or ephrin-Fc ligands (67pg.pL'1) were pre-clustered through incubation with anti-human-Fc antibody (54 pg.pL'1; Sigma; 112136)) at 37°C for 2 hrs and adjusted to the final concentration (10 pg.pL'1) using the appropriate culture medium. Negative controls were performed for all experiments using antibody-clustered rh-IgGi-Fc. Additional positive controls in wound 20 closure and proliferation assays were performed using treatment with the cytokine leukemia inhibitory factor (LIF; lOOOU.mL'1; Sigma) as previously described (Goldshmit etal., Eur J Neurosci. 2014;39(9): 1419-28).

Wound-closure assay 8-well chambers slides were coated overnight with 1 mg.mL'1 poly-L-lysine solution at 25 37°C, rinsed and coated with 1 mg.mL'1 laminin in MEM for 2 hrs at 37°C. Dissociated marmoset neurosphere-derived astrocytes were plated at high density (35x104 cells/well) in NeuroCult NSA medium with proliferation supplement and allowed to recover overnight. Cells were incubated for 45 mins with 10μΜ CellTracker Green CMFD (Life; #7025) diluted in the fresh culture medium. A longitudinal scratch was then 30 performed across the astrocyte monolayer using a sterile 200pL pipette tip, making sure 43 1001272724 not to disturb the laminin coating. Wells were rinsed once before the addition of 200μΙ_ of NeuroCult NSA medium containing proliferation supplement and the pre-clustered EphA4, ephrin ligands, human-Fc and LIF controls. Slides were transferred to a humidity-controlled chamber (37°C, 5% CO2) fitted on a Leica AXF600 LX inverted 5 fluorescent microscope and multi-positional time-lapsed imaging was performed over 48 hrs at 30 min intervals for temporal qualitative and quantitative analyses. 2015252036 03 Nov 2015

Proliferation assay

Chambers slides were coated overnight with Img.mL'1 poly-L-lysine and laminin as described. Astrocytes were plated at low density (2.625 x104 cells/well) in NeuroCult I0 NSA medium with proliferation supplement and allowed to recover overnight. 200pL of NeuroCult NSA medium containing the pre-clustered ephrin ligands, human-Fc and LIF controls were added in each well in the presence of 0.2μΜ BrdU. Cells were incubated at 37°C in 5% CO2 for 48 hrs and fixed in 4% PFA for subsequent qualitative and quantitative analyses. I5 Stripe assay 13mm diameter glass coverslips were treated overnight in a solution of 2M HCI at 60°C and sonicated in successive baths of H2O and graded concentrations of EtOH. Coverslips were coated with poly-L-lysine and laminin (see above) were rinsed with H20, dried and positioned coated side down on a silicone matrix with 50pm wide ?0 alternating grooves 50pm apart. Pre-clustered ephrin ligands or human Fc were injected into the matrix and incubated for 2 hrs at room temperature. Following a PBS flush, a solution of 2% BSA-Alexa488 (Invitrogen) in PBS was injected into the matrix and incubated for 2 hrs to visualise the stripes. Following a second PBS flush, coverslips were place in 24-well plate, coated with Laminin (Img.mL'1 in MEM) for 2 hrs at 37°C. 25 Astrocytes were plated (4 x104 cells) on each coverslip in NeuroCult NSA with differentiation supplement and incubated for 18 hrs. Cells were fixed in 4% PFA after 18 hrs incubation for downstream qualitative and quantitative analyses.

Microscopy

Standard brightfield and epifluorescent microscopic examination of processed sections 30 were conducted using Zeiss SteREO discovery (V20) and Axio Imager Z1 microscopes 44 1001272724 (Zeiss). Images were obtained with a Zeiss Axiocam (HR rev3) using Axiovision software (V4.8.1.0; Zeiss). Tile-scanning and high magnification confocal microscopy was performed using a Zeiss LSM5 (Zeiss) and Leica TCS SP5 (Leica Microsystems) and images obtained using the Zeiss Efficient Navigation (ZEN; Zeiss) and Leica 5 Application Suite Advanced Fluorescence (LAS AF; Leica Microsystems) softwares, respectively. All photomicrographs were cropped and resized using Photoshop (CS6 v13.0.0, Adobe) and GNU Image Manipulation Program (GIMP; V2.8.10). Necessary manipulation (eg. Resolution adjustment, pseudocolouring, brightness/contrast adjustments, image cropping, photomerging and the addition of legends, arrows and I0 bounding boxes) was performed to aid in analysis and improve quality of final images. 2015252036 03 Nov 2015

Quantitative and statistical analyses

Protein band fluorescent intensity analysis was performed using Image Studio Software (Li-Cor; version 4.0.21), as previously described (Falivelli etal., 2013). Protein band signal was calculated as: Signal = Total pixel intensity - (background intensity x area). 15 Quantitative analysis of protein band signals was performed on triplicated experiments for each sample and comparisons were made within age groups only. No comparisons were made between age groups. Protein electrophoresis and immunoblots for each experiment used for quantitative analysis were performed concurrently. Data was presented in histogram form as a ratio of signal over background ± standard error of ?0 mean (SEM) for statistical analyses.

The degree of receptor / ligand puncta colocalisation was calculated as the Pearson’s coefficient (Rr) which describes the puncta correlation between two channels (Teo et al., The Journal of comparative neurology. 2012;520(13):2941-56). In brief, the red (EphA4) and green (ephrins) channels from confocal microscopy were input into 25 CoLocalizer Pro software (V2.6.1-198; Colocalizstion Research Software Inc. & Sente, Zurich, Switzerland), background value normalised and the Pearson’s coefficient (Rr) was calculated. Rr = 1 represents high linear correlation (direct colocalisation) between red and green channels and Rr = -1 represents low or no correlation (no colocalisation).

Wound closure assay analyses were performed using time-lapsed images of > 2 non-30 overlapping positions per experiment (n=3) over 48 hrs. A 2000pm2 grid was superimposed over each time-lapsed stack and boxes within the scratch wound were 45 1001272724 counted. Boxes were omitted from counts if the fluorescently labelled astrocyte cell bodies were either partly or completely within its boundary. The progress of wound-closure was analysed from T0 to T48 at 1 hr intervals. Results were represented in a line graph representing the mean percentage of wound closure (Tx/T0 x 100%) over time ± 2015252036 03 Nov 2015 5 SEM. Additionally, the median % wound-closure ± interquartile range at the experiment end point (48 hrs) was presented in a histogram for statistical analyses.

Cell counts for stripe assays were performed using Fiji (NIH; version 1.44o). A 500x500pm (250000pm2) grid was superimposed over 10 non-overlapping fluorescent photomicrographs of repeated stripe assay experiments (n=4). Only GFAP+ astrocytes I0 with Floechst-labelled nuclei completely within the boundary of stripes or completely in the inter-stripe spaces were counted. Astrocytes with nuclei that were only partially on the stripes were excluded. Data were presented in histograms as the median percentage of GFAP+ nuclei in contact with stripes over total nuclei counted / 0.25mm2 ± interquartile range followed by statistical analyses. 15 Cell counts for proliferation assays were conducted using Fiji, over 10 non-overlapping photomicrographs per experiment (n=3). A 1000pm2 grid was superimposed over each photomicrograph. Total Floechst+ nuclei were counted in addition to BrdU+ / Hoechst+ nuclei for each treatment condition. Results were presented in histograms as the median percentage of BrdU+ nuclei over total Floechst+ nuclei counted / 1000pm2 ± >0 interquartile range for statistical analyses.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism (version 6). D’Agostino and Pearson normality tests were performed on all quantitative data to determine normality of data distribution prior to statistical analyses. A parametric Student’s t-test was used to 25 determine statistical significance in fluorescent protein band. Statistical analysis of wound-closure, stripe and proliferation assays were performed using the non-parametric analysis of variance (ANOVA; Kruskal-Wallis test; KW) followed by the Dunn’s multiple comparisons post-hoc test. A non-parametric Mann-Whitney U test (MW) was used to determine statistical significance of ephrin-A1 inhibition of astrocyte reactivity compared 30 to non-treated ephrin-A2 and -A5 groups. 46 1001272724

Example 2 2015252036 03 Nov 2015

The infant and adult V1 exhibit different glial scar extent in the chronic period after focal ischemia

The aim of this study was to determine if the sub-acute astroglial difference observed 5 continued into the chronic period (> 1 year) following infant and adult stroke. In both cases, dense immunofluorescent glial fibrillary acidic protein (GFAP)+ reactive astrocyte labelling was detected proximal to the infarct core (Fig. 1C-F) compared to adult control V1 (Fig. 1A-B). The results revealed that the glial scar >1 year after infant stroke retained the discrete astrocyte distribution observed at 21 DPI, i.e. concentrated I0 proximal to the infarct core (Fig. 1C-D). In contrast, the chronic adult scar also retained the denser and more widespread distribution of reactive astrocytes (Fig. 1E-F) observed at 21 DPI. This result demonstrates that the reactive astrogliotic responses are different between the infant and adult brain after stroke. Specifically, in contrast to infants the adult brain undergoes a more prolific, long-lasting reactive astrocyte response after 15 focal ischemia that is sustained by the presence of a glial scar 1 year post-injury.

Example 3

EphA4 is increased on reactive astrocytes after both infant and adult stroke

Flere is revealed an increase in EphA4 expression on GFAP+ reactive astrocytes distributed proximally to the ischemic core in infants (Fig. 1H-J) and adults (Fig.1L-N) at 20 21 DPI, compared to their respective controls (Fig. 1G, K). Further analysis confirmed the higher EphA4 immunofluorescent intensity was detected on the morphologically distinct GFAP+ reactive astrocytes (infant, Fig. 1P; adult, Fig. 1R) compared to control/ resting astrocytes (infant, Fig 10; adult, Fig. 1Q). Immunoblot analysis revealed no significant change in EphA4 expression levels at 1 DPI, compared to controls. However, 25 a statistically significant increase in EphA4 expression levels was detected in the infant (Fig. 1S, U) and adult (Fig. 1T, V) post-ischemic V1 tissue lysates by 21 DPI, compared to their respective controls (p <0.05).

Example 4

Different ephrin ligands are expressed in the infant and adult at the site of injury 47 1001272724

To identify candidate ephrin ligands involved in EphA4 signalling on reactive astrocytes after focal ischemia, a Western blot screen was performed to investigate the expression profiles of ephrin-A1, -A2, A3, -A5, -B1, -B2 and -B3 in the normal and post-ischemic infant and adult marmoset V1 (results summarised in Table 3). Ephrin-A4, -A6 and -A7 5 was excluded from this screen; ephrin-A4 and -A6 has never been reported beyond the developing retina, and ephrin-A7 has never been reported in the CNS. 2015252036 03 Nov 2015 A Western blot screen revealed that baseline levels of ephrin-A1, -A2, A3, -A5, -B1, -B2 and -B3 were detected in control infant V1 (Table 3). After focal ischemia, ephrin-A1 expression was significantly increased at 1 DPI with a more profound increase detected I0 at 21 DPI (Fig. 2A-B). No significant change in ephrin-A2 and -A5 expression was detected at either 1 DPI or 21 DPI, relative to control levels (Fig. 2C-F; Table 3). In adults, baseline levels of only ephrins-A2, -A3, -A5 and -B1 were detected in controls (Table 3). Adulthood expression of ephrin-A1 was not detected in either control or postischemic V1 (Fig. 2A-B). At both post-ischemic time points tested, significant increases 15 in ephrin-A2 and -A5 were detected, contrary to that observed in the post-ischemic infant V1 (Fig. 2C-F; Table 3).

Table 3: Expression of ephrins in the infant and adult revealed through Western blot analysis. ephrins Infant Adult Control 1 DPI 21 DPI Control 1 DPI 21 DPI A1 I + ++ O 0 0 A2 I I I I ++ A3 I I I I 1 I A5 I I I I ++ B1 I I I I 1 I B2 I I I 0 0 0 48 1001272724 0 0 0 2015252036 03 Nov 2015 B3

Legend: |: Baseline. +: increased. 0: not detected.

Example 5

Ephrin-A1, -A2 and -A5 interacts with EphA4 in the normal and post-ischemic V1 at different stages of life 5 To determine if the candidate ephrins were responsible for EphA4 receptor activation, co-immunoprecipitation experiments were performed on post-ischemic infant and adult V1 lysates at 21 DPI. Following antibody mediated pull-down of EphA4, immunoblots were performed to detect for ephrin-A1 in infant lysates as well as ephrin-A2 and -A5 in adult lysates. In infants, ephrin-A1 was detected on immunoblots from eluate of control I0 and 21 DPI V1 lysates (Fig. 2H), which implied interaction with EphA4. Ephrin-A1 was not detected in eluate from either control or post-ischemic adult lysates (Fig. 2H), consistent with Western blot data. In adults, V1 lysates revealed the presence of both ephrin-A2 (Fig. 2I) and ephrin-A5 (Fig. 2J) after EphA4 pull-down, confirming EphA4 interaction. 15 Example 6

Ephrin-A1 and ephrin-A2/ -A5 immunoreactive cells are distributed proximal to the ischemic core after stroke

Further investigation was conducted into the distribution of ephrin-A1 and ephrin-A2/ -A5 immunoreactive cells in the post-ischemic infant and adult V1, respectively. 20 Immunoperoxidase experiments revealed that in infants ephrin-A1 expression was detected at very low levels in control V1 (Fig. 3A) but was markedly increased at 21 DPI proximal to the injury site (Fig. 3B-C). Immunoperoxidase labelling intensity of ephrin-A1 was greatest proximal to the ischemic core with a gradual decline distally (Fig 3i-iv). In the normal adult V1, cellular labelling of ephrin-A2 (Fig. 3D) and -A5 (Fig. 3E) was 25 detected at baseline levels in V1. At 21 DPI, ephrin-A2 and -A5 immunohistochemistry revealed a striking increase in cellular labelling intensity, compared to their respective controls (Fig. 3H-J), particularly in the white matter underlying the ischemic core. 49 1001272724

Ephrin-A2 (Fig. 3v-viii) and -A5 (Fig. 3ix-xii) immunopositive cells were similarly distributed at higher density proximal to the infarct core, diminishing distally. 2015252036 03 Nov 2015

Example 7

Ephrin-A1 (infants), ephrin-A2 and -A5 (adults) are increased on reactive astrocytes 5 after focal ischemia

Immunofluorescence double labelling was performed to identify the neural cell types associated with the increased expression of ephrin-A1 and ephrin-A2/ -A5 in the normal and post-ischemic infant and adult V1, respectively. In control infant V1, immunofluorescent labelling revealed low levels of ephrin-A1 expression on GFAP+ I0 astrocytes (Fig. 4A). Ephrin-A1 immunofluorescent labelling remained specific to GFAP+ reactive astrocytes after V1 stroke but was accompanied by marked increase in labelling intensity proximal to the ischemic core (Fig. 4B), consistent with increased ephrin-A1 expression. In adult controls, immunofluorescent analysis revealed expression of both ephrin-A2 and -A5 ligands on a subset of cortical interneurones 15 (combination of calbindin, parvalbumin and calretinin labelling), with no expression detected on cortical or subcortical astrocytes (Fig. 3F-G). After focal ischemia, no change in ephrin-A2/ -A5 labelling intensity was detected on cortical interneurones. However, expression of ephrin-A2 (Fig. 4C) and -A5 (Fig. 4D) was present on GFAP+ reactive astrocytes proximal to the ischemic core, with distribution pattern consistent >0 with immunoperoxidase data (Fig. 3I, J; v-xii).

Example 8

EphA4 and ephrins are co-expressed on reactive astrocytes in the post-ischemic infant and adult V1

The results herein suggests that the EphA4 receptor and upregulated ligands: ephrin-A1 25 (infants) and ephrin-A2/ -A5 (adults), are expressed on reactive astrocytes in near identical distribution, proximal to the infarct core. It was subsequently examined if the expression of EphA4 and ephrins are segregated into distinct receptor/ ligand expressing sub-populations or co-expressed in identical populations. Double-immunofluorescent detection of ephrin-A1 and EphA4 was performed on post-ischemic 30 infant V1 sections. High-magnification confocal imaging revealed co-expression of 50 1001272724 ligand and receptor on reactive astrocytes proximal to the ischemic core (Fig. 4E). In adults, a triple-immunofluorescent detection of ephrin-A2 (or ephrin-A5), EphA4 and GFAP was performed on post-ischemic V1 sections, proximal to the ischemic core. Analysis revealed that in both cases, ephrin-A2 (Fig. 4F) and -A5 (Fig. 4G) was co-5 expressed on EphA4+/ GFAP+ reactive astrocytes proximal to the ischemic core. Colocalisation analysis revealed that while receptor and ligands are co-expressed, the degree of puncta colocalisation (Pearson’s coefficient; Rr) between ephrin-A1 (Fig. 4E; Rr = -0.61), ephrin-A2 (Fig. 4F; Rr = -0.38) and ephrin-A5 (Fig. 4G; Rr = -0.43) with EphA4 was extremely low. This indicates little to no colocalisation of receptor and ligand I0 fluorescent puncta on the same cell. 2015252036 03 Nov 2015

Example 9

Ephrin-A2 and -A5 treatment promotes astrocyte wound closure but eohrin-A1 does not

To determine the effect of ephrin-A1, ephrin-A2 and ephrin-A5 treatment on astrocyte reactivity, wound-closure assays were performed using marmoset neurosphere-derived 15 astrocytes. The results revealed significant differences in astrocyte wound closure capacity over 48 hrs (Fig. 5A). Ephrin-A5 treatment resulted in 100% wound closure (Fig. 5A, E; 6A; p <0.0001) compared to the 25% wound closure achieved in negative controls (Fig. 5A, B; 6A). The ephrin-A5 result was similar to the extent of wound closure achieved in EphA4- (Fig. 5A; 6A; ~94% closure; p <0.001) and leukaemia 20 inhibitory factor (LIF)-treated (Fig. 5A, F; 6A ~92% closure; p <0.001) conditions at 48 hrs. To a lesser extent, ephrin-A2 treatment was also able to increase astrocyte wound-closure (Fig. 5A, D; 6A; 76% closure; p <0.001) compared to negative controls.

Flowever, a similar effect was not observed following ephrin-A1 treatment, which achieved a 36% wound closure that was not significantly different to that of the negative 25 controls (Fig. 5A, C; 6A; p >0.05).

Example 10

Ephrin-A2 and -A5 attracts but ephrin-A1 repels astrocytes in vitro

Next investigated was the effects of the ephrins on astrocyte guidance, which potentially contributes to the astrocyte behaviour observed in the wound-closure assay. Stripe 30 assays were performed to investigate the effects of ephrin-A1, -A2 and -A5 forward 51 1001272724 signalling on the guidance of marmoset astrocytes (Fig. 6B-E). Migration of astrocytes onto ephrin-containing stripes constitutes cell attraction and vice versa. Initial fluorescent immunocytochemistry revealed that virtually all GFAP+ astrocytes detected were also immunopositive for EphA4 expression (Fig. 6B-E). Quantitative analysis 5 revealed that compared to control stripes, on which astrocytes exhibited no preferential distribution (Fig. 6B, F; -49% on stripes), a significantly lower proportion of GFAP+ astrocytes were distributed on ephrin-A1 stripes (Fig. 6C, F; -39%; p <0.0001). The majority of astrocytes were preferentially distributed in between ephrin-A1 stripes, consistent with cell repulsion. In contrast, a significantly larger proportion of astrocytes I0 were preferentially distributed on ephrin-A2 (Fig. 6D, F; -80%; p <0.0001) and -A5 (Fig. 6E, F; -69%; p <0.0001) stripes compared to controls, consistent with cell attraction. 2015252036 03 Nov 2015

Example 11

Astrocyte proliferation is increased by eohrin-A2 and -A5 treatment but is reduced by ephrin-A1 in vitro 15 To investigate the effect of ephrin-A forward signalling on astrocyte proliferation, marmoset astrocytes were treated with ephrin-A1, -A2 and -A5 in the presence of 5-bromo-2'-deoxyuridine (BrdU). Quantitative analysis revealed an increase in the percentage of BrdU+ astrocytes over total cell population following ephrin-A2 (67.5%; p <0.05) and -A5 (71.4%; p <0.0001) treatment, compared to negative controls (Fig. 6G; 20 58%). The degree to which ephrin-A5 treatment increased astrocyte proliferation was equal to that of EphA4 (71.5%; p >0.05) and LIF-treated positive controls (Fig. 6G; 74.1%; p >0.05). Additionally, the effect of ephrin-A1 on astrocyte proliferation was investigated. In contrast to ephrin-A2 and -A5, only 39.0% of astrocytes counted were BrdU+ following ephrin-A1 treatment, which was significantly lower to that of negative 25 controls (Fig. 6G; p <0.0001).

Example 12

Ephrin-A1 treatment inhibits astrocyte reactivity induced bv eohrin-A2 and -A5

Based on the results herein, it is postulated that ephrin-A1 signalling most likely functions to limit astrocyte reactivity in the post-ischemic infant brain, correlating to a 30 more discrete glial scar outcome. To test this the astrocyte wound-closure experiment 52 1001272724 was repeated in the presence of ephrin-A2 and -A5, respectively in combination with ephrin-A1. Quantitative analysis revealed a significant reduction in astrocyte wound-closure capacity when ephrin-A1 was administered in combination with either ephrin-A2 (Fig. 7A, B; KW p < 0.0001) or A5 (Fig. 7C, D; KW p < 0.0001). In ephrin-A2 treated 5 groups, the addition of ephrin-A1 reduced the final extent of wound closure by ~42%, achieving only 44.5% closure (Fig. 7E; MW p < 0.01), compared to the initial 76.3% achieved with ephrin-A2 treatment alone. Ephrin-A1 treatment in combination with ephrin-A5 similarly reduced the extent of final wound closure by ~37%, achieving only 62.7% closure (Fig. 7F; MWp < 0.01) compared to the initial 100% closure achieved I0 with ephrin-A5 treatment alone. 2015252036 03 Nov 2015

In this study, the ephrin ligands involved in the EphA4-mediated reactive astrocyte response and glial scar formation following ischemic stroke in the infant and adult primate V1 were identified. In infants, ephrin-A1/ EphA4 signalling most likely limits reactive astrogliosis after ischemic stroke, whereas in adults, ephrin-A2 or -A5/ EphA4 15 signalling promotes and exacerbates the astrogliotic response. It was also demonstrated that ephrin-A1 treatment reduced astrocyte reactivity induced by ephrin-A2 and -A5 forward signalling in vitro, suggesting a potential target for future drug therapy development.

This study demonstrates for the first time the differences in chronic glial scarring after 20 ischemic stroke in the infant and adult NHP brain. The adult primate brain undergoes a more prolific reactive astrocyte response after ischemic stroke compared to infants. The more discrete glial scar in the post-ischemic infant V1 after 1 year recovery reflects the lesser degree of astrocyte involvement. The reduced glial scarring is notable when compared to the post-ischemic adult, where reactive astrogliosis leads to a denser, 25 more widespread scar formation. This clearly indicates that regulatory factors, such as the molecular guidance cues, differentially modulate the astrogliotic response in the post-ischemic infant and adult brains.

The receptor tyrosine kinase (RTK) EphA4 was observed to be increased on reactive astrocytes after focal ischemic stroke in the infant and adult NFIP at 21 DPI. The timing 30 of EphA4 upregulation coincided with the peak of reactive astrocyte accumulation observed in NFIP after ischemic stroke. The results demonstrate that the ephrin ligands 53 1001272724 responsible for EphA4-mediated reactive astrogliosis after ischemic stroke differs depending on the age at which the injury occurred. In infants, ephrin-A1/ EphA4 forward signalling limits reactive astrocyte accumulation proximal to the ischemic core through cell repulsion and suppression of astrocyte proliferation. Conversely, ephrin-A2 and -A5 5 forward signalling after adulthood stroke promotes astrocyte reactivity by increasing proliferation and conferring cell attraction that potentially induces astrocyte recruitment and rapid wound closure. This would certainly account for the sparse and discrete subacute distribution of reactive astrocytes after infant stroke compared to the more severe astrogliotic response observed here after adulthood stroke. In addition, ephrin-A2/ 2015252036 03 Nov 2015 I0 EphA4 signalling is a motogenic factor and may be required for the rapid wound closure and recruitment reactive astrocytes to the ischemic core. Ephrin-A2 and -A5 induced astrocyte attraction may also contribute to establishment of the dense, entangled and tightly woven meshwork of astrocytic processes that is an integral component of glial scarring. 15 The reasons as to why and when the shift from ephrin-A1 to ephrin-A2/ -A5 expression on reactive astrocytes occurs in the primate brain across postnatal life remain unclear. The heterogeneity of astrocytes may contribute, in part, to the discrepancy in reactive astrogliotic responses throughout life. For example, glial scar borders are formed by newly proliferated astrocytes in response to injury. This is consistent with the >0 observation that ephrin-A2/ -A5 induced astrocyte proliferation contributed to a more severe astrogliotic response and glial scarring. The reduction in reactive astrocyte proliferation in the post-ischemic infant, through ephrin-A1 forward signalling, would also account for the less severe glial scarring observed. In addition, previous work has demonstrated that lower levels of CSPGs are secreted during glial scarring after early-25 life CNS injuries, resulting in a glial scar that is less inhibitory to neurite outgrowth compared the adult. This suggests that the infant glial scar is more permissive towards regeneration. While ephrin-A1 is an important prognostic factor in cancer research, less is known about its role in the CNS. The expression of ephrin-A1 on immature astrocytes in the uninjured infant V1 might indicate a role for the postnatal maturation of V1. 30 Without being bound by any theory or mode of action, ephrin-A1 may potentially be involved in the migration of immature astrocytes into the neocortex during early postnatal stages by modulating their migration or regulating astrocyte adhesion site 54 1001272724 dynamics. The absence of ephrin-A1 in adults, even after ischemic stroke, suggests a role that it is limited to developmental periods, subsequently decreased as the CNS matures and but can be upregulated in response to injury during this period. Similarly, the expression of ephrin-A2/ -A5 by adult but not infant astrocytes further supports the 5 notion that reactive astrocyte populations in the infant and adult brains may be functionally distinct. 2015252036 03 Nov 2015

The co-expression of EphA4 and its ligands on reactive astrocytes after infant and adulthood stroke implies a role for Eph/ ephrin bidirectional signalling in astrocyte-astrocyte communication after injury. Eph/ ephrin signalling can occur in either a cis I0 (same-cell interaction) or trans (cell-cell interaction), although a cis interaction inhibits trans interaction. Minimal cis interaction was observed through lack of receptor-ligand colocalisation after stroke in infants and adults, suggesting the subcellular localisation of EphA4 and its ligands on separate membrane micro-domains. In addition, administration of EphA4-Fc in functional assays resulted in increased astrocyte 15 proliferation and rapid wound closure, most likely through ephrin-A2 and -A5 reverse signalling. This suggests that this co-expression facilitates astrocyte-astrocyte communication to enable simultaneous, bidirectional trans-activation of adjacent astrocytes upon the onset of injury. While the exact mechanism of ephrin-A2/ -A5 reverse signalling through EphA4 activation is beyond the scope of this study, it is likely >0 that ephrin-A reverse signalling partners such as Ret or p75NTR may be involved. An EphA4/ ephrin-A2 or -A5 bidirectional signalling cascade could account for the exaggerated astrogliotic response that often occurs after adulthood CNS injuries, resulting in more severe glial scarring.

The results of this present study indicate that ephrin-A1 would be beneficial to reduce 25 glial scarring following ischemic stroke in adults. Based on the functional assays, it is postulated that ephrin-A1 treatment will attenuate EphA4-mediated astrogliosis induced by ephrin-A2/ -A5 signalling after injury. The exact mechanisms by which ephrin-A1 inhibits or disrupts EphA4 signalling in the presence of ephrin-A2/ -A5 post-stroke is unclear. One explanation for this inhibition is through competitive binding of exogenous 30 ephrin-A1 to astrocytic EphA4, similar to the previously demonstrated competitive inhibition of EphA4 signalling through monomeric ephrin-A5. Ephrin-A1 binds EphA4 at higher affinity compared to ephrin-A2, while ephrin-A5 retains the highest EphA4- 55 1001272724 binding affinity overall. This is consistent with the functional analysis demonstrating a greater reduction in astrocyte reactivity following ephrin-A1 administration in the ephrin-A2 treated group. 2015252036 03 Nov 2015

Previous studies have demonstrated that reactive astrocytes support surviving 5 neurones by scavenging excessive excitotoxic compounds as well as playing a neuroprotective and neurotrophic role in the acute period after injury. However, their exaggerated response, especially after adulthood injuries, hinders regenerative processes. Most treatment strategies targeting astrocyte reactivity and glial scarring currently being trialled are administered soon after the injury, which may impede the I0 neuroprotective function of the astrocyte in the acute period post-injury. The advantage of reinstating ephrin-A1 signalling in the adult after ischemic stroke may induce ‘infantlike’ astrogliotic responses without compromising neuroprotection, leading to a more discrete ‘infant-like’ scar that is more permissible towards functional recovery.

Example 12 15 Eohrin-A1 in vivo rat trial

The results of the following in vivo experiments show that administration of ephrin-A1 after a needle-stick injury in the rat brain resulted in: 1. Reduction in overall GFAP+ immunoreactivity proximal to the injury core - implies reactivity and density of reactive astrocyte response is reduced 20 2. Statistically significant reduction in overall glial scar volume; 3. Statistically significant changes in the distribution and density of reactive astrocyte population in both proximal and distal portions; 4. Statistically significant reduction in astrocyte density in proximal and distal portion -ephrin-A1 treatment successfully reduced the secondary migration / recruitment of 25 reactive astrocytes from distal portion into the injury core (a major hallmark of glial scarring); 5. Statistically significant reduction in apoptotic (dying) cells at the ischemic core -implies a reduction in secondary injury severity and may play neuroprotective roles. 56 1001272724

Summary: Ephrin-A1 treatment in vivo successfully suppressed the reactive astrocyte response after brain injury resulting in a reduction of the density and severity of glial scar, as well as an important suppression of secondary recruitment. 2015252036 03 Nov 2015

Materials and Methods 5 A total of 5 adult male Long-Evans rats were used in this experiments (n=2 treated, n=2 untreated, n-1 normal control). Animals were housed individually after surgical procedures in 12:12 hrs light/dark cycle. Experiments were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Monash University Animal Ethics Committee. I0 Surgical procedures

Rats will be anesthetised with ketamine (87mg/kg) and xylazine (13mg/kg) administered IP. Blood oxygen, respiration and core body temperature were non-invasively monitored throughout the surgery. Dexamethasone (0.4mg/kg) and atropine (0.2mg/kg) were administered IM to supressed bronchiole secretions and stabilise heart rate, 15 respectively. A midline incision was performed followed by a circular craniotomy (2mm diameter) created using a dental drill. Once the target brain tissue has been visualised, a fine biopsy needle was used to pierce the cerebral cortex to the depth of ~2mm to generate the needle stick injury. Any bleeding at this stage will be arrested using vitamin K administered topically. Reproducibility of the injury is defined by the pre-specified 20 location of injury, which is sterotaxically identified, use of a consistent gauge biopsy needle and the depth of needle penetration. For the untreated cohort, the craniotomy was closed using bone wax and the incision site was sutured closed.

In the treated cohort, rats underwent a micro-infusion pump implantation following needle stick. An MRI-compatible intra-cerebral cannula (3280PM; 2mm length; 30G 25 needle; Plastics One; Figure 8) was installed in the craniotomy hole and adjusted to 1.5-2mm needle penetration depth and affixed to the surrounding cranium through orthopaedic bone cement. Subsequently, a cutaneous incision of 25 mm was made perpendicular to the midline of the back, between the shoulder blades. A sterile preprogrammed micro-infusion pump (iPrecio; SMP-300; Figure 9; designed for mice) with 30 tubing primed with saline and reservoir filled with recombinant mouse ephrin-A1 57 1001272724 (0.6mg/ml in injectable saline) was inserted into a pocket of connective tissue/ fascia, and secured using anchoring sutures. The connecting silicon tubing was fed subcutaneously towards the exposed cranium, and connected to the inlet tube on the cannula. Finally, the incision sites were sutured closed (refer to II.k). The infusion 5 protocol was programmed to deliver 1 pL/hr for 2 days during the recovery period at which point saline in the tubing was delivered, followed by a continuous infusion of ephrin-A1 at 2.5pl_/hr (0.1mg/kg/day) for 6 days. 2015252036 03 Nov 2015

Animals were allocated a 10-day survival period which coincided with the end of the astrogliotic peak in rats (7-9days). At the end of the survival period, Animals were I0 administered an overdose of pentobarbitone sodium and transcardially perfused with heparinised saline and 4% paraformaldehyde (as described herein). Tissue was treated for cryopreservation and cryosectioning as described herein.

Immunohistochemistrv

One full series of brain sections representing the entire brain of the rat was used for 15 Glial fibriliary acidic protein (GFAP) immunolabeling. In rodents, GFAP strongly labelled reactive astrocytes, resting and normal astrocytes are weakly or not labelled. - protocol is described herein. Activated caspase 3 labelling was also performed on sections containing the lesion core. aCas3 labelling on cells indicate on-going apoptosis (cell death). 20 Analyses

Glial scar volume was measured using the Cavalieri method (Gundersen, H.J. and E.B. Jensen, The efficiency of systematic sampling in stereology and its prediction. J Microsc, 1987. 147(Pt 3): p. 229-63) on ImageJ (NIH). Full GFAP immunolabelled series representing the entire brain was imaged. A 5000pm2 grid was superimposed on 25 the resulting images of sections (90pm separation) and volume estimation was performed on the glial scar as visualised by dense GFAP immunolabelling. Data was presented in a histogram. Reactive astrocyte counts were performed on sagittal sections containing the lesion core. Using ImageJ, A 100pm buffer was superimposed on the lesion core and reactive astrocytes within this buffer were not counted due to 30 their high density. A 500x500pm (0.25mm2) box was superimposed onto the image 58 1001272724 subsequently at a distance of ΙΟΟμιτι, 600pm, 1100μηη, 1600μιτι and 2100μιτι from the buffer. All GFAP immunoreactive reactive astrocytes within each 0.25mm2 box were counted. Data was presented as the density of reactive astrocytes / 0.25mm2 over distance. Counts of apoptotic cells were performed in a similar manner. A 104pm2grid 5 area was superimposed on images of aCas3 labelled cells proximal to the injury core and cells within the grid was counted. Data was presented as the density of apoptotic cells/104pm2. 2015252036 03 Nov 2015

Statistical analyses

All statistical analyses were performed using Prism6 (GraphPad). The non-parametric I0 T-test (Mann-Whitney U Test) was used to determine statistical significance of glial scar volume, reactive astrocyte density / distance and apoptotic cell counts between ephrin-A1 treated and untreated rat brains. Statistical significance was defined as P< 0.05.

Results

Ephrin-A1 treatment significantly reduced the severity and volume of the glial scar 15 The analysis revealed that the immunoreactivity of GFAP+ reactive astrocytes proximal to the injury core was predictably increased as a result of the needle stick injury (Fig. 10C) compared to uninjured controls (Fig. 10A, B). Floweverthe GFAP immunoreactivity was significantly reduced in ephrin-A1 treated rats (Fig. 10D) compared to the untreated controls (Fig. 10C). This could be attributed to a reduction in 20 astrocyte density as well an overall reduction in recruitment of reactive astrocytes to the injury core and their proliferation in situ. A statistically significant decrease (p <0.05) in glial scar volume was also observed in the ephrin-A1 treated rats (Fig. 10E; 2.3mm3) compared to the untreated control (Fig. 10E; 1.2mm3). This result clearly demonstrates the efficacy of ephrin-A1 treatment in reducing the severity of glial scarring after brain 25 injury.

Eohrin-A1 treatment supress secondary reactive astrocyte migration to the injury core A hallmark of reactive gliosis and glial scarring is the secondary recruitment of reactive astrocytes to the core of injury, which increases the overall density of the local population (Fig. 10F). The migration of reactive astrocytes from distal portions of the 59 1001272724 brain, which may not be directly affected by the initial injury, is guided by molecular guidance cues. The result of the GFAP immunolabelling on ephrin-A1 treated brains revealed an overall reduction in reactive astrocyte density at the injury core (Fig. 10G). More importantly, the observed density of reactive astrocytes distal to the injury core 5 was significantly reduced (Fig. 10G) compared to the untreated control (Fig. 10F). Subsequent analysis of reactive astrocyte density / distance from core through cell counts revealed significant difference in the pattern of reactive astrocyte distribution after ephrin-A1 treatment (Fig. 10H). In untreated control, a high proximal density of reactive astrocytes initially decreases (600pm-1100pm) followed by a subsequent 10 increase (1600pm-21 OOpm), which is consistent with a secondary wave of astrocytes migrating towards the injury core (Fig. 10H, I). Flowever in ephrin-A1 treated brains, a statistically significant (p <0.05) reduction in reactive astrocyte density was observed proximal to the injury core (100pm) compared to untreated controls (Fig. 10H, I). More significantly, the density of reactive astrocyte underwent a steady decline distally 15 without a secondary increase (Fig. 10H, I). This result demonstrates the capacity for ephrin-A1 treatment to reduce the density of reactive astrocytes proximal to the injury core. Ephrin-A1 treatment was also successful in supressing the secondary wave of reactive astrocyte recruitment to the core of injury, contributing to the reduction in overall glial scar volume and severity (Fig. 10A-E). 2015252036 03 Nov 2015 >0 Ephrin-A1 treatment significantly reduced apoototic cell death at the injury core

Apoptotic cell death, as detected by activated caspase-3 (aCas3) labelling, occurs as a result of CNS injury (Fig. 10J). The analysis revealed that ephrin-A1 treatment was able significantly reduce (p <0.05) the density of aCas3+ cells at the injury core (Fig. 10K-L), compared to untreated controls (Fig. 10J). This reduction in apoptotic cell death 25 indicates a reduction in the secondary injury cascade that results in further loss of neurones in the sub-acute period after brain injury. This result demonstrates that treatment of ephrin-A1 after brain injury was able to significantly reduce the density of dying cells proximal to the injury core, which may have neuroprotective implications.

In summary, administration of ephrin-A1 in vivo after brain injury results in the reduction 30 in overall reactive astrocyte reactivity and density at the injury core as well as supressing secondary recruitment resulting in a significantly smaller glial scar. Taken 60 1001272724 together, these results demonstrate that ephrin-A1 treatment successfully reduced the severity of glial scarring after brain injury in vivo and may play additional neuroprotective roles. 2015252036 03 Nov 2015

It will be understood that the invention disclosed and defined in this specification 5 extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. 61 1001272724

Claims

1. A method for minimising glial scarring in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby minimising glial scarring.

2. A method for promoting the recovery of central nervous system function in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby enhancing recovery of central nervous system function.

3. A method for reducing reactive astrogliosis in an individual having CNS injury or damage, the method comprising administering ephrin-A1, variant or analog thereof to the individual, thereby reducing reactive astrogliosis.

4. A method according to any one of claims 1 to 3, wherein the method further comprises the step of - identifying an individual with a condition associated with, or arising from, CNS injury or damage.

5. A method of reducing astrocyte reactivity in an individual having CNS injury or damage, the method comprising administering ephrin-A1 to the individual, thereby reducing astrocyte reactivity in the individual.

6. A method according to any one of claims 1 to 5, wherein the ephrin-A1, variant or analog thereof is administered directly at the site of injury or damage.

7. A method according to claim 6, wherein the ephrin-A1, variant or analog thereof is applied topically proximal to the injury core.

8. A method according to claim 6 or 7, wherein the ephrin-A1, variant or analog thereof is administered by osmotic or peristaltic pump locally to the site of injury.

9. A method according to any one of claims 1 to 8, wherein the administration of ephrin-A1, variant or analog thereof commences between 3 to 7 days post-injury.

10. A method according to any one of claims 1 to 9, wherein ephrin-A1, variant or analog thereof is administered for a period of 7, 8, 9, 10, 11, 12, 13 or 14 days.

11. A method according to any one of claims 1 to 10, wherein the CNS injury or damage is associated with, or arising from, astrocyte dysfunction or glial scar formation or progression.

12. A method according to any one of claims 1 to 11, wherein the CNS injury or damage is a brain or spinal cord injury.

13. A method according to claim 13, wherein the CNS injury or damage has arisen from physical force trauma, chemical injury or irradiation.

14. A method according to any one of claims 1 to 12, wherein the CNS injury or damage has arisen from any one or more of the conditions selected from the group consisting of haemorrhagic strokes, ischemia (including global ischemia and focal ischemia), hypoxia, stroke, seizure, epilepsy, status epilepticus, CNS vascular disease and neuroocular disease.

15. A method according to any one of claims 1 to 14, wherein the individual is at least 10 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, at least 60 years, at least 70 years, at least 80 years or at least 90 years old.

16. A pharmaceutical composition for minimising glial scarring in an individual comprising ephrin-A1, variant or analog thereof and a pharmaceutically acceptable diluent, excipient or carrier.

17. Ephrin-A1, variant or analog thereof for use in the treatment of an individual with CNS injury or damage.

18. A pharmaceutical composition comprising ephrin-A1 and a pharmaceutically acceptable diluent, excipient or carrier for use in minimising glial scarring, or reducing astrocyte reactivity, in an individual.

19. Use of ephrin-A1 in the manufacture of a medicament for minimising glial scarring, or reducing astrocyte reactivity, in an individual in need thereof.

20. A surgical or implantable device for use in surgery of the CNS coated or impregnated with an ephrin-A1, variant or analog thereof.

21. A surgical or implantable device according to claim 20, wherein the device is a neuroprosthetic or implantable microelectrode.

22. A surgical or implantable device according to claim 20, wherein the device is a Deep Brain Stimulating micro electrode or microelectrode array.

23. A composition for use in coating a surgical or implantable device, the composition comprising an ephrin-A1, variant or analog thereof.

24. A composition according to claim 23, wherein the composition is a slow-dissolving hydrogel or foam.

25. A kit or article of manufacture comprising ephrin-A1, variant or analog thereof, and/or pharmaceutical composition comprising ephrin-A1, variant or analog thereof.

26. A method, composition, use, surgical or implantable device according to any one of claims 1 to 25, wherein the ephrin-A1 has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1.

27. A method, composition, use, surgical or implantable device according to any one of claims 1 to 25, wherein ephrin-A1 is human.

28. A method, composition, use, surgical or implantable device according to claim 27, wherein human ephrin-A1 has an amino acid sequence shown in SEQ ID NO: 1.