PRIORITY PARAGRAPH

[0001] This application claims priority to U.S. Provisional Application No.

62/840,739 filed on April 30, 2019 titled“METHODS AND COMPOSITIONS FOR

TREATING CNS INJURY” and is incorporated herein by reference.

GOVERNMENT INTERESTS

[0002] This work was supported by the National Institutes of Health (NS042291). Core infrastructure support for the primate spinal cord research facility was provided by the Veterans Administration (Gordon Mansfield Spinal Cord Injury Collaborative Consortium IP50RX001045, RR&D B7332R, RR&D 1I01RX002245). The California National Primate Research Center was funded by the NIH (NCRR P51 OD011107-56).

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 27, 2020, is named 127304_4200_SL.txt and is 207,032 bytes in size.

BACKGROUND

[0004] A number of mechanisms limit the regenerative capacity of the injured adult mammalian central nervous system (CNS), including insufficient trophic support and the presence of inhibitors to axon growth associated with both myelin and the extracellular matrix (ECM). Among the ECM molecules that inhibit axon growth, a major contribution is made by chondroitin sulphate proteoglycans (CSPGs). The predominant CSPG molecules that are present in the CNS include NG2, neurocan and aggrecan, which consist of a core protein with long sulfated glycosaminoglycan (GAG) side chains. The side chains are the primary determinants of axon inhibition. As the period of robust developmental plasticity of the nervous system closes, CSPGs form around neurons and synapses in so-called“peri- neuronal nets” that are postulated to play a role in influencing the function of the CNS by controlling plasticity and sprouting. In addition, CSPGs are newly synthesized at sites of CNS injury and are believed to directly block axon growth.

[0005] In a spinal cord injury, the axons of ascending sensory and descending motor neurons are disrupted, that can result in the loss of sensation and paralysis. These axons fail to regenerate successfully leading to permanent disability. A scar envelopes the site of the injury which is believed to wall off the area of fragile tissue, stabilize the blood brain barrier, and prevent an overwhelming cascade of uncontrolled tissue damage. This scar is composed of hypertrophic glial cells and an extracellular matrix (ECM). Chondroitin sulfate proteoglycans (CSPGs) are one important component of the scar. They are expressed by glial cells and deposited in the ECM in regions of blood brain barrier breakdown. In vitro evidence demonstrates that these CSPGs are potently inhibitory for the growth of axons and without wishing to be bound by theory, are believed to contribute to the failure of the spinal cord axons to regenerate and reform functional synapses. In vivo studies have demonstrated that regenerating axons are able to grow into and even beyond the scar.

SUMMARY

[0006] Disclosed herein are methods and compositions for treating a subject with central nervous system injury, including but not limited to spinal cord injury. In some embodiments, a method of treating a subject with CNS injury comprises administering to the subject in need thereof a composition comprising an effective amount of a proteoglycan degrading domain. In some embodiments, the proteoglycan degrading domain is selected from the group consisting of chondroitinase ABC I, chondroitinase ABC II, chondroitinase B, chondroitinase AC, hyaluronidase-1, hyaluronidase-2, hyaluronidase-3, hyaluronidase-4, PH- 20, and a combination thereof. In a preferred embodiment the proteoglycan degrading domain is chondroitinase ABC I.

[0007] In some embodiments, a method of treating a subject with CNS injury comprises administering to the subject in need thereof a composition comprising an effective amount of a proteoglycan degrading domain and a domain that promotes neural regeneration.

[0008] In some embodiments, the composition is administered at or near the site of injury. In some embodiments, the composition is administered at a single site close to the injury. In other embodiments, the composition is administered at multiple sites close to the injury. In other embodiments, the composition is administered as multiple injections at each site, wherein the composition is injected at different depths.

[0009] In some embodiments, treating a subject comprises injecting at or near the site of injury a first amount of the composition at a first depth, and injecting a second amount of the composition at a second depth, wherein the second depth is more than the first depth, and wherein the first depth and the second depth are at the same site of administration.

[0010] In some embodiments, treating a subject comprises injecting at or near the site of injury a first amount of the composition at a first depth, and injecting a second amount of the composition at a second depth, wherein the second depth is less than the first depth, and wherein the first depth and the second depth are at the same site of administration.

[0011] In one embodiment, the CNS injury in a subject is a spinal cord injury.

[0012] Other embodiments of the present invention relate to methods for promoting neurological functional recovery, including sensory, motor and autonomic function, after central nervous system injury or disorder. In some embodiments, a method of promoting neurological function in a subject with CNS injury comprises administering to the subject in need thereof a composition disclosed herein.

[0013] Further embodiments of the present invention relate to methods for promoting neurite outgrowth. In some embodiments, a method of promoting neurite outgrowth in a subject having a spinal cord injury comprising administering to the subject in need thereof a composition disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1 depicts the experimental approach. (A) Timeline of Chase (cABC I) efficacy experiment. (B) Schematic of experimental approach. The right hemisection lesion at segment C7 severs 90% of CST axons originating in the left hemisphere (traced with BDA, blue), and 10% of CST axons originating in the right hemisphere (traced with A488, brown). Chase is injected into 10 sites (1.5 mm apart) in the gray matter below the lesion.

[0015] Figure 2 depicts the degradation of proteoglycans by intraparenchymal Chase I injections. (A-C) Comparison of Chase-treated (right) and untreated (left) sides of the spinal cord in a rhesus monkey 2 wk after Chase administration (1 mo after lesion). WFA labeling reveals CSPG degradation on the treated side. Boxed regions are shown at higher magnification in (B), (C). Zone of peri-neuronal net CSPG degradation indicated by dashed lines. (D) Tissue section from C7, above the lesion and the treated region, has intact CSPGs. (E) Series of tissue sections at 2 mm intervals moving caudally from lesion site reveals effective CSPG degradation from C7 through Tl. Scale bars: (A) 500 pm; (B),(C), 1 mm.

[0016] Figure 3 depicts the functional outcomes. (A) Combining all behavioral data into a principal components analysis (PCA) demonstrates a significant interaction of Chase treatment with time on PCI (P=0.001). Plot shows the change in PCI scores over time. PCI explains 68% of the variance in the behavioral data (see Supp. Fig. 5 for loadings). (B- C) Example of food reward retrieval on one of the tasks comprising behavioral testing, Brinkman 5. Arrow indicates pincer motion used to retrieve food reward from well. (D-L) Recovery curves for the individual tasks that comprise PCI. These individual task plots show that, although single functional outcome metrics may not be sensitive on their own, combining all metrics in a PCA reveals a robust effect of chondroitinase treatment at the multivariate level. Object manipulation score (D) reflects use of the impaired forelimb to manipulate a large piece of fruit and retrieve food items from inside a“Kong” toy. Brinkman 1 through Brinkman 5 (E-I) indicates progressively more difficult versions of food retrieval. Locomotion score (J) reflects use of the ipsilesional limbs for moving around the exercise enclosure. (K) Climbing score reflects use of ipsilesional limbs for vertical climbing in the exercise enclosure. (L) General Score is a composite of multiple measures that describe forelimb and hind limb function, as described in. Generally, the Chase group exhibited superior recovery on tasks reflecting hand use (the spinal cord region targeted with Chase injections), and no difference in measures that included hind limb function (i.e., those not targeted by Chase treatment). N=6 Chase and N=5 controls. Data points show group means, error bars represent SEM.

[0017] Figure 4 shows the effect of Chase administration on corticospinal axon and synapse density. (A) BDA-traced CST axons descending in the intact white matter (WM; left dorsolateral quadrant of spinal cord), crossing the spinal cord midline near the central canal (CC, dashed line in inset), and terminating in the gray matter (GM) on both sides of the spinal cord. Dashed line shows WM-GM boundary. Chase-treated subject, spinal level C8. (B) Representative images of gray matter from Chase-treated and control subjects at C8; note the qualitatively greater CST density in Chase-treated tissue. (C) Quantification of CST density in lesion-side gray matter indicates greater CST axon length in treated subjects (P=0.036, linear mixed model). (D) Synapse frequency was assessed by counting bouton-like portions of CST axons that co-labeled with synaptophysin through a confocal stack of a coronal section at C8. Top panel is a flattened stack of five 0.75 pm optical sections from a confocal microscope, and shows two presynaptic boutons (arrows). Bottom panels are higher magnifications of single optical sections, demonstrating co-localization of synaptophysin in a bouton-like CST axon. (E) Quantification of synaptophysin bouton-like density identifies significantly more synapses per mm3 in Chase-treated subjects (P=0.001, linear mixed model). N=6 Chase and N=5 controls. Bar graphs show means, data points are individual subjects, error bars represent linear mixed model SEM. Scale bars: A, 1 mm; D, 10 pm.

[0018] Figure 5 depicts Chase intrathecal infusions in pig. (A) 2B6 labeling of digested stubs of chondroitin sulfate proteoglycans (CSPGs) in transverse spinal cord sections of the porcine spinal cord at C8. Following intrathecal Chase infusions, there is an approximate 1 mm rim of CSPG degradation in the peripheral white matter of Chase-treated subjects. (B) Saline-infused control. (C) Wisteria Floribunda Agglutinin (WFA) labeling of peri-neuronal net CSPGs in gray matter reveals intact peri-neuronal net CSPGs in all Chase- infused subjects, which appear identical to (D) saline-infused controls. Scale bars: 500 pm.

[0019] Figure 6 shows parenchymal integrity at Chase injection sites (Nissl). Intraparenchymal injections of Chase in the monkey spinal cord do not cause extensive damage or inflammation. Images of transverse sections very near injection sites at C8-T1. Left column of images is from a short-term Chase subject (2 wks post injection, 6 wks post SCI); middle column is from a saline-injected subject (4 mos post injection, 5 mos post SCI); right column is from a Chase-injected subject (4 mos post injection, 5 mos post SCI). (A) Labeling for Nissl substance reveals mild hypercellularity along the injection track

(arrowheads) 2 weeks after Chase injection. (B-C) By 4 months after injections, there are no evident differences in Nissl stains comparing saline and Chase injection sites. (D-F) Higher magnifications of sections in A-C. (G-I) Further magnification of panels D-F. Scale bars: A- C, 500 pm; D-F, 100 pm; G-I, 50 pm.

[0020] Figure 7 depicts parenchymal integrity at Chase injection sites (C8, GFAP, and motor neurons). Intraparenchymal injections of Chase in the monkey spinal cord do not cause extensive damage or inflammation. Images of transverse sections midway between injection sites at C8-T1. (A-C) CD8 labeling in gray matter reveals rare T-cells 2 weeks after Chase injection; these cells are not detected 4 months after injection. CD3 and CD45 labeling showed the same pattern (data not shown). (D-F) Labeling for GFAP (astrocytes) in gray matter shows no qualitative differences between short-term Chase, long-term saline, and long-term Chase. (G) Spinal motor neurons (MNs) were counted at sequential rostro-caudal distances from the injection site on both injected and uninjected sides of the spinal cord. Injections of either Chase or saline result in no loss of motor neurons (expected proportion is 0.5). Moreover, there was no drop in MN numbers at the actual injection site, nor when comparing motor neuron numbers in Chase vs. control animals (Chase proportion: 0.53+0.04; Control proportion: 0.51+0.01; F _(i 0.00055) = 0.11, P=0.75). Lines show group means, data points are individual tissue sections, 5-6 sections per subject. Scale bars: 100 pm.

[0021] Figure 8 shows the temporary microglial activation at Chase injection sites. Images of transverse sections immediately adjacent to injection sites (or similar regions in C8-T1 spinal cord for uninjected subjects), labeled for IBAl (microglia) at the indicated time points. There are significant differences in density of IBAl labeling across groups (Overall ANOVA: F _{(5 197.30)} = 4.43, P = 0.009). Analyses include sections from animals involved in a previous study ( N=3 Intact, N=4 Lesion 2wk, N=3 Lesion 5mo); sections were immunolabeled for IBAl concurrently with sections from present experiment. IBAl pixel density was calculated using the ImageJ autothresholding function on images of the intermediate zone of the gray matter. Main panels show actual images used for quantification; insets are 3x zooms. Images shown are from subjects whose results were closest to their group's mean value. Quantifications in panel G. (A) Intact subjects show the baseline level of microglial labeling. (B) Two weeks after lesion, a trend toward increased microglial labeling is observed compared to baseline (N=4, F _{(i 144.57)} = 2.79, P = 0.16, compared to Intact group). (C) Two weeks after Chase injections (six weeks after lesion), significantly increased IBAl labeling is evident compared to the intact baseline immediately adjacent to the injection site (arrow) (N=2, F _{(1 835.30)} = 11.81, P = 0.04). (D-F) At longer time points after lesions, increased IBAl labeling remains evident and statistically significant compared to intact baseline (Lesion 5mo, N=5, F ₍₁ 435.10) = 6.74, P = 0.04; Saline 4mo, N=3, F ₍₁ 277.44) =10.34, P = 0.03; Chase 4mo, N=6, F ₍₁ 479.47) = 35.19, P = 0.0006). However, there are no significant differences among animals with lesions alone, saline injections or Chase injections (Lesion 5mo vs. Saline 4mo, vs. Chase 4mo, ANOVA: F _{(2 3.78)} = 0.11, P=0.89). (G) Bar graphs show means, data points are individual subjects, error bars represent SEM, asterisks indicate statistical difference from intact baseline. Scale bar: 200 pm.

[0022] Figure 9 shows degradation of chondroitin sulfate by Chase injections up to the caudal lesion border. (A) 2 wk after Chase administration (1 mo after lesion), WFA labeling reveals that CSPGs are intact at the rostral end of the lesion site. (B) In contrast, CSPG degradation is evident up to the caudal lesion border. However, because no“bridge” was placed in the lesion site to allow the growth of axons through the lesion, this CSPG degradation likely had no impact on experimental outcomes. Approximate zone of peri- neuronal net CSPG degradation indicated by dashed lines. Labeling experiment included 6 control subjects, and 6 Chase-injected subjects. Scale bar: 1 mm.

[0023] Figure 10 shows objection manipulation studies. (A) Intact subject manipulating an apple bimanually. Note palmar contact of right hand (arrow) with object, digit extension and thumb apposition, all features of normal object manipulation. (B) Manipulation by a lesioned subject early after injury. Note the flexed digits of the impaired right hand (arrows). (C) Partial recovery of the impaired right hand during object

manipulation. The hand is more normally positioned and less tightly flexed, but the dorsal aspect of the digits are still in contact with the fruit, and the thumb remains close to digit two (arrows). The score for object manipulations is based on these features in combination with evaluations of movement of the wrist and fingers during manipulation, and the presence of pincer grasp.

[0024] Figure 11 shows the lesion constructions. Extent of lesion is shown in red shading. Control subject #1 was considered an‘overlesion’ and was excluded from behavioral analyses (but not CST anatomical analysis, as the left dorsolateral tract, which carries nearly all of the spared CST axons, was unaffected by the overlesion). Control subject #3 did not receive cortical tracer due to weight loss, and was therefore excluded from CST density analyses.

[0025] Figure 12 depicts behavioral task loadings on Principal Component Analysis. Non-linear principal components analysis of all behavioral tasks (open field tasks and Brinkman board) across all time points revealed strong loadings of all behavioral components on PCI, which accounted for 67.7% of the variance. For PCI, positive loadings (in red) indicate increased behavioral score, and negative loadings (in blue) are found on Brinkman board clearance time measurements (where lower values indicate improved performance). The loading pattern for PCI indicates that this component broadly reflects improved behavioral performance. Two more principal components were extracted, with much lower percent variance accounted for (10.4% for PC2 and 5.1% for PC3). The loading patterns for these components did not indicate clearly definable eigenmodules, and scores on these components were therefore not tested. N = 11 subjects, with results confirmed through 1000 iterations of bootstrap subsampling at 60%.

[0026] Figure 13 shows that Chase increases the density of axons originating in the right motor cortex. (A) A488-traced CST axons originating in the right motor cortex; 90% of these axons cross midline at the medullary pyramids and descend in the intact left hemicord. (B) Some of the axons cross midline near the central canal (CC) and (C) terminate primarily in the ventromedial gray matter. (D) Ventromedial gray matter from control subject. (E) Quantification shows that, just as with axons originating in the left motor cortex, Chase increases the density of these CST axons caudal to the spinal cord hemisection (total length in lesion-side GM per 40pm section; P=0.001, linear mixed model). N=6 Chase and N=5 controls. Bars represent means, data points are individual subjects, error bars represent linear mixed model SEM. Scale bar: 1 mm.

[0027] Figure 14 shows that Chase had no effect on raphespinal density.

Serotonin labeling (5HT) in lesion-side gray matter in transverse sections at C8 in (A) control and (B) Chase-treated subjects. Higher magnification shown in insets. (C) Quantification reveals no differences between treated and control subjects in raphespinal density in motor neuron pools (P=0.924, generalized estimating equation). N=5 Chase and N=4 controls. Bars represent means, data points are individual subjects, error bars represent generalized estimating equation SEM. Scale bars: 200 pm.

[0028] Figure 15 shows the non-linear PC A workflow. Analysis was divided into two distinct stages. In Stage I, we compiled the full behavioral data outcome matrix (open cage measures and Brinkman board measures), and created a cross-correlation matrix of data from all animals at each time point, regardless of condition. Non-linear principal component analysis then determined the extent to which each outcome measure correlated with the variance explained by the principal component (PC Loadings). The PC loadings were then examined and cross-validated to confirm the resulting PC scores serve as legitimate multidimensional outcome metrics. A PC score was then generated for each animal at each time point. In Stage II, we performed a directed, single hypothesis test (linear mixed model), unblinded to the effect of Chase on PCI score.

DETAILED DESCRIPTION

[0029] As used in this application and in the claims, the singular forms“a,”“an,” and“the” include the plural forms unless the context clearly dictates otherwise.

[0030] The term“about” when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g.,“about 50” means 45 to 55,“about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation.

[0031] The transitional term“comprising,” which is synonymous with “including,”“containing,” or“characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase“consisting essentially of’ limits the scope of a claim to the specified materials or steps“and those that do not materially affect the basic and novel

characteristic(s)” of the claimed invention. In embodiments or claims where the term comprising is used as the transition phrase, such embodiments can also be envisioned with replacement of the term“comprising” with the terms“consisting of’ or“consisting essentially of.”

[0032] An“effective amount” of a composition is a predetermined amount calculated to achieve the desired effect, i.e., to ameliorate, prevent or improve an unwanted condition, disorder or symptom of a patient. The activity contemplated by the present methods may include both therapeutic and/or prophylactic treatment, as appropriate. The specific dose of the agent administered according to this disclosure is to obtain therapeutic and/or prophylactic effects. These will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. The effective amount administered may be determined by a physician in light of the relevant circumstances, including the patient’s make-up, the condition to be treated, the choice of the effective agent to be administered, the chosen route of administration, and other factors. [0033] The term“patient” and“subject” are interchangeable and may be taken to mean any living organism which may be treated with compounds of the present invention. As such, the terms“patient” and“subject” may include, but are not limited to, any non-human mammal, primate or human. In some embodiments, the“patient” or“subject” is a mammal, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, or humans. In some embodiments, the patient or subject is an adult, child or infant. In some embodiments, the patient or subject is a human.

[0034] The term“treating” is used herein, for instance, in reference to methods of treating a CNS injury, such as a spinal cord injury, and generally includes the administration of a therapeutic composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition or in a subject relative to a subject not receiving the therapeutic compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition.

[0035] Disclosed herein are methods and compositions for treating a central nervous system (CNS) injury.

[0036] In some embodiments, the composition comprises a proteoglycan degrading domain. Non-limiting examples of proteoglycan degrading domains include chondroitinases such as chondroitinase ABC I, chondroitinase ABC II, chondroitinase B, chondroitinase AC, and hyaluronidases such as hyaluronidase-1, hyaluronidase-2, hyaluronidase-3, hyaluronidase-4, PH-20, and a combination thereof. In a preferred embodiment the proteoglycan degrading domain is chondroitinase ABC I.

[0037] In some embodiments, the chondroitinase enzyme family comprises chondroitinase ABC I (EC 4.2.2.20), chondroitinase ABC II (EC 4.2.2.21),

chondroitinase AC (EC 4.2.2.5), and chondroitinase B (EC 4.2.2.19). Chondroitinase AC shows activity against chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C4S), while chondroitinase B cleaves dermatan sulfate (DS) as its sole substrate. Chondroitinase ABC I and chondroitinase ABC II act on a variety of substrates including C4S, C6S, DS, and hyaluronan.

[0038] In some embodiments, the compositions may comprise chondroitinase ABC I. Chondroitinase ABC I (cABC I) can be obtained from any source, such as human, bacteria ( Proteus Vulgaris ), or can be produced by recombinant techniques. Chondroitinase ABC I can also be obtained commercially. In some embodiments, the chondroitinase ABC I has an amino acid sequence as represented by SEQ ID NO: 1. In some embodiments, the chondroitinase ABC I has an amino acid sequence that is at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 1.

[0039] In some embodiments, the proteoglycan degrading domain may be a deletion mutant or a substitution mutant. Because of their smaller size compared to the full length enzyme, the deletion and or substitution mutants may be easier to make and easier to deliver to target cells and tissues. These and other even smaller deletion or substitution mutants of proteoglycan degrading domain could be used as potential therapeutics with lesser immunogenicity and similar or higher tissue penetration ability for the treatment of CNS injury.

[0040] In some embodiments, chondroitinase ABC I may be an N-terminal deletion mutant, with deletion of about 1 amino acid to about 120 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 40 amino acids, or about 1 amino acid to about 20 amino acids from the N- terminus. In other embodiments, chondroitinase ABC I may be a C-terminal deletion mutant, with deletion of about 1 amino acid to about 20, about 1 amino acid to about 40, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 60 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 120 amino acids, or about 1 amino acid to about 275 amino acids from the C-terminus. In some embodiments, chondroitinase ABC I may comprise both N-terminal and C-terminal deletions. Non-limiting examples of cABC I deletion mutants include deletion of 20 amino acids at N-terminus (cABC I ND20; SEQ ID NO: 2), deletion of 60 amino acids at N-terminus (cABC I ND60; SEQ ID NO: 3), deletion of 60 amino acids at N-terminus and 80 amino acids at C-terminus (cABC I ND60, CA80; SEQ ID NO: 4). Other examples of cABC I deletion mutants are described in U.S. Patent Nos. 8,906,363 and 7,429,375, and are incorporated herein by reference.

[0041] In some embodiments, the compositions may comprise cABC I substitution mutants that exhibit enhanced resistance to inactivation, including inactivation from UV, pH, salt, or heat exposure. Such cABC I mutants include cABC I BC6 (SEQ ID NO: 5), cABC I BE7 (SEQ ID NO: 6), cABC I BF4 (SEQ ID NO: 7), cABC I BC9 (SEQ ID NO:8), cABC I BC7 (SEQ ID NO:9), cABC I RD4 (SEQ ID NO: 10), cABC I BE11 (SEQ ID NO: 11), cABC I 055D2-3 (SEQ ID NO: 12), cABC I 079B6-2 (SEQ ID NO: 13), cABC I 023G6-4 (SEQ ID NO: 14), cABC I 005B12-3 (SEQ ID NO: 15), and combinations thereof. Other examples of cABC I mutants are described in U.S. Patent Nos. 7,485,295 and

7,722,864, and are incorporated herein by reference.

TABLE 1

[0042] In some embodiments, the compositions may comprise chondroitinase ABC II (cABC II). cABC II can be obtained from any source, such as human, bacteria ( Proteus Vulgaris ), or can be produced by recombinant techniques. Chondroitinase ABC II can also be obtained commercially. In some embodiments, the chondroitinase ABC II has an amino acid sequence as represented by SEQ ID NO: 16. In some embodiments, the chondroitinase ABC II has an amino acid sequence that is at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 16. In other embodiments, chondroitinase ABC II may be an N-terminal deletion mutant, with deletion of about 1 amino acid to about 120 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 40 amino acids, or about 1 amino acid to about 20 amino acids from the N-terminus. In other embodiments, chondroitinase ABC II may be a C-terminal deletion mutant, with deletion of about 1 amino acid to about 20, about 1 amino acid to about 40, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 60 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 120 amino acids, or about 1 amino acid to about 275 amino acids from the C-terminus, or combinations thereof. In some embodiments, chondroitinase ABC II may comprise both N-terminal and C-terminal deletions. Other examples of cABC II deletion mutants are described in U.S. Patent Nos. 8,906,363 and 7,429,375.

[0043] In some embodiments, the compositions may comprise chondroitinase B. Chondroitinase B can be obtained from any source, such as human, bacteria {Flavobacterium Heparinum ), or can be produced by recombinant techniques. Chondroitinase B can also be obtained commercially. In some embodiments, the chondroitinase B has an amino acid sequence as represented by SEQ ID NO: 17. In some embodiments, the chondroitinase B has an amino acid sequence that is at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 17. In other embodiments, chondroitinase B may be an N-terminal deletion mutant, with deletion of about 1 amino acid to about 120 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 40 amino acids, or about 1 amino acid to about 20 amino acids from the N- terminus. In other embodiments, chondroitinase B may be a C-terminal deletion mutant, with deletion of about 1 amino acid to about 20, about 1 amino acid to about 40, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 60 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 120 amino acids, or about 1 amino acid to about 275 amino acids from the C-terminus. In some embodiments, chondroitinase B may comprise both N-terminal and C-terminal deletions. Non-limiting examples of chondroitinase B deletion mutants include deletion of 80 amino acids at N-terminus (chondroitinase B ND80; SEQ ID NO: 18), deletion of 120 amino acids at N-terminus (chondroitinase B ND120; SEQ ID NO: 19), deletion of 19 amino acids at C-terminus (chondroitinase B CA19; SEQ ID NO: 20), deletion of 120 amino acids at C- terminus (chondroitinase B OD120; SEQ ID NO: 21), deletion of 120 amino acids from N- terminus and 120 amino acids from C-terminus (chondroitinase B ND120, CA120; SEQ ID NO: 22), and combinations thereof. Other examples of chondroitinase B deletion mutants are described in U.S. Patent Nos. 8,906,363 and 7,429,375.

[0044] In some embodiments, the compositions may comprise chondroitinase AC. Chondroitinase AC can be obtained from any source, such as human, bacteria

( Flavobacterium Heparinum ), or can be produced by recombinant techniques.

Chondroitinase AC can also be obtained commercially. In some embodiments, the chondroitinase AC has an amino acid sequence as represented by SEQ ID NO: 23. In some embodiments, the chondroitinase AC has an amino acid sequence that is at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NO: 23. In other embodiments, chondroitinase AC may be an N-terminal deletion mutant, with deletion of about 1 amino acid to about 120 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 40 amino acids, or about 1 amino acid to about 20 amino acids from the N-terminus. In other embodiments, chondroitinase AC may be a C-terminal deletion mutant, with deletion of about 1 amino acid to about 20, about 1 amino acid to about 40, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 60 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 120 amino acids, or about 1 amino acid to about 275 amino acids from the C-terminus. In some embodiments, chondroitinase AC may comprise both N-terminal and C-terminal deletions. Non-limiting examples of chondroitinase AC deletion mutants include deletion of 200 amino acids at C- terminus (chondroitinase AC CA200; SEQ ID NO: 24), deletion of 220 amino acids at C- terminus (chondroitinase AC CA220; SEQ ID NO: 25), deletion of 20 amino acids from N- terminus and 200 amino acids from C-terminus (chondroitinase AC ND20, CA200; SEQ ID NO: 26), deletion of 50 amino acids from N-terminus and 200 amino acids from C-terminus (chondroitinase AC ND50, CA200; SEQ ID NO: 27), deletion of 100 amino acids from N- terminus and 200 amino acids from C-terminus (chondroitinase AC ND100, CA200; SEQ ID NO: 28), deletion of 50 amino acids from N-terminus and 275 amino acids from C-terminus (chondroitinase AC ND50, CA275; SEQ ID NO: 29), and combinations thereof. Other examples of chondroitinase AC deletion mutants are described in U.S. Patent Nos. 8,906,363 and 7,429,375.

[0045] In some embodiments, the composition comprises hyaluronidases. The hyaluronidase enzyme family consists of enzymes capable of hydrolyzing or“breaking down” the polysaccharide hyaluronic acid. Hyaluronic acid is an important constituent of connective tissue. Hyaluronidases can be broadly classified into three groups: mammalian- type hyaluronidases (EC 3.2.1.35) are endo-beta-N-acetylhexosaminidases that produce tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), specifically C4-S and C6-S. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, and to various extents, CS and DS. They are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products.

Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-beta- glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta 1-3 linkage. The hyaluronidase disclosed herein can be derived from any source whatsoever and, for instance, may be recovered from bovine protein (bovine type), leeches or bacteria (e.g. in the form of hyaluronate lyase). The hyaluronidase can also be of vegetable origin. Genetic engineering techniques in the art can likewise be used to produce hyaluronidase. Various types of hyaluronidase can be also obtained commercially, e.g.. from Wyeth-Ayerst (Wydase®), Abbot (Hyazyme), Bristol-Myers Squibb (Enzodase), and Ortho Pharmaceuticals (Diffusin). Non-limiting examples of hyaluronidases that can be used in the compositions are human hyaluronidase- 1 (SEQ ID NO: 30), human

hyaluronidase-2 (SEQ ID NO: 31), human hyaluronidase-3 (SEQ ID NO: 32), human hyaluronidase-4 (SEQ ID NO: 33), and human PH20 (SEQ ID NO: 34). In some

embodiments, hyaluronidases may an amino acid sequence that is at least 90% identical, at least 95% identical or at least 99% identical to SEQ ID NOs: 30-34. In other embodiments, hyaluronidase may be an N-terminal deletion mutant, with deletion of about 1 amino acid to about 120 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 40 amino acids, or about 1 amino acid to about 20 amino acids from the N-terminus. In other embodiments, hyaluronidase may be a C- terminal deletion mutant, with deletion of about 1 amino acid to about 20, about 1 amino acid to about 40, about 1 amino acid to about 50 amino acids, about 1 amino acid to about 60 amino acids, about 1 amino acid to about 80 amino acids, about 1 amino acid to about 100 amino acids, about 1 amino acid to about 120 amino acids, or about 1 amino acid to about 275 amino acids from the C-terminus, or combinations thereof. In some embodiments, hyaluronidase may comprise both N-terminal and C-terminal deletions.

[0046] In some embodiments, deletion mutants of chondroitinases and

hyaluronidases can be obtained by recombinant techniques. Expression of a mutant chondroitinase or a mutant hyaluronidase of the invention can be performed by ligating a nucleic acid encoding the protein, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Procedures for ligation are well known to those of ordinary skill in the art. Expression vectors for production of recombinant forms of the polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of a chondroitinase ABCI mutant polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. A number of vectors exist for the expression of recombinant proteins in yeast and could be used to express mutant proteins of the invention. For instance, YEP24, YIP 5, YEP5I, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae . In some instances, it may be desirable to express a recombinant mutant polypeptide of the invention by the use of an insect expression system such as the baculovirus expression system. Examples of such baculo virus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the b-gal containing pBlueBac III). The expression vectors and host cells listed herein are provided by way of example only and represent the well-known systems available to those of ordinary skill in the art that may be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other systems suitable for maintenance propagation or expression of the nucleic acid molecules described herein.

[0047] In some embodiments, the composition further comprises a domain that promotes neural regeneration. It is believed that these neural regenerating proteins may stimulate glial cells and block axon growth inhibitors. Non-limiting examples of proteins that promote neural regeneration include neural cell adhesion molecules (N-CAM), LI CAM, myelin-associated glycoproteins, laminins, fibronectins, cadherins, Tenascins, fibronectin type-III (FN-III) domain, netrins, BSP-2 (mouse N-CAM), neural antigen D-2, neural antigen 224-1A6-A1, NILE (nerve growth factor-inducible large external glycoprotein), Nr-CAM (neuronal cell adhesion molecule), TAG-1 (axonin-1), Ng-CAM (neuron-glia cell adhesion molecule), F3/F11 glycoprotein, integrins, Fasciclin III, Nogo-A antagonist peptides (eg., NgR-27-3ii), neurotrophic factors, and combinations thereof. In some embodiments, neurotrophic factor is selected from the group consisting of NGF (nerve growth factor), BDNF (brain derived neurotrophic factor), NT-3 (neurotrophin-3), IGF (insulin-like growth factor), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), PDGF (platelet-derived growth factor), TGF (transforming growth factor) a and b, and GGF2 (glial growth factor-2). In some embodiments, cadherins are selected from N-cadherin, E-cadherin, P-cadherin, L-CAM, B-cadherin, and T-cadherin. [0048] In some embodiments, the domain that promotes neural regeneration may be present as a chimeric protein along with proteoglycan degrading domain. In some embodiments, the domain that promotes neural regeneration and the proteoglycan degrading domain may be present in the composition as individual proteins or polypeptides.

[0049] In some embodiments, the compositions may comprise other suitable molecules that overcome the inhibition of neuronal growth, and include but are not limited to Protein Kinase C family inhibitors, Rho Kinase family inhibitors, agents such as

phosphodiesterase inhibitors that increase intracellular cyclic AMP.

[0050] In some embodiments, the proteoglycan degrading domain may further comprise a protein transduction domain (PTD). Protein transduction domains may be used to transport polypeptides and polynucleotides cargo across anatomical barriers and into cells. Examples of PTDs include HIV TAT protein PTD, antennapedia homeodomain, and others. PTD can be linked to proteins and facilitate the transduction of the proteins into cells. In some embodiments, the composition comprises a proteoglycan degrading domain and a PTD. In other embodiments, the composition comprises a chimeric protein comprising a proteoglycan degrading domain, a domain that promotes neural regeneration, and a PTD.

[0051] In some embodiments, the composition comprises the proteoglycan degrading domain alone or in combination with a domain that promotes neural regeneration in an effective amount of about 0.1 to about 200 mg/ml, about 0.1 to about 180 mg/ml, about 0.1 to about 160 mg/ml, about 0.1 to about 140 mg/ml, about 0.1 to about 120 mg/ml, about 0.1 to about 100 mg/ml, about 0.1 to about 80 mg/ml, about 0.1 to about 60 mg/ml, about 0.1 to about 40 mg/ml, about 0.1 to about 20 mg/ml, about 0.1 to about 10 mg/ml, about 2 to about 40 mg/ml, about 4 to about 35 mg/ml, about 6 to about 30 mg/ml, about 8 to about 25 mg/ml, about 10 to about 20 mg/ml, about 12 to about 15 mg/ml, or any of about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, or 2.5 mg/ml. In some embodiments, the composition disclosed above may be administered at each site from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is administered as multiple injections at each site, and the composition is injected at different depths.

[0052] In some embodiments, the composition comprises the proteoglycan degrading domain in an effective amount of about 0.1 U/ml to about 100 U/ml, about 0.1 U/ml to about 80 U/ml, about 0.1 U/ml to about 60 U/ml, about 0.1 U/ml to about 40 U/ml, about 0.1 U/ml to about 20 U/ml, about 0.1 U/ml to about 10 U/ml, about 0.1 U/ml to about 5 U/ml, or about 0.1 U/ml to about 1 U/ml. Non-limiting examples include about 0.1 U/ml, about 0.2 U/ml, about 0.5 U/ml, about 1 U/ml, about 10 U/ml, about 50 U/ml, about 100 U/ml, and ranges in between these values. The units are known in the art and may be defined by the rate of conversion of 1 micromole of the substrate per minute under specific conditions suited for the assay method. For example, 1 U of cABC I is the amount that catalyzes the conversion of 1 micro-mole of chondroitin sulfate A or C per minute at 37°C and pH 8.0. In some embodiments, the composition disclosed above may be administered at each site from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is administered as multiple injections at each site, and the composition is injected at different depths.

[0053] In some embodiments, the composition comprises a proteoglycan degrading domain in combination with a domain that promotes neural regeneration (as separate domains or as a chimeric protein), wherein the proteoglycan degrading domain is present in an effective amount from about 0.1 U/ml to about 100 U/ml, about 0.1 U/ml to about 80 U/ml, about 0.1 U/ml to about 60 U/ml, about 0.1 U/ml to about 40 U/ml, about 0.1 U/ml to about 20 U/ml, about 0.1 U/ml to about 10 U/ml, about 0.1 U/ml to about 5 U/ml, or about 0.1 U/ml to about 1 U/ml. Non-limiting examples include about 0.1 U/ml, about 0.2 U/ml, about 0.5 U/ml, about 1 U/ml, about 10 U/ml, about 50 U/ml, about 100 U/ml, and ranges in between these values. In some embodiments, the composition disclosed above may be administered at each site from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is administered as multiple injections at each site, and the composition is injected at different depths.

[0054] In some embodiments, the composition comprises the domain that promotes neural regeneration in an effective amount of about 0.1 to about 200 mg/ml, about 0.1 to about 180 mg/ml, about 0.1 to about 160 mg/ml, about 0.1 to about 140 mg/ml, about 0.1 to about 120 mg/ml, about 0.1 to about 100 mg/ml, about 0.1 to about 80 mg/ml, about 0.1 to about 60 mg/ml, about 0.1 to about 40 mg/ml, about 0.1 to about 20 mg/ml, about 0.1 to about 10 mg/ml, about 2 to about 40 mg/ml, about 4 to about 35 mg/ml, about 6 to about 30 mg/ml, about 8 to about 25 mg/ml, about 10 to about 20 mg/ml, about 12 to about 15 mg/ml, or any of about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, or 2.5 mg/ml. In some embodiments, the composition disclosed above may be administered at each site from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is administered as multiple injections at each site, and the composition is injected at different depths.

[0055] In some embodiments, the composition comprises a domain that promotes neural regeneration in combination with a proteoglycan degrading domain (as separate domains or as a chimeric protein), wherein the domain that promotes neural regeneration is present in an effective amount from about 0.1 to about 200 mg/ml, about 0.1 to about 180 mg/ml, about 0.1 to about 160 mg/ml, about 0.1 to about 140 mg/ml, about 0.1 to about 120 mg/ml, about 0.1 to about 100 mg/ml, about 0.1 to about 80 mg/ml, about 0.1 to about 60 mg/ml, about 0.1 to about 40 mg/ml, about 0.1 to about 20 mg/ml, about 0.1 to about 10 mg/ml, about 2 to about 40 mg/ml, about 4 to about 35 mg/ml, about 6 to about 30 mg/ml, about 8 to about 25 mg/ml, about 10 to about 20 mg/ml, about 12 to about 15 mg/ml, or any of about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, or 2.5 mg/ml. In some embodiments, the composition disclosed above may be administered at each site from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is administered as multiple injections at each site, and the composition is injected at different depths.

[0056] In some embodiments, the compositions may comprise nucleic acid molecules that encode the proteoglycan degrading domain. In other embodiments, the compositions may comprise nucleic acid molecules that encode a proteoglycan degrading domain and a domain that promotes neural regeneration.

[0057] In some embodiments, the composition further comprises pharmaceutical acceptable carriers, such as solvents, suspending agents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, vehicle, such like materials and combinations thereof, for delivering the complexes of the present invention to the patient, as would be known to one of ordinary skill in the art.

Specifically, pharmaceutical carriers that may be used are dextran, sucrose, lactose, maltose, xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin, gelatin, creatinine, polyethlene glycol, non-ionic surfactants (e.g. polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardened castor oil, sucrose fatty acid esters, polyoxyethylene

polyoxypropylene glycol) and similar compounds. A pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable (e.g., due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0058] In some embodiments, the compositions may be formulated for or are administered via parenteral administration by injection or a catheter, such as intravenous, intramuscular, intratumoral, intrathecal, intraparenchymal, peritoneal, or subcutaneous injection. Formulations for injection may be presented in unit dosage form e.g. in ampoules or in multi-dose containers, optionally with an added preservative. The compositions for parenteral administration may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in dry form such as a powder, crystalline or freeze-dried solid for constitution with a suitable vehicle, e.g. sterile pyrogen-free water or isotonic saline before use. They may be presented, for example, in sterile ampoules or vials.

Methods of Using the Compositions

[0059] Also disclosed herein are methods of treating a subject with central nervous system injury. In some embodiments, a method of treating a subject with CNS injury comprises administering to the subject in need thereof a composition comprising an effective amount of a proteoglycan degrading domain. In some embodiments, the proteoglycan degrading domain is selected from the group consisting of chondroitinase ABC I,

chondroitinase ABC II, chondroitinase B, chondroitinase AC, hyaluronidase-1,

hyaluronidase-2, hyaluronidase-3, hyaluronidase-4, PH-20, and a combination thereof. By way of example, the proteoglycan degrading domain is chondroitinase ABC 1.

[0060] In some embodiments, a method of treating a subject with CNS injury comprises administering to the subject in need thereof a composition comprising an effective amount of a proteoglycan degrading domain and a domain that promotes neural regeneration. In some embodiments, the domain that promotes neural regeneration is selected from the group consisting of neural cell adhesion molecules (N-CAM), LI CAM, myelin-associated glycoproteins, laminins, fibronectins, cadherins, Tenascins, fibronectin type-III (FN-III) domain, netrins, BSP-2 (mouse N-CAM), neural antigen D-2, neural antigen 224-1 A6-A1, NILE (nerve growth factor-inducible large external glycoprotein), Nr-CAM (neuronal cell adhesion molecule), TAG-1 (axonin-1), Ng-CAM (neuron-glia cell adhesion molecule),

F3/F11 glycoprotein, integrins, Fasciclin III, Nogo-A antagonist peptides, neurotrophic factors, and combination thereof.

[0061] In some embodiments, the method comprises administering a composition comprising a proteoglycan degrading domain. In some embodiments, the method comprises administering a composition comprising a domain that promotes neural regeneration. In some embodiments, the method comprises administering a composition comprising a chimeric protein comprising a proteoglycan degrading domain and a domain that promotes neural regeneration. In other embodiments, the method comprises administering separately a composition comprising a proteoglycan degrading domain and a composition that comprises a domain that promotes neural regeneration.

[0062] In some embodiments, the proteoglycan degrading domain and the domain that promotes neural regeneration can be administered as a gene delivery. Any technique or method known in the art for direct gene delivery to the central nervous system may be employed. For example, the genes expressing the proteoglycan degrading domain and the domain that promotes neural regeneration according to the present invention can be administered by viral vectors or by other means such as liposomes. Cells in the neighborhood will take up the DNA molecule and express proteoglycan degrading domain and/or the domain that promotes neural regeneration locally. Non-limiting examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like.

[0063] In some embodiments, CNS injuries and disorders may include but are not limited to contusion injury, traumatic brain injury, stroke, multiple sclerosis, brachial plexus injury, amblioplia, and spinal cord injuries. In one embodiment the CNS injury or disorder is a spinal cord injury. Spinal cord injuries includes disorders and traumatic injuries, such as the crushing of neurons brought about by an auto accident, fall, contusion, or bullet wound. Spinal cord injuries also include complete spinal cord injury or incomplete spinal cord injury. In even more particular embodiments, the spinal cord injury is caused by contusion of the spinal cord, bruising of the spinal cord, loss of blood to the spinal cord, pressure on the spinal cord, anterior cord syndrome, central cord syndrome, Brown-Sequard syndrome, injuries to individual nerve cells, or spinal contusion. Spinal cord injuries also include partial transectional injury or complete transectional injury.

[0064] Other embodiments of the present invention relate to methods for promoting neurological functional recovery, including sensory, motor and autonomic function, after central nervous system injury or disorder. In some embodiments, a method of promoting neurological function a subject with CNS injury comprises administering to the subject in need thereof a composition disclosed herein.

[0065] Further embodiments of the present invention relate to methods for promoting neurite outgrowth. In some embodiments, a method of promoting neurite outgrowth in a subject having a spinal cord injury comprising administering to the subject in need thereof a composition disclosed herein.

[0066] Various routes of administration are contemplated in aspects of the invention. In a particular embodiment, the composition is administered to a subject intraparenchymally. In other embodiments, methods of administration may include, but are not limited to, intravascular injection, intravenous injection, intraarterial injection, intratumoral injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, transmucosal administration, oral administration, topical administration, local administration, or regional administration.

[0067] In some embodiments, the composition is administered at or near the site of injury. In some embodiments, the composition is administered at a single site close to the injury. In other embodiments, the composition is administered at multiple sites close to the injury. Administration near or close to the site of injury may mean about 0.5 mm away from the injury site, about 1 mm away from the injury site, about 1.5 mm away from the injury site, about 2 mm away from the injury site, or about 3 mm-5 mm away from the injury site. The multiple site administration may be, for example administering at 2 sites, at 3 sites, at 4 sites, at 5 sites, at 10 sites, at 15 sites, or at 20 sites or more. The multiple sites are preferably spaced such that the points of application are separated by about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, or about 4 mm. In some embodiments, the first administration site is 1 mm away from the injury, and the subsequent administration sites are 2 mm apart from each other. In some embodiments, the administration is

intraparenchymal. In some embodiments, the administration is by injection or by infusion. In some embodiments, the injury is a spinal cord injury.

[0068] In some embodiments, the composition is administered into the spinal cord parenchyma at up to about 10 sites, each of which is caudal to the spinal cord injury site. In some embodiments, the first administration site is about 1 mm caudal to the injury border, and the second administration site is about 2 mm away from the first administration site, and each subsequent additional site (up to about 8 more) is about 2 mm further away from the previous administration site and towards caudal.

[0069] In some embodiments, it is preferred to administer the composition at multiple depths at each site as it enables delivery of the composition to all depths of the tissue. For example, in some embodiments the composition is administered at a first depth and a second depth, wherein the first depth is shallower than the second depth. In some embodiments, the composition is administered one, two, three or more depths at the same site. In some embodiments, the composition is administered at multiple depths at multiple sites, as set forth above.

[0070] In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is

administered as multiple injections at each site, wherein the composition is injected at different depths. For example, a first amount of the composition is administered as a first injection, and a second amount of the composition is administered as a second injection at the same site, and wherein the depth of second injection is greater than the depth of the first injection. For example, the depth of the first injection is about 2.5 mm and the depth of the second injection is about 3.5 mm, and both are administered at the same site. In other embodiments, the depth of the first injection is about 2 mm and the depth of the second injection is about 3.5 mm. In some embodiments, the depth of the first injection is about 2.5 mm and the depth of the second injection is about 3 mm. In some embodiments, the depth of the first injection is about 2 mm and the depth of the second injection is about 3 mm. In some embodiments, the depth of the first injection is about 2.5 mm and the depth of the second injection is about 4 mm. In some embodiments, the depth of the first injection is about 2.5 mm and the depth of the second injection is about 5 mm.

[0071] In some embodiments, the composition is administered as a single injection at each site of administration. In other embodiments, the composition is

administered as multiple injections at each site, wherein the composition is injected at different depths. For example, a first amount of the composition is administered as a first injection, and a second amount of the composition is administered as a second injection at the same site, and wherein the depth of second injection is lesser than the depth of the first injection. For example, the depth of the first injection is about 4.5 mm and the depth of the second injection is about 3 mm, and both are administered at the same site. In other embodiments, the depth of the first injection is about 4 mm and the depth of the second injection is about 3 mm. In some embodiments, the depth of the first injection is about 3.5 mm and the depth of the second injection is about 3 mm. In some embodiments, the depth of the first injection is about 5 mm and the depth of the second injection is about 3.5 mm. In some embodiments, the depth of the first injection is about 5 mm and the depth of the second injection is about 4 mm. In some embodiments, the depth of the first injection is about 6 mm and the depth of the second injection is about 3 mm.

[0072] In some embodiments, when the composition is injected at different depths at the same site, the difference in the depth of the first injection to the second injection is about 0. 1mm, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, about 0.4 mm, about 0.45 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm or about 2 mm, or ranges between any two of these values.

[0073] In some embodiments, the composition is administered near the site of spinal cord injury at two dorsoventral depths at each site of administration, the first injection targeting the intermediate gray matter (4.5 mm depth) and the second injection targeting the ventral gray matter(3 mm depth). In other embodiments, the administration at a medio-lateral position immediately medial to the medial border of the dorsal root entry zone, a distance of about 3 mm from the spinal cord midline. The needle is inserted into the spinal cord to a first depth of about 4.5 mm, and the second depth of about 3.0 mm.

[0074] In some embodiments, the composition is administered near the site of spinal cord injury at two dorsoventral depths at each site of administration, the first injection targeting the intermediate gray matter (3 mm depth) and the second injection targeting the ventral gray matter(4.5 mm depth). In other embodiments, the administration at a medio- lateral position immediately medial to the medial border of the dorsal root entry zone, a distance of about 3 mm from the spinal cord midline. The needle is inserted into the spinal cord to a first depth of about 3 mm, and the second depth of about 4.5 mm.

[0075] In some embodiments, the depths of the first injection and the second injection are same. In some embodiments, it is preferred to administer the first injection at a shallow depth when compared to the second injection as this would reduce the reflux of the injected composition out of the tissue.

[0076] In further embodiments, the amount or the dose of the composition delivered at each site may be from about 1 microliter to about 20 microliters, about 1 microliter to about 18 microliters, about 1 microliter to about 15 microliters, about 1 microliter to about 10 microliters, or about 1 microliter to about 5 microliters of the composition. Specific examples include about 1 microliter, about 2 microliters, about 2.5 microliters, about 5 microliters, about 10 microliters, about 15 microliters, about 20 microliters, and ranges in between any of these values. As set forth above, the composition may be administered as a single injection at each site of administration. In other

embodiments, the composition may be administered as multiple injections at each site, and the composition is injected at different depths. The composition that is administered may comprise any concentration of the proteoglycan degrading domain and/or domain that promotes neural regeneration, as disclosed herein. For example, the composition may comprise a proteoglycan degrading domain at a concentration of about 0.1 U/ml to about 100 U/ml, about 0.1 U/ml to about 80 U/ml, about 0.1 U/ml to about 60 U/ml, about 0.1 U/ml to about 40 U/ml, about 0.1 U/ml to about 20 U/ml, about 0.1 U/ml to about 10 U/ml, about 0.1 U/ml to about 5 U/ml, or about 0.1 U/ml to about 1 U/ml.

[0077] In some embodiments, the first injection and the second injection (when injected at the same site) may deliver same or different amount of the composition. For example, the first injection may deliver 2.5 microliters of the composition, and the second injection may deliver 3 microliters of the composition at the same site of administration. In other embodiments, the first injection may deliver 2.5 microliters of the composition, and the second injection may deliver 2.5 microliters of the composition at the same site of

administration.

[0078] In some embodiments, the composition is administered at a rate of about 0.1 microliter/min to about 20 microliters/min, about 0.1 microliter/min to about 15 microliters/min, about 0.1 microliter/min to about 10 microliters/min, about 0.1

microliter/min to about 8 microliters/min, about 0.1 microliter/min to about 5 microliters/min, or about 0.1 microliter/min to about 1 microliters/min. Specific examples include about 0.1 microliter/min, about 0.2 microliter/min, about 0.3 microliter/min, about 0.5 microliter/min, about 1 microliter/min, about 2 microliters/min, about 5 microliters/min, about 10

microliters/min, about 20 microliters/min, and ranges in between any of these values.

[0079] Another parameter of the multiple administration which can be

manipulated is the time differential between each administration. Preferably, each of the multiple administration is administered within about 30 minutes (e.g., about 0.5-30 minutes) of each other, more preferably within about 20 minutes (e.g., about 0.5-20 minutes) of each other, and even more preferably within about 10 minutes (e.g., about 1-10 minutes) of each other. In some embodiments, a time interval of about 0.5 min, about 1 min, about 2 min, about 3 min, about 4 min, or about 5 min is preferred between the first injection and the second injection, when multiple injections are administered at the same site. In some embodiments, both the first injection and the second injection may be administered at the same site without any time intervals between the injections.

[0080] In some embodiments, the total dose of the protein (proteoglycan degrading domain alone or in combination with a domain that promotes neural regeneration) that may be administered to a subject (including administration at single site or at multiple sites) is in an amount from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350

microgram/kg/body weight, about 500 microgram/kg/body weight, about 1

milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500

milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.

[0081] In some embodiments, the total amount of protein (proteoglycan degrading domain alone or in combination with a domain that promotes neural regeneration) that may be administered to a subject (including administration at single site or at multiple sites) is in an amount from about 1 milliunit/kg body weight to about 10 units/kg body weight, about 1 milliunit/kg body weight to about 5 units/kg body weight, about 1 milliunit/kg body weight to about 2 units/kg body weight, about 1 milliunit/kg body weight to about 1 unit/kg body weight, or about 1 milliunit/kg body weight to about 0.2 units/kg body weight.

[0082] In some embodiments, the compositions disclosed herein may be administered (including administration at single site or at multiple sites) once, as needed, once daily, twice daily, three times a day, once a week, twice a week, every other week, every other day, or the like for one or more dosing cycles. A dosing cycle may include administration for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks.

After this cycle, a subsequent cycle may begin approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks later. The treatment regime may include 1, 2, 3, 4, 5, or 6 cycles, each cycle being spaced apart by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

EXAMPLES

Example 1: Chondroitinase Improves Anatomical and Functional Outcomes

After Primate Spinal Cord Injury

[0083] The natural bacterial enzyme chondroitinase ABC I (Chase) can degrade the inhibitory carbohydrate side chains on CSPGs. Indeed, Chase administration to rats after spinal cord injury (SCI) enhances growth of both corticospinal and sensory axons, and improves functional outcomes. These growth-promoting effects of Chase result from removal of perineuronal nets, which increases sprouting from spared axons below the injury site and formation of new synaptic connections. Alternatively, when a cellular or peripheral nerve bridge is placed into a spinal cord lesion site, Chase can promote axonal regeneration of cut axons into the bridge. Beneficial effects of Chase administration have been reported in rodent models of SCI, nigrostriatal injury and stroke and cats with SCI.

[0084] Novel therapies to promote CNS sprouting and regeneration are needed to improve outcomes after human neurotrauma and stroke. Given the replication of Chase efficacy across multiple experimental models by independent laboratories, together with the identification of compelling candidate mechanisms underlying its beneficial effects, we advanced Chase therapy to a non-human primate model of SCI as a potential prelude to human clinical trials. Primates, both human and non-human, exhibit potentially important differences from rodents in size, anatomy, systems function and immunology that make predictions of human safety and benefit based solely on rodent studies uncertain, a concern highlighted by numerous failed trials in stroke, spinal cord injury and other disorders. We first employed a porcine model to test methods for Chase administration in the large animal spinal cord, and found that intrathecal infusions failed to degrade CSPGs in spinal cord gray matter. We accordingly moved to intraparenchymal spinal cord injections of Chase; these effectively degraded CSPGs in our rhesus monkey model of C7 spinal cord lateral hemisection. This experiment hypothesized that degradation of perineuronal nets surrounding neurons in spinal cord gray matter below the lesion would enhance sprouting of spared host axonal systems, including the corticospinal tract (CST), the most important motor system for voluntary movement in primates. Previously, we reported spontaneous sprouting of spared corticospinal axons in monkeys after C7 lesions, supporting their potential relevance as a target for enhanced growth after human SCI, where the majority of lesions spare a superficial rim of white matter including corticospinal axons. Thus, using intraparenchymal injections, we tested Chase administration in rhesus monkeys with hemisections, comparing

experimental and control lesion subjects.

RESULTS:

[0085] Route of Chase Administration: Successful Delivery by Intraparenchymal but Not Intrathecal Infusion

[0086] In a preliminary study, we determined whether intrathecal injections of Chase, a frequent and effective route of administration in rat studies, would effectively diffuse into the spinal cord of a larger animal. We used pigs for this study because their spinal cords measure roughly 8-11mm in diameter (compared to rat 3.5mm width and human 12.5mm width). Seven adult American Yorkshire-Landrace-Duroc pigs received intermittent (every other day for two weeks) intrathecal injections of 2 ml of saline (N=l), 5 Units Chase enzyme/ml (5U/ml, 10 U/dose; N=3 animals; For Chase, 1 U is the amount that catalyzes the conversion of 1 micro-mole of chondroitin sulfate A or C per minute at 37°C and pH 8.0), or 25 Units Chase enzyme/ml (50 U/dose; N=3 animals). Injections were made at 0.2 ml/min via an indwelling catheter into the intrathecal space between vertebrae T11-13. Pigs were sacrificed two days after the last dose of intrathecal Chase. With this infusion method, 2B6 immunohistochemistry revealed degradation of GAG side chains in both low-dose and high- dose groups in a 1mm rim of the superficial circumferential thoracic spinal cord white matter (Figure 5 A, B). However, peri-neuronal nets in spinal cord gray matter were not attenuated, as revealed by wisteria floribunda agglutinin (WFA) lectin- histochemistry (Figure 5 C, D). Because a prime target of Chase therapy is removal of peri-neuronal inhibitory CSPGs in gray matter to enhance plasticity and new synapse formation, we concluded that intrathecal infusions of the current formulation of Chase in the larger spinal cord were not an effective delivery method to enable hypothesis testing in the primate model, and (in the absence of a more parenchymal-penetrant formulation or method for Chase administration) we moved to intraparenchymal injections in monkeys.

[0087] In a preliminary study to test the delivery efficacy of intraparenchymal Chase administration in monkeys, two adult rhesus male monkeys underwent C7 spinal cord hemisection lesions followed four weeks later by intraparenchymal injections of Chase. Four weeks post lesion is a time point at which spontaneous plasticity in the primate is occurring (see below). Monkeys received injections of 20 U/ml Chase starting at spinal cord level C7 (1 mm caudal to the lesion). 5 mΐ of Chase were injected at each of 10 sites (spaced 1.5 mm apart in the rostrocaudal axis) on the right side of the spinal cord from C7-T1. Chase was infused at two dorsoventral depths per injection site, the first targeting the intermediate gray (2.5mm depth) and the second targeting the ventral gray (3.5mm depth). 2.5 mΐ were injected at each depth (infusion rate 0.5 mΐ/min) for a total volume of 5 mΐ per site (Figure 1). As noted above, the primary objective of this injection design is to degrade peri-neuronal CSPG“nets” that surround adult neurons in host gray matter below the lesion site, to enhance sprouting from spared host axons that occurs spontaneously after SCI. We did not attempt to promote regeneration into the lesion site by grafting cellular or peripheral nerve bridges into the lesion cavity. Subjects were transcardially perfused 2 weeks later to assess CSPG degradation by WFA histochemistry. CSPGs were completely degraded in the spinal cord gray matter over a distance of at least 2mm from each injection site without evident damage to the spinal cord or neuronal loss (Figures 2, 6-8). We therefore used the same injection design to deliver Chase to the subjects in the therapeutic efficacy study (see below). CSPGs were also degraded at the caudal aspect of the C7 spinal cord lesion site (Figure 9).

[0088] Chase Therapeutic Efficacy Study in Non-Human Primates

[0089] A total of 12 adult male rhesus monkeys underwent C7 right-sided complete spinal cord hemisection lesions, which result in persistent deficits in right hand dexterity but retention of bowel/bladder function and locomotion. While this lesion results in extensive impairment in right hand function immediately after the lesion, there is consistent and predictable but incomplete recovery from 4-8 weeks post-lesion, with little further improvement thereafter. Hypothesizing that the period of recovery from 4-8 weeks is mediated by spontaneous anatomical plasticity, we injected Chase four weeks post-injury to target enhancement of endogenous plasticity. This delay was also chosen because it is a clinically practical time to intervene after SCI, when most medical complications in humans have stabilized and patients are better candidates to undergo a surgical intervention.

Moreover, the extent of functional deficit and probability of further spontaneous clinical recovery can be assessed more reliably in humans four weeks post-injury than within days of injury, considerations that are helpful in designing a clinical trial.

[0090] The primate C7 lesion model was designed to assess potential therapies for improving hand function, since neural circuitry for hand control is located in the C7-T1 spinal levels ranging from 1mm to 15mm below the lesion. These segments received intraparenchymal injections of Chase (20 U/ml) four weeks after lesions were placed, as indicated above (Figure 1). Four days later, monkeys underwent daily half-hour exposure to a large testing enclosure enriched with numerous objects, perches and food rewards to encourage use of both forelimbs and hindlimbs. Monkeys engage in naturalistic and reward- driven behaviors in this environment. Once per week, several functions in the enclosure were evaluated and scored on an ordinal scale by an observer blinded to treatment condition. Some of these functional measures are sensitive to forelimb performance (Figure 10) and others to hindlimb performance; yet other scores represent composites of overall function (Figure 3). Separately, monkeys underwent daily exposure to a cage-based Brinkman board task with graded levels of difficulty, requiring retrieval of small food items from baited wells using only the affected right arm (Figure 3). There were five levels of difficulty on this task, and monkeys were scored weekly on each level. A total of 12 monkeys underwent C7 lesions: 6 received injections of Chase and 6 were lesioned controls. Of the lesioned controls, 4 monkeys received injections of 5 mΐ/site of saline; 1 underwent needle entry only into each site; and 1 monkey underwent sham surgery to expose the spinal cord, without needle entry or saline injection. No differences were noted between these control subjects on any measured parameter, and their data were combined in the subsequent analyses. Three months after Chase administration or control injections, subjects underwent anterograde tracing of the corticospinal projection with biotinylated dextran amine in the left hemisphere and dextran- conjugated Alexa-Fluor 488 in the right hemisphere, and were transcardially perfused 6 weeks later (5.5 months after the initial lesion). One control subject was excluded post-hoc from behavioral analyses on the basis of over-lesion that extended onto the right side of the spinal cord (see Figure 11). An additional control subject did not undergo corticospinal tracing due to weight drop below the minimum level required to allow surgery.

[0091] Chase Administration Improves Functional Outcomes

[0092] We performed a non-linear principal components analysis (NL-PCA) on the ensemble of all behavioral outcome data from the exercise enclosure and the Brinkman board (Fig. 3; for further methodology see Figure 15). Behavioral measures loaded highly on the first principal component (PCI), which accounted for 68% of the variance in recovery (Figure 12). The resulting weights for each measure were then extracted to create PC scores for each animal at each timepoint. The PCI scores were then used to test for group differences in performance over time. Overall, PCI showed a significant difference between lesioned controls and Chase-treated animals over time, significantly favoring the Chase- treated group (condition x time, F(9, 23.87) = 5.15, P = 0.001, AIC = 94.21, linear mixed model; Figure 3 A). Lesioned control animals in this study followed a previously reported pattern (noted above) of marked impairment in function post-lesion relative to their pre-lesion baseline for the first four weeks after C7 hemisection (Figure 3). This was followed by a spontaneous recovery of function (reflected by the PCI scores) over weeks 5-8 (Figure 3 A). The function of lesioned controls did not significantly improve further, from weeks 5-8 through weeks 17-20 (main effect of time, F(3, 12) = 1.87, P = 0.189, hr2 = 0.319, repeated measures ANOVA; Figure. 3A).

[0093] Animals treated with Chase also exhibited marked impairment in hand function over the first 4 weeks post-lesion, followed by early spontaneous improvement by weeks 5-8. However, in contrast to the lesioned control group, animals in the Chase treatment group exhibited continued improvement from weeks 5-8 to weeks 17-20 (main effect of time, F(3, 15) = 7.53, P = 0.003, hr2 = 0.601, repeated measures ANOVA; Figure 3A). Analysis of performance on individual tasks showed divergence in performance that favored Chase- treated subjects on measures sensitive to right hand use (Figure 3 D-I) but not locomotion (Figure 3 J), an observation consistent with the delivery of Chase to spinal cord segments influencing hand function. In terms of absolute performance levels, monkeys that received Chase exhibited an overall final food object retrieval success rate of 47 ± 12% across all difficulties of the Brinkman board in the final testing period, compared to nearly 100% pre injury performance, and an inability to retrieve objects in the first four weeks after injury. In comparison, lesioned control monkeys recovered to 31 ± 14% final food object retrieval on the Brinkman board, compared to 5 ± 3% performance in the first four weeks after injury.

This is a 51 % difference between groups in the extent of recovery, favoring Chase. The difference in absolute performance between groups is significant (P = 0.014, linear mixed model, condition x time, F(37, 12.48) = 3.26, AIC = 1461.5).

[0094] PC A also generated a second and third principal component that accounted for 10.5 % and 5.1% variance respectively (Figure 12). However, loading patterns for these components did not indicate clearly definable eigenmodules, and scores on these components were therefore not tested.

[0095] Chase Administration Improves Anatomical Outcomes [0096] We hypothesized that, by degrading peri -neuronal nets in the gray matter surrounding neurons and synapses caudal to the C7 lesion site, Chase administration would increase axonal sprouting and synaptogenesis from spared axonal systems. Previously we reported that spared components of the corticospinal system in rhesus monkeys that decussate across the spinal cord midline undergo spontaneous axonal sprouting after C7 hemisection lesions. Following Chase administration, we found greater total length (compared to control subjects) of corticospinal axons in gray matter caudal to the injury at C8, indicative of sprouting of spared corticospinal axons that deccusate across the spinal cord midline

(F(l,30.02) = 4.81, P = 0.036, AIC = 693.89, linear mixed model; Figure 4C). The same Chase-associated significant increase in axon density (compared to control subjects) was observed in CST axons originating in the right motor cortex (F(l,43.13) = 13.15, P = 0.001, AIC = 1239.11, linear mixed model; Figure 13). All measures were made in a blinded fashion, and analysis of corticospinal axons arising from the left hemisphere was done in one laboratory, while analysis of axons arising from the right hemisphere was done independently in a second laboratory.

[0097] In addition, Chase treatment was associated with a significant increase, compared to lesioned controls, in the number of corticospinal synapses in gray matter caudal to the injury (F (1,38.12) = 12.06, P = 0.001, AIC = 119.18, linear mixed model; Figure 4). CST synaptic connectivity was assessed by quantifying the number of CST terminal boutons that co-localized with synaptophysin immunoreactivity, divided by the sampled volume of gray matter (Figure 4E).

[0098] We also assessed whether serotonergic axons respond to chondroitinase treatment. In a previous study of lesioned animals lacking experimental treatment, serotonergic axons, in contrast to corticospinal axons, did not exhibit detectable sprouting after C7 hemisection lesions. In the present study, we observed no difference between Chase and control subjects in total serotonergic axon length in motor neuron pools caudal to SCI (Wald x2 = 0.009, QIC = 4.86, P=0.924, generalized estimating equation; Figure 14). All anatomical measures were made by observers blinded to treatment group.

[0099] Safety

[0100] Anatomical analysis of injection sites with Nissl stain revealed no detectable toxicity after intraparenchymal spinal cord injections (Figure 6). We also quantified motor neurons in a series of sections straddling an injection site in each monkey, and found no neuron loss in Chase-injected vs. lesion control monkeys (Figure 7). In addition, we quantified IBA-1 labeling in gray matter adjacent to injection sites to assess microglial responses, and found no difference between Chase-injected, saline-injected, and lesion control groups (ANOVA, F(2, 3.78) = 0.11, P=0.89; Figure 8). Moreover, cellular- mediated inflammation in gray matter, assessed by labeling for CD8, CD3, and CD45, was extremely mild 2 weeks after Chase injection (Figure 7; CD8 labeling shown). Labeling for CD8, CD3, and CD45 was no longer detectable 4.5 months after injections of either Chase or saline (Figure 7). Systemically, Chase-treated and lesioned control monkeys exhibited no notable differences in weight, activity or post-lesion complications. Although formal pain tests were not employed, monkeys exhibited no behaviors that suggested pain (e.g., reduced activity, enhanced startle).

DISCUSSION:

[0101] Chondroitinase treatment showed efficacy on both anatomical and functional outcome measures. On functional assays, effects were only suggested on forelimb measures (which were anatomically targeted by Chase injections into cervical segments mediating hand control), whereas effects were not evident on hindlimb measures.

Anatomically, Chase treatment significantly increased the length of corticospinal axons in cervical spinal cord segments below the lesion, representing sprouting of axons spared by the lesion, and also significantly increased the number of corticospinal terminals colocalizing with a synaptic marker, indicating a likely increase in the number of corticospinal synapses.

[0102] In contrast to studies in rats and cats, effects of Chase on serotonergic axons were not detected. Interestingly, serotonergic axons do not detectably sprout following C7 hemisection lesions in rhesus monkeys. A number of mechanisms could potentially account for this observation, including the possibility that serotonergic axons are highly branched and sustaining collaterals may minimize injury responses in the hemisection model.

[0103] The overall functional success of food object retrieval on the Brinkman board task, a test of finger use, was 47 + 12% among Chase-injected animals compared to 31 + 14% in lesioned controls, a difference of 51% favoring Chase. Chase-treated subjects also showed better recovery than lesioned controls on an object manipulation scale in the testing enclosure (21.25 pt. improvement vs. 12.5 pt. improvement; Figure 3D). On anatomical measures, CST axon terminal innervation was 50% denser after Chase treatment (Figure 4C) and the number of putative synapses on corticospinal axons increased 2-fold compared to lesioned controls (Figure 4E). The reticulospinal system was not traced or quantified in this study. It is possible that, like the CST, reticulospinal axons might sprout in response to Chase treatments and mediate functional recovery.

[0104] We developed the non-human primate model of SCI to enable testing of potential translational therapies, invasive therapies in particular, prior to human translation. The rhesus monkey represents the most proximate animal model of the human nervous system that is experimentally testable. We have found that rhesus macaques exhibit variation in motivation, task engagement and response to injury that constitutes a source of variability that exceeds that encountered in rodent models. This variability may result from the increased complexity of the rhesus brain compared to the rodent and the considerable genetic differences in primates that exceed variation found even in outbred rat strains. Consequently, it is likely that this non-human primate model, while less variable than human injuries, incorporates some features of variability that are typically encountered in the human clinical setting. Thus, the ability to detect significant differences on several measures following Chase administration despite inter-subject variability in this model provides support for the initiation of human clinical trials. While the sample size in this study is small, the costs and duration of this work constitute a relative barrier to studying larger numbers of animals. Our use of data-driven multivariate statistical analysis prior to hypothesis testing allowed us to efficiently test recovery in ensemble across multiple tasks, thereby maximizing information gain with small N, while remaining sensitive to therapeutic effects. Notably, multivariate statistics are not typically employed in clinical trials, which instead generally rely on one or two pre-defmed outcome measures to establish clinical efficacy. Interestingly, had we used classical univariate statistics in the present study, the functional benefit of Chase therapy would likely not have been appreciated. This raises the possibility that a treatment of potential functional benefit might be unappreciated in a clinical trial, despite the presence of a clear biological benefit at the level of axonal growth and synaptogenesis. This finding highlights the great importance of continued consideration and development of multivariate outcome measures in clinical trials, a point that is receiving increasing attention.

[0105] There are important limitations to this non-human primate model of SCI. First, the injury itself is a hemisection, rather than a contusion, which is generally considered more representative of human injuries. We have, in fact, developed a contusive model of SCI in the non-human primate, which could be employed in future pre-clinical studies. Second, the C7 level of the hemisection SCI is optimally placed for recovery of hand function. New studies would be required to assess potential benefit to humans with higher cervical injuries or with thoracic injuries, although preceding rat SCI studies have demonstrated functional benefits of Chase after C4 and thoracic SCI.

[0106] In addition to presenting evidence of efficacy, this study also preliminarily demonstrates that multiple parenchymal injections of Chase appear to be well-tolerated. The outcome of the present study, taken together with beneficial effects observed by many independent research groups in various rodent models of SCI, support the concept of translation of Chase treatment to human clinical trials.

[0107] The large-animal porcine model revealed that intrathecal infusions of the native Chase enzyme at even relatively high concentrations did not reach gray matter target regions. Thus, to advance these proof-of-concept studies into the clinically-relevant primate model of SCI, we turned to more invasive intraparenchymal injections of Chase. The injections appeared to be well-tolerated anatomically based on inflammatory markers and cell counts. Chemical modifications to Chase may enhance its penetrant properties after intrathecal administration, a possibility that merits further study for potential future clinical translation.

METHODS:

[0108] Subjects

[0109] We studied a total of 14 rhesus macaques (Macaca mulatta, aged 6-10 years) and seven pigs (American Yorkshire-Landrace-Duroc pigs, adult). Power analysis: two of the macaques were utilized to establish the delivery efficacy of intraparenchymal Chase administration. The remaining 12 macaques were split into two groups of 6. At a standard deviation of 20% (an estimate drawn from our previous work) and effect size of 40%, N=6 per group gives a computed power of 0.89 at an alpha level of 0.05. Power analyses were not performed for the porcine preliminary study of intrathecal Chase delivery. All surgical and experimental procedures adhered to the principles outlined by American Association for the Accreditation of Laboratory Animal Care. Porcine subjects were housed and tested at MPI, Inc., which is USDA registered and compliant with the Animal Welfare Act (AW A), and has Assurance of Compliance with the Public Health Service Policy for the Humane Care and Use of Laboratory Animals (PHS/OLAW Assurance). Non-human primates were housed and surgeries performed at the California National Primate Research Center (CNPRC, Davis,

CA); all primate procedures were approved by the CNPRC Institutional Animal Care and Use Committee (IACUC). Subsequent tissue processing and analysis was performed at the Center for Neural Repair (University of California, San Diego; La Jolla, CA) or at Cambridge University..

[0110] Porcine Study

[0111] Spinal cord lesions were not made. All subjects underwent intrathecal catheter placement at T11-13 and Chase provided by Acorda was infused through this indwelling catheter every other day for a total of 14 days (7 doses). Subjects were

transcardially perfused two days later. Three animals received low-dose Chase (5 Units/ml x 2 ml per infusion, total dose over 14 days of 70 Units), and three animals received high-dose Chase (25 Units/ml x 2 ml per infusion, total dose over 14 days of 350 Units). Note that because Chase is an enzyme, it is packaged and administered according to units of enzymatic activity (U). For Chase, 1 U is the amount that catalyzes the conversion of 1 micro-mole of chondroitin sulfate A or C per minute at 37°C and pH 8.0. The low dose was comparable to rat intrathecal studies, scaled according to CSF volume. The high dose was meant to test whether better CSPG degradation could be achieved with higher Chase concentration, but showed no difference in CSPG degradation compared to the low dose (see Results). Subjects were sacrificed 2 days after the last infusion (16 days after the first infusion). Because Chase achieves its maximum digestion range within 24 hours, this time point should reflect the extent of CSPG digestion achievable through intrathecal administration. Spinal cords with dura intact were removed, sectioned into 1 cm blocks and fixed in 4% cold paraformaldehyde for 24 hours. Tissue was transferred to 10% glycerol/2% DMSO in PBS overnight and then transferred to 20% glycerol/ 2% DMSO in PBS (pH 7.4) and was stored refrigerated (2 to 8°C). Tissue was sent to UCSD and sectioned in the coronal plane on a freezing microtome set at 35 ^A m intervals. Sections were then labeled as detailed below for primate studies.

[0112] Primate Lesion Surgery

[0113] Animals were sedated with 1 mg/kg ketamine intramuscularly and anesthetized with 1.5-2.5% isoflurane. The caudal half of the C5 dorsal lamina and the entire C6 dorsal lamina were removed. The dura was slit longitudinally along the midline and retracted gently. A surgical micro-knife was mounted on a stereotaxic arm positioned at the spinal midline midway between the C5 and C6 dorsal laminae. This rostrocaudal position corresponds to the C7 spinal cord segment. The stereotaxic manipulator was used to lower the blade through the entire dorsoventral extent of the spinal cord without severing the ventral artery. This initial cut established the medial position of the lesion. The lesion was then completed using microscissors under microscopic observation by the surgeon to ensure lesion completeness laterally and ventrally. The dura was sutured, then the overlying muscle and skin were sutured in layers. Animals retained bowel, bladder, and autonomic function after SCI. Lesion reconstructions are shown in Figure 11.

[0114] Chondroitinase Injections

[0115] Subjects were assigned to Chase-treated or control groups according to post-lesion performance on functional tests (see below) such that pre-treatment functional deficits were equivalent across groups. Four weeks after SCI, the lesion site was re-exposed, and additional laminectomy and longitudinal dural incision was performed to expose the C7- C8-T1 spinal cord. Hand-assembled nested silica cannulae (240 and 150 pm outside diameter) were attached to a NanoFil 100 pi syringe filled with 20 U/ml chondroitinase ABC I in ice-cold saline solution. This concentration of Chase is at the higher end of the spectrum of doses used in the literature. Because Chase is an enzyme, it tends to be active upon reaching a threshold activity concentration, and higher concentrations are typically ineffective in generating a greater effect. The volume injected (5 pi per site) was scaled from previous rat studies according to spinal cord cross-sectional area (monkey ~= lOx rat). The syringe was mounted in a syringe pump attached to a stereotaxic frame. 5 pi of

chondroitinase was injected into the spinal cord parenchyma at each of 10 sites caudal to the SCI site. The first site was 1 mm caudal to the lesion border, each additional site was 1.5 mm further caudal. Each site was 0.7 medial to the medial edge of the dorsal root entry zone (DREZ) on the right side of the spinal cord. At each site, the pia was nicked with a 25G needle to allow cannula entry. Chondroitinase was injected at two depths (2.5 mm and 3.5 mm; 2.5 pi each) at a rate of 0.5 pl/min. To minimize Chase reflux, after the second injection at each site we waited 2 min before withdrawing the cannula. As with the initial SCI, the dura was sutured, then the overlying muscle and skin were sutured in layers. No adverse effects were observed following Chase administration.

[0116] Corticospinal tracing [0117] CST axons were traced to assess regeneration into the graft. As previously described, six weeks prior to sacrifice (4 months after SCI), the subject was anesthetized and a craniotomy was performed to expose the right and left motor cortex. Using a pulled glass micropipette attached to a picospritzer (Parker Hannifin, Fairfield, NJ), 300 nl of the anterograde neuronal tracer biotinylated dextran amine (BDA; 10,000 MW, 10% in water, Thermo Fisher) was injected at each of 127 sites (59 different surface locations) in the left motor cortex. These sites included motor cortex innervating the hand, arm, trunk, leg, and foot. Identical injections of the anterograde tracer dextran-conjugated Alexa Fluor-488 (A488, 10,000 MW, 5% in water, Thermo Fisher) were made in the right motor cortex. After tracer injection, the excised piece of cranium was replaced, cemented in place with dental acrylic, and the incision closed in layers.

[0118] Tissue processing

[0119] Subjects were sacrificed 5.5 months after SCI. Subjects were deeply anesthetized and transcardially perfused with a 4% solution of paraformaldehyde, and the spinal cord was dissected out of the spinal column. Spinal cord dura was removed, and the spinal cord was cut in the transverse plane into 1.5-cm-long blocks as detailed previously. Blocks from segments C3, C7, C8, Tl, and T2 were cut into 40- pm-thick transverse sections using a freezing microtome. Tissue sections were stored at -20 °C in cryoprotectant (25% glycerin (v/v), 30% ethylene glycol (v/v) in 0.05 M phosphate buffer).

[0120] Fluorescent immunolabeling

[0121] Transverse sections were pre-treated with 50% methanol for 20 min at 22- 24°C, washed in tris-buffered saline (TBS) and blocked for 1 hr in TBS containing 5% normal donkey serum and 0.25% Triton X-100. Sections were incubated in streptavidin 488 (1 :400, Life Tech) and/or primary antibodies against serotonin (1 :5000, Immunostar), synaptophysin (1 :300, Sigma), IBAl (1 : 1500, Wako), GFAP (1 : 1500, Encor Bio). Sections were washed with TBS and then incubated in Alexa-Fluor 488- or 594- conjugated secondary antibodies (Thermo Fisher, 1 :500) for 1 hour. Sections were washed with TBS, mounted on slides, and coverslipped with Mowiol mounting medium. All antibodies were used previously in monkeys.

[0122] Light-level labeling [0123] For WFA and BDA detection, sections were washed in TBS and then quenched in 0.6% H202 in 50% methanol in TBS for 30 minutes. Sections were then washed with TBS and incubated overnight at 4°C in WFA-biotin (1 : 1000; reduced form; Sigma L1766) or Vectastain Elite ABC solution (Vector Labs) and 0.25% Triton-X100 in TBS. WFA sections were washed with TBS, then incubated with Vectastain Elite ABC solution for 1 hour. All sections were then washed again and developed with diaminobenzidine (DAB) and NiC12. Sections were mounted on gelatin-subbed glass slides, dehydrated, and coverslipped with DPX mounting medium.

[0124] For A488, CD8, CD3,and CD45 detection, sections were washed in TBS and then quenched in 0.6% H202 in 100% methanol in TBS for 30 minutes. Sections were then washed with TBS, blocked for 1 hr in 5% horse serum and 0.25% Triton-X100 in TBS (TBS++), and incubated at 4°C in TBS++ with rabbit anti-A488 (1 :5000, ThermoFisher) for two nights, or mouse anti-CD8 (1 :500, BD Pharmingen), mouse anti-CD3 (1 :500, BD Pharmingen), or mouse anti-CD45 (1 :500, BD Pharmingen) for one night. Sections were washed in TBS, then incubated for 45 minutes in, as appropriate, horse-anti-rabbit or horse- anti-mouse Vector ImmPRESS (1 : 1, Vector Labs) in TBS. Sections were washed again and developed with DAB and NiC12. Sections were mounted on gelatin-subbed glass slides, dehydrated, and coverslipped with DPX mounting medium.

[0125] For 2B6 detection, pig tissue sections were washed in TBS and then quenched in 0.6% H202 in TBS for 30 minutes. Sections were then washed with TBS, blocked for 1 hr in 5% horse serum and 0.25% Triton-X100 in TBS (TBS++), and incubated at 4°C in TBS++ with mouse anti-2B6 (1 :2000, Seikagaku, now available from Amsbio) for one night. Sections were washed in TBS, then incubated for 1 hour in biotinylated horse-anti mouse lgG (1 :200, Vector) in TBS++. Sections were washed again with TBS, then incubated with Vectastain Elite ABC solution for 45 minutes. Sections were then washed again and developed with diaminobenzidine (DAB) and NiC12. Sections were mounted on gelatin- subbed glass slides, dehydrated, and coverslipped with DPX mounting medium.

[0126] Nissl substance was labeled in sections from a 1 : 12 series covering spinal cord segments C3, C7, C8, Tl, and T2. Sections were washed in TBS, fixed in buffered 4% paraformaldehyde for 1 hour at room temperature, mounted on gelatin-subbed glass slides, and dried overnight. Sections were then defatted in a 1 : 1 mixture of chloroform and ethanol, rehydrated, placed briefly in 0.25% thionin, dehydrated, cleared, and coverslipped. [0127] Quantification of axons, synapses, motor neurons, microglia

[0128] Axon density was quantified with ImageJ 1.41c (Wayne Rasband,

National Institutes of Health) and a custom -written script, derived from previous methods. 20x images of the entire spinal cord were obtained, and loaded into ImageJ. All images were corrected for brightness and contrast equally to reduce background noise. For CST axons, regions of interest (ROIs) were drawn around the right gray matter. For 5HT-labeled raphespinal axons, ROIs were drawn around the right and left motor pools. The image was auto-thresholded, the detected fiber profiles were skeletonized (so that sum of pixels = total axon length) and measurements for each ROI were recorded in the right gray matter.

[0129] Synapse counts (colocalization of BDA and synaptophysin) were performed with 60x confocal image stacks loaded into ImageJ. Three non-overlapping image stacks were obtained in the right IZ of each of 4 tissue sections per subject. The experimenter systematically sampled the entire image stack for clearly co-localized BDA and Syn in bouton-like swellings connected to BDA-labeled axons. The number of putative synapses was divided by the volume of the image stack (-900,000 pm ³ ) and converted to

synapses/mm ³ .

[0130] Spinal motor neurons (MNs) were counted in Nissl-labeled sections at sequential rostro-caudal distances from a sample injection site on both injected and uninjected sides of the spinal cord. The number of MNs on the injected side was divided by the sum of the number of MNs on injected and uninjected sides, such that the expected proportion was 0.5. Five to six total sections were counted per subject (up to one section per x-axis position).

[0131] Microglial density was quantified using IBAl labeling. IBAl pixel density was calculated using the ImageJ autothresholding function on images of the intermediate zone of the gray matter in tissue sections immediately adjacent to injection sites (or similar regions in C8-T1 spinal cord for uninjected subjects). Tissue sections from subjects in the present experiment were immunolabeled for IBAl concurrently with sections from subjects involved in a previous study ([30]; N=3 Intact subjects, N=4 subjects 2 weeks after C7 hemisection SCI, N=3 subjects 5 months after C7 hemisection SCI);

[0132] Experimenters were blind to group membership during all quantitative analyses. [0133] Functional testing

[0134] Functional outcomes were assessed in an open-field paradigm using an ordinal scale. In the open field testing enclosure (5 x 7 x 10ft ³ ), monkeys can access perches at various levels, climb along the enclosure walls, and manipulate objects containing small food rewards using dexterous hand movements. Use of each hindlimb and forelimb on the tasks is rated by an observer over the 30 min observation period; video recordings allow re assessments as necessary. A total of 72 points on the scale are possible. We also rated monkeys on a 22-point forelimb sub score that focuses on arm, hand and digit use during object manipulation. Monkeys underwent pre-lesion baseline training, followed by three half hour exposures per week to the enclosure to encourage activity and limb use. Performance was videotaped and rated once weekly. We compared the level of functional performance of lesioned control monkeys (N=5) to Chase-treated monkeys (N=6).

[0135] Subjects’ ability to perform fine motor functions with the affected hand were also assessed using a modified, cage-mounted Brinkman board, which requires use of the affected (right) hand to retrieve small food items. This custom-made plexiglas box is hung at the front of each animal’s cage. The left hand pushes a spring-mounted lever, which moves a plexiglas occlusion plate, giving the affected hand access to the Brinkman board under the occlusion plate. This configuration allows the subject to perform the task in the home cage, and requires use of the affected hand, ensuring optimal rehabilitation and functional testing. Five different boards of increasing difficulty were presented sequentially in each testing session. Difficulty is increased by: 1) increasing the depth of wells from which monkeys need to extract the food reward, 2) altering the angle of the well relative to the monkey’s position, and 3) increasing the number of reward wells (Figure 3). Monkeys were scored on the number of rewards obtained from each board, the time to clear the board, and whether a pincer grasp was used.

[0136] All functional outcomes were measured and recorded by observers blinded to treatment group.

[0137] Statistical Analysis

[0138] For statistical analysis of functional data, we employed an a priori established, blinder, data-driven statistical workflow, (Figure 15). Analysis was divided into two distinct stages. First, we compiled the full behavioral data outcome matrix (animals x tests x time points) and performed non-linear PCA (NLPCA) on whole matrix from all animals (both treated and untreated). The PC loadings were then examined and cross- validated to confirm the resulting PC scores serve as legitimate multidimensional outcome metrics . This was done in a computationally unsupervised manner by applied biostatisticians blind to experimental condition. Second, we performed a directed, single hypothesis test (linear mixed model, Figure 3 A), unblinded to the effect of Chase on PCI. This staged statistical analysis approach brings together machine-learning-based pattern detection (stage 1), with hypothesis testing (stage 2) to maximize outcome information without relying on multiple hypothesis testing, thereby limiting the statistical false discovery rate. Technically, the NL-PCA approach (stage 1) reduces the large amount of behavioral data (open cage and each level of brinkman board for each animal at each timepoint) into an overall integrated behavioral score, while taking into account level of measurement of each measure (ordinal, continuous etc) through optimal scaling transformations and pattern detection using the alternating least squares algorithm. The resulting principal components are orthogonal, with the first PC accounting for the most variance. The PC loading reflects the correlation between each measure and the non-linear PC. The squared factor loading for each of these measures indicates the percentage of variance in a principal component that is explained by that measure. A bootstrapping procedure was then used for internal validation of the loading pattern. The bootstrapping procedure resampled the dataset 1,000 times, with each iteration producing loadings from a slight variation of the original dataset. This step was followed by a pattern matching algorithm to test the similarity between the original and bootstrapped loading patterns. After confirmation of loading pattern stability, PC loading weights were then applied to derive a PC score for each animal at each individual time-point. In stage 2, we applied a linear mixed model (LMT M) to assess the impact of chase on PC scores over time for each group, using a diagonal covariance matrix structure with individual animal as a random factor, thereby standardizing scores for each animal according to their pre-drug baseline function Stage 1 and stage 2 were implemented in IBM SPSS Statistics v.25 using CATPCA and LMM subcommands, respectively. Repeated measures analysis of variance (ANOVA) was used to test for main effects of time for each group in instances where data had no missing values, with sphericity assumed. CST and synapse measurements were also analyzed using an LMM, with histological section set as a repeated measure for each subject, using a diagonal covariance matrix structure and subject as a random factor. A generalized estimating equation (GEE) model was used for non-linear 5HT ratio data. Linear mixed models and GEEs are modem approaches that do not suffer from the violations of 'independence' and 'sphericity' assumptions that plague general linear modeling approaches popularized in the 20th century. F values and degrees of freedom are reported for all ANOVA and LMM (precise, fractional df reported) and Wald Chi squared value is reported for the GEE model. Effect sizes and fit metrics for each analysis are reported throughout (partial eta squared (hr2) for F tests, Akaike information criteria (AIC) for linear mixed models, quasi likelihood under the independence model criterion (QIC) for GEE). As main effects were between only two conditions throughout, no post hoc corrections for multiple comparisons were necessary. Statistical significance for all effects was assessed at a p value below 0.05.

All analyses were run using SPSS v.25 (IBM).