AU2016204464B2 - Proteoglycan degrading mutants for treatment of cns - Google Patents

Abstract

The present disclosure relates to the preparation and deletion mutants of chondroitinase proteins and their use in methods for promoting the diffusion of therapeutic composition into tissues and their use for neurological functional recovery after central nervous system ('CNS') injury or disease. See Fig 7. WO 2004/110360 PCT/US2004/015662 IdD 2b0 3b0 60 6 doo bOo 800 m 9 WT-ABCI - 1 1 . 1 - '11, - " 1 NA2O(.p oplpl--lw -11 (A45-N1 023) NA60 [7- (F85-N1023) NA60 CA80 (F85-A942)

Description

COMPLETE SPECIFICATION

DIVISIONAL APPLICATION

APPLICANT: ACORDA THERAPEUTICS, INC.

Invention Title: PROTEOGLYCAN DEGRADING MUTANTS FOR

TREATMENT OF CNS

The following statement is a full description of this invention, including the best method of performing it known to me:

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PROTEOGLYCAN DEGRADING MUTANTS FOR TREATMENT OF CNS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit and priority of U.S. Provisional

Application Serial No. 60/471,240, Attorney Docket No. 127304,01300, filed May 16, 2003; U.S. Provisional AppEcation Serial No. 60/471,239, Attorney Docket No. 127304.01200, filed May 16,2003; U.S. Provisional Application Serial No. 60/471,300, Attorney Docket No. 127304.01400, filed May 16,2003; U.S. Provisional Application Serial No. 60/474,372 i

Attorney Docket No. 127304.01700, filed May 16, 2003; and U.S. Pat. Application entitled FUSION PROTEINS FOR TREATMENT OF CNS, filed cocurrently herewith on May 17, 2004, U.S. Patent Application Serial No. [not yet assigned] Attorney Docket No, 127304.01201 the contents of each of these references incorporated herein by reference in their entirety.

BACKGROUND AND SUMMARY [0002] Chondroitinases are enzymes of bacterial origin that act on chondroitin sulfate, a component of the proteoglycans that are components of the extracellular matrix of a wide variety of tissues such as the central nervous system and for example they can mediate the attachment between the retina and the vitreous body of the human eye. Examples of chondroitinase enzymes are chondroitinase ABC I, SEQ ID NO: 37, which is produced by the bacterium Proteus vulgaris (P. vulgaris), and chondroitinase AC, SEQ ID NO: 5, which is produced by Flavobacterium heparinum. Chondroitinases ABC I SEQ ID NO: 37, and chondroitinase AC SEQ ID NO: 5, function by degrading polysaccharide side chains in protein-polysaccharide complexes, without degrading the protein core.

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2016204464 29 Jun 2016 [0003] Yamagata et al. (J. Biol. Chem. 243:1523-1535,1968) describe the purification of the chondroitinases like ABC I SEQ ID NO: 37 from extracts of j°. vulgaris. This enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate, and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B, and C which are side chains of proteoglycans) at pH 8 at higher rates than it degrades chondroitin or hyaluronic acid. The products of the degradation are high molecular weight unsaturated oligosaccharides and an unsaturated disaccharide. However, chondroitinase ABC I, SEQ ID NO: 37, does not act on keratosulfate, heparin or heparitin sulfate.

[0004] Uses of chondroitinases include rapid, specific and non-surgical disruption of the attachment of the vitreous body to the neural retina of the eye, thereby facilitating removal of the vitreous body.

[0005] P. vulgaris chondroitinase, for example ABC I SEQ ID NO: 37 migrates with an apparent molecular mass of about 110 kDa when resolved by SDS-PAGE. The appearance of a doublet in SDS-PAGE resolution of chondroitinase ABC has been reported (Sato et al., Agric. Biol. Chem. 50:4,1057-1059,1986). However, this doublet represents intact chondroitinase ABC and a 90 kDa degradation product Commercial chondroitinase ABC protein preparations contain variable amounts of this 90 kDa degradation product and an additional 18 kDa degradation product also derived from chondroitinase ABC I, SEQ ID

NO: 37.

[0006] Chondroitinase ABC H, SEQ ID NO: 27, has also been isolated and purified from P. vulgaris, Chondroitinase ABC Π, SEQ ID NO: 27, is a polypeptide of 990 amino acids with an apparent molecular mass by SDS-PAGE of about 112 kDa. Its molecular mass as determined by electrospray and laser desorption mass spectrometry is about 111,772 daltons. Chondroitinase ABC II, SEQ ID NO: 27, has an isoelectric point of 8.4-8.45. Its enzymatic activity is distinct from, but complementary to, that of chondroitinase ABC I SEQ

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ID NO: 37. Chondroitinase ABC I, SEQ ID NO: 37, endolytically cleaves proteoglycans to produce end-product disaccharides, as well as at least two other products which are thought to be tetrasaccharides, Chondroitinase ABC II, SEQ ID NO: 27, digests at least one of these tetrasaccharide products from the chondroitinase ABC I (SEQ ID NO: 37) digestion of proteoglycan.

[0007] After a injury in the adult mammalian central nervous system (CNS), the inability of axons to regenerate may lead to permanent paralysis. An injury-caused lesion will develop glial scarring, which contains extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). CSPGs inhibit nerve tissue growth in vitro, and nerve tissue regeneration fails at CSPGs rich regions in vivo.

[0008] A number of molecules, and specified regions of them, have been implicated in the ability to support the sprouting of neurites from a neuronal cell, a process also referred to as neurite outgrowth. The term neurite refers to both axon and dendrite structures. This process of spouting neurites is essential in neural development and regeneration, especially after physical injury or disease has damaged neuronal cells. Neurites elongate profusely during development both in the central and peripheral nervous systems of all animal species. This phenomenon pertains to both axons and dendrites. However, neurite regrowth in the CNS decreases as the animal’s age increases.

[0009] Chondroitinase enzymes have shown efficacy in improving functional outcomes in several in vivo models of spinal cord injury. Recombinantly produced chondroitinases AC (SEQ ID NO: 5) and chondroitinase B (SEQ ID NO: 12) polypeptides have shown efficacy in vitro by overcoming the barrier of an inhibitory substrate border, such as aggrecan, resulting in neurite extension for rat cortical neurons.

10010} The inventors have discovered through a deletion analysis based on the available crystal structures, mutant polypeptides capable of degrading chondroitin sulfate

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2016204464 29 Jun 2016 proteoglycans (CSPGs). The cleavage activity of all these mutants have been screened in vitro by zymographic assay using aggrecan as a substrate. A truncated polypeptide of chondroitinase AC (nA50-cA275), (SEQ ID NO: 11), lacking 50 and 275 amino acids from the amino and carboxy termini respectively and having a molecular weight of 38 kDa compared to 75kDa of the full length protein, was found to be the minimal size that retained activity as tested by a zymographic assay. The deletion mutant of chondroitinase B (nA120-c Δ120), (SEQ ID NO: 17), lacking 120 amino acids from each of the amino and carboxy termini and having a molecular weight of 26 kDa compared to 52kDa of the full, length protein, was shown to retain activity as well in a zymographic assay. Reduction in the size and complexity of the molecule may facilitate diffusion to the site of action and potentially reduce immunogenicity for prolonged therapeutic use. These smaller chondroitinases could be potential therapeutics for spinal cord injury.

[0011] The present disclosure relates to mutants of chondroitinase genes, polypeptides and proteins derived therefrom, and their use in methods for promoting neurological functional recovery after central nervous system (“CNS”) injury or disease. The mutant genes, polypeptides and proteins derived from them preferably include deletion, substitution, or a combination of these from the structural units the mature gene or polypeptide; more preferably the mutant genes or polypeptides are deletion mutants of the mature gene or polypeptide. These mutant genes or polypeptides, preferably biologically active, may be used in various pharmaceutical compositions.

[0012] Polypeptide mutants of chondroitinases, for example chondroitinase ABC Type I, SEQ ID NO: 1 or 37, Chondroitinase ABC Type Π, SEQ ID NO: 27,

Chondroitinase AC, SEQ ID NO: 5, and Chondroitinase B, SEQ ID NO: 12, are provided. Other mammalian enzymes mutants with chondroitinase-like activity may independently include such enzymes as hyaluronidase 1, SEQ ID NO: 30, hyaluronidase 2, SEQ ID NO:

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S

31, hyaluronidase 3, SEQ ID NO: 32, hyaluronidase 4, SEQ ID NO: 33, and optionally PH20, SEQ ID NO: 34. These deletion or substitution mutant may be used alone or in combination with chondroitinases or their deletion or substitution mutants as therapeutic compositions and mixtures. Further provided is the use of these mutants, and preferably the chondroitinase deletion or substitution mutants to promote neurological functional recovery in mammals following injury to the CNS, including but not limited to contusion injury, [0013] One embodiment ofthe present invention are isolated nucleic acid molecules consisting of, and preferably comprising, a nucleotide sequence encoding the amino acid sequence of polypeptides that are deletion and or substitution mutants of proteoglycan degrading molecules. Independently, nucleic acid molecules of the present invention may encode for mutant proteoglycan degrading polypeptides of chondroitinase, for example chondroitinase ABC Type I, SEQ ID NO: 1 or 37, Chondroitinase ABC Type II, SEQ ED NO: 27, Chondroitinase AC, SEQ ID NO: 5, and Chondroitinase B, SEQ ID NO: 12, hyaluronidase 1, SEQ ID NO: 30, hyaluronidase 2, SEQ ID NO: 31, hyaluronidase 3, SEQ ID NO: 32, hyaluronidase 4, SEQ ID NO: 33, or optionally PH-20, SEQ ID NO: 34 and combinations of these. Preferably the nucleic acids encode for chondroitinase deletion and or substitution mutants, most preferably the nucleic acids encode for chondroitinase ABC type I or H polypeptides. The invention is also directed to nucleic acid molecules consisting of, and preferably comprising, a nucleotide sequence complementary to the above-described nucleic acid sequences. Also provided for are nucleic acid molecules at least 80%, preferably 85% or 90%, still more preferably 95%, 96%, 97%, 98%, or 99% identical to any of the abovedescribed nucleic acid molecules. Also provided for are nucleic acid molecules which hybridize under stringent conditions to any of the above-described nucleic acid molecules. The present invention also provides for recombinant vectors comprising these nucleic acid molecules, and host cells transformed with such vectors.

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2016204464 29 Jun 2016 [0014] Also provided are isolated polypeptides consisting of, and preferably comprising, the amino acid sequence of deletion and or substitution mutants of proteoglycan degrading polypeptides. Independently, proteoglycan degrading polypeptides can include chondroitinases, for example ABC Type I, SEQ ID NO: X or 37, Chondroitinase ABC Type II, SEQ ID NO: 27, Chondroitinase AC, SEQ ID NO: 5, and Chondroitinase B, SEQ ID NO: 12, hyaluronidase 1, SEQ ID NO: 30, hyaluronidase 2, SEQ ID NO: 31, hyaluronidase 3, SEQ ID NO: 32, hyaluronidase 4, SEQ ID NO: 33, optionally PH-20, SEQ ID NO: 34. Preferably the polypeptides are deletion mutants of chondroitinases. Pharmaceutical compositions may be prepared from the mutant proteoglycan degrading molecules such as these chondroitinases and or hyaluronidases; the composition may include one or more of the deletion and substitution mutants from different proteoglycan degrading polypeptides.

[0015] In one aspect of the invention, biologically active proteoglycan degrading polypeptide are provided having a deletion or substitution of at least one amino acid. The mutant proteoglycan degrading polypeptides include those having the minimal size yet retain a degree of activity as determined by the enzyme assays described in the specification. .Preferred deletion or substitution mutants of the proteoglycan degrading molecule are those which degrade chondroitin and have one or more amino acid deletions from the N-terminus, about 1 to at least aboutl20 amino acids and/or the C-terminus, about 1 to at least about 275 amino acids, more preferably the deletions are from a chondroitinase or a substituted chondroitinase, and even more preferably chondroitinase ABC I or II or a substituted chondriotinase ABC I or II.

10016] One aspect of this invention are deletion and or substitution mutants of proteoglycan degrading polypeptides, preferably deletion mutants of chondroitinase polypeptides, that promote neurite regeneration and or plasticity in the CNS and or promote or inhibit the diffusion of therapeutic molecules into tissues by degradation of proteoglycans.

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2016204464 29Jun2016 [0017] The mutant proteoglycan degrading polypeptides, preferably deletion and or substitution mutants of chondroitinases, may promote neurite regeneration in the CNS and or promote or inhibit the diffusion of therapeutic molecules into tissues by degradation of proteoglycans and can be obtained through expression of suitably modified DNA sequences. Thus, the present invention also provides suitable expression vectors and host cells compatible with them, [0018] In yet other aspects, the invention comprises pharmaceutical compositions that include biologically active polypeptide of deletion and or substitution mutants of proteoglycan degrading molecules, and preferably deletion or substitution mutants of chondroitn degrading polypeptides as described above, in combination with a pharmaceutically acceptable carrier.

[0019] The deletion mutants and or substitution mutants of the proteoglycan degrading polypeptides of the present invention may be used to promote the regeneration of neurites in nerve tissue. These mutants might also be useful in the treatment of other CNS disorders in which plasticity, regeneration, or both might be beneficial. For example CNS injuries and disorders may include but not limited to contusion injury, traumatic brain injury, stroke, multiple sclerosis, brachial plexus injury, amblioplia. Because of their proteoglycan degrading properties, they may be used to promote the delivery of theraprutic compositions and diagnostics to tissues and cells that are normally impermeable to them. Alternatively, they may be used to inhibit penetration of therapeutic compositions, diagnositics or cells to tissues that use part of the extracellular matrix to enter tissues. Because of their smaller size compared to the full length enzyme, the deletion and or substitution mutants are easier to make and easier to deliver to target cells and tissues. These and other even smaller deletion or substitution mutants of proteoglycan degrading molecules could be used as potential

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2016204464 29 Jun 2016 therapeutics with lesser immunogenicity and similar or higher tissue penetration ability for the treatment of CNS injury.

[0020] The deletion mutants may offer significant advantages over the full length proteins in the therapeutic development process. The tissue penetration of the enzymes may be significantly effected by the protein size. The effect of protein size on tissue penetration is difficult to predict, but dependent on size and charge. The rate of penetration depends on tissue composition, charge interactions and hydration effects. Having active enzymes of widely ranging size may allow selection of an enzyme based on optimal tissue penetration properties, perhaps maximizing effective concentrations or limiting peripheral exposure to the enzyme. ) [0021] The immune response of a mammal to a bacterial protein may or may not limit the ability to use the protein or polypeptide as a therapeutic. The generation of antibodies to the protein can restrict repeated exposures, as well as potentially inactivate the protein therapeutic making it ineffective. The smaller mutant proteoglycan degrading enzymes, preferably mutant chondroitinase enzymes, may limit the antigenic sites, limit an immune response or at least simplify the process of engineering an enzyme with reduced immunogenicity.

[0022] The release rate of proteins from matrices often used in sustained release formulations can be dependent upon size and cross-linking. The effective release rate of deletion mutants of proteoglycan degrading polypeptide from the matrix can be engineered through the manipulation of the size of the enzyme. Having a repertoire of chondroitinase enzymes of various size and charge will give an significant advantage for the development of a sustained release formulations.

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A BRIEF DESCRIPTION OF THE FIGURES [0023] FIG. 1(A) shows Anti-His-tag Western Blot (top) and zymogram (bottom) demonstrating chondxoitinase B deletion ΝΔ120 CA120 mutant (SEQ ID NO: 17) expression activity; FIG. 1(B) shows Anti-His-tag Western Blot (top) and zymogram (bottom) demonstrating chondroitinase AC deletion NASO CA275 mutant (SEQ ID NO: 11) expression activity;

[0024] FIG. 2 shows illustrates the relative substrate degrading activity of various deletion mutant polypeptides of Chondroitinase AC (SEQ ID NO: 6-11) relative to the full length Chondroitinase AC SEQ ID NO: 5 ;

[0025] FIG. 3(A)shows a schematic of deletion mutant polypeptides of chondroitinase AC (SEQ ID NO: 6-11); FIG. 3(B) shows confirmation of chondroitinase AC deletion mutants by Western blotting;

[0026] FIG. 4. shows confirmation of protein expression and catalytic activity of Chondroitinase AC deletion mutants (SEQ ID NO: 6-ll)by (A) Western Blotting and (B) zymography;

[0027] FIG. 5 shows a schematic of deletion mutant polypeptides (SEQ ID NO: 1317) of chondroitinase B (SEQ ID NO: 12);

[0028] FIG. 6 shows confirmation of protein expression and catalytic activity of Chondroitinase B and deletion mutants (SEQ ID NO: 12-17) by (A) Western Blotting and (B) zymography;

[0029] FIG. 7 shows a schematic of Chondroitinase ABC I deletion mutant polypeptides (SEQ ID NO: 2-4) of Chondroitinase ABC I SEQ ID NO: 1;

DETAILED DESCRIPTION

2016204464 29 Jun 2016 [0030] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0031] It must also be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Throughout this specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

[0032] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs or material is present and instances where the event does not occur or where the material is not present.

[m3] One aspect of the present disclosure relates to a series of deletion and or substitution mutants of chonchoitinase genes that can be used to generate deletion mutant enzymes with substantially lower molecular weight, but modified, and preferably equivalent

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2016204464 29 Jun 2016 or superior proteoglycan degrading catalytic activity compared to the wild type enzymes.

The deletion and or substitution mutants can be generated by polymerase chain reaction. The resulting mutants are expressed and then enzymatic activity of the mutant polypeptide can be confirmed by using zymography.

[0034] The mutants of the proteoglycan degrading molecules can be used to treat mammalian CNS injuries, typically caused by trauma or disease. In particular, a deletion mutant of a proteoglycan degrading polypeptide molecule like chondroitinase, for example ABC Type I, (SEQ ID NO: 1 or 37), Chondroitinase ABC Type Π, (SEQ ID NO: 27), Chondroitinase AC, (SEQ ID NO: 5), and Chondroitinase B, (SEQ ID NO: 12), or mammalian enzymes with chondroitinase-like activity such as hyaluronidase 1, (SEQ ID NO: 30), hyaluronidase 2, (SEQ ID NO: 31), hyaluronidase 3, (SEQ ID NO: 32), hyaluronidase 4, (SEQ ID NO: 33), and optionally PH-20, (SEQ ID NO: 34), or mixtures of any of these may be used to provide a therapeutic treatment for CNS injuries and disorders which may include but not limited to contusion injury, traumatic brain injury, stroke, multiple sclerosis, brachial plexus injury, amblioplia, spinal cord injuries. Spinal cord injuries includes disease and traumatic injuries, such as the crushing of neurons brought about by an auto accident, fall, contusion, or bullet wound, as well as other injuries. Practice of the present methods can confer clinical benefits to the treated mammal, providing clinically relevant improvements in at least one of the subject’s motor coordination functions and sensory perception. Clinically relevant improvements can range from a detectable improvement to a complete restoration of an impaired or lost function of the CNS.

[0035] Mutants of proteoglycan degrading molecules, for example the deletion mutants of Chondroitinase AC (SEQ ID NO: 5), may have their enzyme activity stabilized by the addition of excipients or by lyophilization. Stabilizers may include carbohydrates, amino acids, fatty acids, and surfactants and are known to those skilled in the art. Examples

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2016204464 29 Jun 2016 include carbohydrates such as sucrose, lactose, mannitol, and dextran, proteins such as albumin and protamine, amino acids such as arginine, glycine, and threonine, surfactants such as TWEEN® and PLURONIC®, salts such as calcium chloride and sodium phosphate, and lipids such as fatty acids, phospholipids, and bile salts. The stabilizers may be added to the proteoglycan degrading polypeptide deletion mutants in a ratio of 1:10 to 4:1, carbohydrate to polypeptide, amino acids polypeptide, protein stabilizer to polypeptide, and salts to polypeptide 1:1000 to 1:20; surfactant to polypeptide; and 1:20 to 4:1, lipids to polypeptide. Other stabilizers include high concentrations of ammonium sulfate, sodium acetate or sodium sulfate, based on comparative studies with heparinase activity. The stabilizing agents, preferably the ammonium sulfate or other similar salt, are added to the enzyme in a ratio of 0.1 to 4.0 mg ammonium sulfate/IU enzyme.

[0036] The proteoglycan degrading mutant polypeptides may be formulated as compositions and can he administered topically, locally or systemically to a subject or patient. Preferably the subject is a mammal and even more preferably a human in need of a proteoglycan degrading composition such as one of the chondroitinases. Topical or local administration is can be used for greater control of application. One or more proteoglycan degrading mutant polypeptides, singularly or in combination, can be mixed with an appropriate pharmaceutical carrier prior to administration. Examples of generally used pharmaceutical carriers and additives are conventional diluents, binders, lubricants, coloring agents, disintegrating agents, buffer agents, isotonizing agents, preservants, anesthetics and the like. Specifically pharmaceutical carriers that may be used are dextran, serum albumin, gelatin, creatinine, polyethylene glycol, non-ionic surfactants (e.g. polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardened castor oil, sucrose fatty acid esters, polyoxyethylene polyoxypropylene glycot) and similar compounds.

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2016204464 29 Jun 2016 [0037] Compositions of the present invention having a proteoglycan degrading polypeptide or a nucleic acid for expressing it may also include theraptutic molecules, diagnostics, and agents for promoting neurite growth and regeneration. Examples of diagnostic molecules may include but are not limited to fluorescent probes, radioisotopes, dyes, or magnetic contrast agents. Compounds that facilitate plasticity, neurite growth, and regeneration can include but are not limited to molecules that over come neurite out growth inhibition, or promote nerve growth such as soluble NOGO antagonists like NgR.27-.311, neural cell adhesion molecules like LI, neurotrophic factors, growth factors, phosphodiesterase inhibitors, and inhibitors of MAG or MOG. Additionally, deletion mutants may be combined with other compounds that promote remyelination such as neuregulins (GGF2) and antibodies that promote remyelination.

[0038] Plasticity of the nervous system refers to any type of functional reorganization. This reorganization occurs with development, learning and memory and brain repair. The structural changes that occur with plasticity may include synapse formation, synapse removal, neurite sprouting and may even include strengthening or weakening existing synapses. Regeneration is generally differentiated from plasticity by the long range growth of axons in disrupted tracts that is characteristic of regeneration.

[0039] The biological activity of the proteoglycan degrading molecules of the present invention may be used to control the degradation rate of proteoglycans in a tissue, and for example be chosen to have a slower degradation activity for sensitive tissues and a higher degradation rate for degrading potions of tissue which are thicker. The activity may be contolled by one of more amino acid substitutions or deletions in the polypeptide or vectors used to express them; the activity may be controlled by the concentration or combination of proteoglycan degrading polypeptides in a composition. The proteoglycan degrading activity may he made to be greater or less than that of the full length polypeptide. For example, it can

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2016204464 29Jun2016 be made to be less than that of the full length Chondroitinase AC (SEQ Π) NO: 5), and can be made to be less than half as active as the full length polypeptide as shown in FIG. 2. Also, as further illustrated in FIG. 2, the proteoglycan degrading activity can be made to be greater than the full length Chondroitinase AC (SEQ ID NO: 5), it can be made more active than the full length polypeptide by a factor of 1.5 or more; it can be more active than the full length polypeptide by a factor of 2.5 or more.

[0040] Native or wild-type P. vulgaris bacterial strains typically can be used to produce chondroitinases ABC I, (SEQ ID NO: 1 or 37), and chondroitinase ABC H, (SEQ Π) NO: 27), and mutants of these full length polypeptide under ordinary growth conditions. Wild-type strains of A. vulgaris can be induced to produce detectable levels of chondroitinase ABCI and its mutants by providing an inducing substrate, such as chondroitin sulfate, as the sole carbon source.

[0041] Mutant nucleic acids can be used for expressing mutant proteoglycan degrading polypeptides. The expressed polypeptides or the mutant nucleic acids can be used to treat mammalian CNS injuries, typically caused by trauma or disease. In particular, a deletion mutant nucleic acid for expressing proteoglycan degrading polypeptide molecule like chondroitinase ABC Type I, may include but are not limited to cloned chondroitinase ABC I, (SEQ ID NO: 22 or 28), chondroitinase ABC II, (SEQ ID NO: 26), nucleic acids for expressing fusion proteins of deletion mutants TAT-chondroitinase ABC IΝΔ60 (SEQ ID NO: 43) and mutants of these genes in E. coili can be expressed using a heterologous expression system with an artificial inducer. Chondroitinase AC (SEQ ID NO: 22 or 28), and chondroitinase B (SEQ ID NO: 26), and their mutants may be cloned from F. heparinum and expressed in E. coli.

[00421 The full length proteoglycan degrading molecules like Chondroitinase AC (SEQ ID NO: 5), as well as the deletion and or substitution mutants of the proteoglycan

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2016204464 29 Jun 2016 degrading polypeptides may be cloned in a number of bacterial as well as mammalian expression vectors. Non-limiting of these vectors include pET15b, pET14b, pGEX 6P1, pDNA4HisMax, or pSECTag2b. The deletion mutants and substituted polypeptides of the present invention exhibit the ability to degrade proteoglycans such as chondroitin CS and DS, and have a smaller size and molecular weight than the mature enzyme polypeptides which is expected to facilitate their diffusion into cells, tissues and across membranes. Expression vectors can include the nucleic acid sequence that expresses a mutant proteoglycan degrading polypeptide operably linked to an expression control sequence. Operably linked can refer to a linkage between an expression control sequence and coding sequence, where the linkage }

permits the expression control sequence to control the expression of the coding sequence.

[0043] The properties of the naturally occurring, substituted and or deletion mutants of the proteoglycan degrading molecules may be altered by introducing a variety of mutations in the protein. Such alterations are suitably introduced using the mutagenesis techniques, for example but not limited to PRC mutagenesis, and the mutated polypeptides molecules suitably synthesized using the expression vectors.

[0044] Mutant proteoglycan degrading polypeptides of the present invention include deletions and or substitutions of amino acids from mature proteoglycan degrading polypeptides. Preferably the deletions or substitutions include any two consecutive or separated amino acids, N or C terminal amino acid deletions or substitutions, and internal amino acid deletions or substitutions in the polypeptide. The deletions and or substitutions can start with any amino acid in the molecule and it is possible to have two separated deletions in the molecule. The deletion or substitution results in mutant proteoglycan degrading polypeptide that are smaller than the mature enzyme and retain proteoglycan degrading ability. Mutant proteoglycan degrading polypeptides can be fused or linked to another polypeptide. Polypeptide is used to unambigously encompases amino acid sequences

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2016204464 29Jun2016 for mutants of any length which have proteoglycan degrading activity and improve plasticity including those minus the signal sequence that is initially part of polypeptide when it is i

translated and that is cleaved off by a host-translational modification.

[0045] Mutant nucleic acids of the present invention include deletions and or substitutions of nucleotides from genes which express the mature proteoglycan degrading polypeptides. The deletion and substitution mutations at the DNA level are used to introduce amino acid substitutions and or deletions into the encoded protein. These nucleotide deletions and substitutions can be used to introduce deletions and or substitutions into important conformational or active regions of the polypeptide. A nucleic acid fragment is a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of a mature proteoglycan degrading polypeptide, yet which preferably encodes a mutant polypeptide which retains some biological activity of the full length protein, e.g., the expressed polypeptide fragment retains the ability to induce degradation of proteoglycans, promote diffusion of therapeutics into cells and tissue, or promote regeneration of neurites. Genes encoding either N or C terminal mutants of proteoglycan degrading polypeptide domains linked to other polypeptides can also be used in constructs for expression of fusion . proteins linked to mutant proteoglycan degrading polypeptides.

[0046] The deletion and or substitution mutant proteoglycan degrading polypeptides of the present invention may also include derivatives of these polypeptides which have been been chemically or enzymatically modified, but which retain their biological activity to degrade proteoglycans. The proteoglycan degrading activity of these mutants may be controlled depending upon the deletion and or substitution made to the polypeptide or the nucleic acid used to express the polypeptide. Variants, fragments, or analogs of the mature proteoglycan degrading polypeptides or nucleic acids and vectors used to express them include mutant polypeptides and nucleic acids having a sequence which differs from the

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2016204464 29 Jun 2016 mature polypeptide or nucleic acid sequence by one or more deletions, substitutions, or a combination of both such that the mutant proteoglycan degrading polypeptides retain their biological activity and can degrade proteoglycans, and preferably degrade chondroitin sulfate proteoglycans.

[0047] Due to the degeneracy of the genetic code, one of ordinary skill in the art will recognize that a large number of the nucleic acid molecules having a sequence at at least 80%, preferably 85% or 90%, still more preferably 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence encoding for a mutant proteoglycan degrading molecule will encode a mutant polypeptide having proteoglycan degrading activity and preferably chondroitin degrading ability. It will be further recognized that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a mutant polypeptide having proteoglycan degrading activity. This is because amino acid substitutions that are either less likely or not likely to significantly effect polypeptide activity (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid) to degrade proteoglycans and preferably to degrade chondroitin.

[0048] Variants included in the invention may contain individual substitutions, deletions or additions to the nucleic acid or polypeptide sequences. Such changes will alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence. Variants are referred to as conservatively modified variants where the alteration results in the substitution of an amino acid with a chemically similar amino acid.

[0049] The discovery that the proteoglycan degrading activity of the deletion and substitution mutant polypeptides of the present invention can be controlled to be less, about the same, or greater than the full length proteoglycan degrading molecule has another potential advantage. A pharmaceutical composition containing the proteoglycan degrading molecules may he administered parenterally, intravenously or subcutaneously. The use of a

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2016204464 29 Jun 2016 hydrogel composed of biodegradable polymer enclosing the polypeptide and continuously releasing the polypeptide is limited by the amount of polypeptide that can be enclosed in the hydrogel. Using a deletion mutant of the polypeptide with higher specific activity implies that, on a molar basis, more of the active substance can be enclosed in the same volume, thereby increasing the time between successive administrations or possibly avoiding repeated administrations.

[0050] Purification of the polypeptide obtained after expression is dependent on the host cell and the expression construct used. Generally, the purification of proteoglycan deletion or substitution mutants can be performed in the same way as the purification of native full length polypeptides including the use of histidine-tags.

[0051] The deletion or substitution mutant proteoglycan degrading polypeptides and proteins are administered in an amount effective to degrade CSPGs. The polypeptides may be used to aid the diffusion of therapeutic and diagnostic compositions to tissues and and can be used to promote the recovery of neurological function and neurite outgrowth. Once the mutant proteoglycan degrading proteins or polypeptides in the compositions have been purified to the extent desired, they may be suspended or diluted in an appropriate physiological carrier or excipient for SCI treatment or for screening assays of compositions promoting neurite growth in vitro on suitable substrates like aggrecan. In models of SCI, effective intrathecal doses of chondroitinases in rats have been about 0.06 units on alternate days for 14 days. A dose for a 70 kilogram human may be about 17 Units. At about 100 Units / milligram, this would equal about 170 micrograms. Doses of up to 20 Units appear safe in mammalian subjects like rats. Compositions may include a proteoglycan degrading mutant polypeptide, preferably mutant chondroitinase polypeptides, and more preferably still deletion mutant chondroitinase polypeptides. These compositions may also include other proteoglycan degrading molecules and deletion and or substitution mutants of them,

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2016204464 29 Jun 2016 molecules which block the action of neurite growth inhibitors, molecules which promote neurite or axon adhesion, diagnostic, therapeutic, or the proteoglycan degrading molecule mutant as part of a fusion protein. The mixture or fusion protein may be added to a carrier or pharmaceutically acceptable excipient can be injected, generally at concentrations in the range of 1 ug to 500 mg/kg of subject. Administering the agent can be by bolus injection, intravenous delivery, continuous infusion, sustained release from implants, or sustained release pharmaceuticals. Administration by injection, can be intramuscularly, peritoneally, subcutaneously, intravenously, intrathecally. Oral administration may include tablets or capsules, preferably the oral dosage is a sustained release formulation for once or twice daily

I administration. Percutneous administration can be once per day, and is preferably less than once per day administration. Administration to the human patient or other mammalian subject may be continued until a measurable improvement in autonomic or motor function in the patient is achieved.

[0052] The mutant proteoglycan degrading polypeptides or fusion polypeptides that include them may also be expressed or secreted by genetically modified cells. The expressed deletion or substitution proteoglycan degrading polypeptide or fusion polypeptides may be harvested and purified for a therapeutic compositon, or the genetically modified cells can he implanted, either free or in a capsule, at or near the site of CNS injury or a tissue into which the controlled diffusion of therapeutic or diagnostic molecule is desired. Mutant nucleic acids for expressing mutant proteoglycan degrading polypeptides are illustrated by nonlimiting examples of chondroitinase ABC I (SEQ ID NO: 22 and 28) which encode for substituted chondroitinase ABC I polypeptides and those without leader amino acid sequences; chondroitinase B nucleic acid mutant (SEQ ID NO: 21) which encodes for mutant polypeptide ΝΔ120 CA120 of chondroitinase B (SEQ ID NO: 21); and chondroitinase AC nucleic acid mutant (SEQ ID NO: 19) which encodes for mutant

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2016204464 29 Jun 2016 polypeptide ΝΔ50 CA275 of chondroitinase AC (SEQ ID NO: 11). A non-limiting example of a fusion nucleic acid includes a TAT-deletion mutant chondroitinase AB CI fusion DNA construct (SEQ ID NO: 41). Another example would be a nucleic acid for TATchondroitinase ABCl-NAbO (SEQ ID NO: 43) for the expressed polypeptide (SEQ ID NO:

44).

[0053] Once the mutant proteoglycan degrading polypeptide are administered to cells or a tissue with CSPGs, degradation of CSPGs removes the inhibitory molecules that block neurite outgrowth, and allow the regeneration of neurites into the affected area. The removal of CSPG also promotes plasticity in the CNS. For example, the full length polypeptides of chondroitinase AC (SEQ ID NO: 5), and chondroitinase B, (SEQ Π) NO: 12), degrade CS and DS, respectively, resulting in unsaturated sulfated disaccharides. Chondroitinase AC (SEQ ID NO: 5), cleaves CS at 1,4 glycosidic linkages between N~ acetylgalactosamine and glucuronic acid in the polysaccharide backbone of CS, Cleavage occurs through beta-elimination in a random endolytic action pattern. Chondroitinase B (SEQ ID NO: 12) cleaves the 1,4 galactosamine iduronic acid linkage in the polysaccharide backbone of DS. The cleavage of both CS and DS occurs through a beta-elimination process which differentiates these enzymatic mechanisms from mammalian GAG degrading enzymes. Chondroitinase ABC I (SEQ ID NO: 1), chondroitinase ABC Π (SEQ ID NO: 27), are exo and endo lyases that cleave both CS and DS. The removal of CS and DS from a glial scar permits'the regeneration of neurite outgrowths into the injured area and promotes plasticity. For example, the proteoglycan degrading molecules illustrated in FIG. 2, Chondroitinase AC (SEQ ID NO: 5) and various mutant Chondroitinase AC (SEQ ID NO: 6-11) degrade a model proteoglycan substrate at by various amounts. Similar results are shown by in vitro zymograph for chondroitinase B (SEQ ID NO: 12) and illustrative mutants (SEQ ID NO: 13-17) in FIG. 6. It is reasonable to expect that since a proteoglycan

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2016204464 29 Jun 2016 degrading molecule like Chondroitinase ABC I (SEQ ID NO: 1) improves functional recovery in rats with contusive spinal cord injury and also facilitates the diffusion of model compounds into brain tissue, that mutant proteoglycan degrading polypeptides and compositions containing them can also improve functional recovery in mammalian subjects like rats with contusive spinal cord injury and may also facilitates the diffusion of model compounds into brain tissue.

[0054] The regeneration of the nerve cells and restoration of plasticity in the affected CNS area allows the return of motor and sensory function. Clinically relevant improvement will range from a detectable improvement to a complete restoration of an impaired or lost nervous function, varying with the individual patients and injuries. The degree of functional recovery can be demonstrated by improved corticospinal tract conduction, improved tape removal, beam walking, grid walking and paw placement following chondroitinase treatment of a dorsal column lesion. Motor skill improvement as well as autonomic function: bowel, bladder, sensory and sexual function may also be used as measures of function improvement and related to molecular structure and components in the compositions of the present invention.

[0055] A series of polynucleotides that include coding for deletion or substifion mutants of protec'glycan degrading polypeptides may be generated by PCR using the full length cDNAs for the proteoglycans as templates and cloned into an expression vector such as pETl 5b at the Ndel and BamHI sites for expression in E. Coli. After Induction of gene expression with isopropyl -p-D-thiogalactopyranoside (IPTG), the bacteria can lysed by sonication with the concomitant extraction of the mutant polypeptide with a surfactant such as Triton X-l 14/PBS. The majority of recombinant proteoglycan degrading polypeptide may be found in the cytosolic fraction of the bacterial cell lysate and chondroitinase purification protocols can be used to obtain the mutant proteoglycan degrading enzyme with high activity

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2016204464 29 Jun 2016 at high yields. This protocol may include purification by a column having anti-His antibody to selectively bind His-tagged mutant proteoglycan degrading polypeptides and may also includes cation-exchange chromatography as a capture step and gel filtration as a polishing step. After these steps, anion exchange membrane filtration, for example Intercept Q, Millipore, can be used for endotoxin and host DNA removal. Following filtration, the proteoglycan degrading mutant polypeptides can be dialyzed into volatile buffer, pH 8.0 and lyophilized to dryness. The final product is expected to be stable at -70°C for long term storage. The pi of the purified basic proteoglycan degrading mutant polypeptide may be determined by IEF-PAGE analysis of the samples from the crude cell lysate.

[0056] A variety of analytical methods can be used to compare the enzymatic activity of the recombinant version the deletion or substitution mutants of proteoglycan degrading polypeptides with those of full length proteoglycan degrading molecules like chondroitinase ABC I (SEQ Π) NO: 37) or a commercially available form of the enzyme.

The methods may also be adapted to evaluate the activity of fusion proteins including a mutant proteoglycan degrading polypeptide portion. Specific activity measurements may be obtained using an accepted spectrophotometric assay that measures the change in absorbance due to the production of reaction products from the degradation of proteoglycans. Size exclusion chromatography can be used to compare the hydrodynamic properties of the mutant enzymes.

[0057] A form of zymography can used to characterize the mature proteoglycan degrading enzyme and may be adapted for characterization of the mutants proteoglycan degrading polypeptides. Polyacrylamide gels can be polymerized in the presence of aggrecan, a substrate for proteoglycan degrading molecules like chondroitinase ABCI. The mutant proteoglycan degrading polypeptides, enzyme samples, may be resolved on the aggrecan-impregnated gels by electrophoresis in the presence of SDS. The gels can then be

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2016204464 29 Jun 2016 subjected to a renaturation step wherein the SDS can be extracted and the enzymes allowed to refold. The refolded enzyme regains activity then digests aggrecan within the gel and the resulting loss of carbohydrate in that region of the gel that can be visualized by a carbohydrate-specific stain. A similar loss of carbohydrate in the gel would be expected for equally active forms and concentration of the mutant proteoglycan degrading molecules. In fire case of recombinant Chondroitinase AB CI, its activity can be visualized as a clear spot in the zymogram. The zymography results are consistent with the spectrophotometric analysis.

[0058] HPLC methods may be used for detecting the four and six sulphated disaccharides (A4DS and A6DS, respectively) liberated as a result of mutant proteoglycan degrading polypeptide digestion of CSPG. The two disaccharides can be effectively resolved by anion exchange chromatography. The HPLC assay for the quantitation of A4DS and A6DS from chromatograms is expected to yield a linear relationship proportional to the amounts injected into the HPLC. Production of A4DS and A6DS from CSPG digestion is directly related to the amount of chondroitinase specific activity as determined by the spectrophotometric assay. This assay may be used as a sensitive and accurate method to independently quantitate A4DS and A6DS released by mutant proteoglycan degrading polypeptide digestion of a variety of substrates and may also be used to determine the activity of mutant proteoglycan degrading polypeptides and fusion proteins including them.

[0059] Another functional assay that can be performed to characterize mutant proteoglycan polypeptide activity is where dorsal root ganglian (DRG) neurons are plated on aggrecan or aggrecan treated with a deletion or substitution mutant proteoglycan degrading polypeptide. It is expected that neurons plated on aggrecan will fail to adhere to the plate and extend axons. In contrast, neurons plated on aggrecan treated with a mutant proteoglycan degrading polypeptide in a composition or as part of a fusion polypeptide would be expected to adhere to the surface and extend axons. The extensive axon growth, which is observed for

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2016204464 29 Jun 2016 chondroitinase ABC I (SEQ ID NO:37) is believed to be due to the digestion of the carbohydrates on the aggrecan core protein which creates a more permissive substrate for axon growth.

[0060] Various aspects of the invention may be understood with reference to the following non-limiting examples.

EXAMPLE 1 [0061] This phrophetic example illustrates the diffusion of molecules into cells and tissue using a deletion or substitution mutant of a proteoglycan degrading polypeptide in a composition.

[0062] A brain from an adult Sprague Dawley rat may be removed from the skull and hemispheres may be soaked in buffer alone or containing about 33U/ml of a mutant proteoglycan degrading polypeptide such as (SEQ ID NO: 9) NASO CA200 AC (T74-T5O0) protein for 2 hours at 37 °C. Hemispheres can be rinsed and immediately placed in dye such as Eosin Y (Sigma) or a saturated solution of Congo Red (Sigma) in 70% ethanol. Slabs of tissue may he cut and images acquired on a scanner. The penetration of the dyes into the brain tissue may be used as an indication of the proteoglycan degrading activity of a mutant proteoglycan degrading molecule and expectant penetration or diffusion of therapeutic and diagnostic molecules into the same type of tissue.

EXAMPLE 2 [0063] This prophetic example illustrates a Chondroitinase ABC I Assay Protocol which may he modified to measure the activity of a mutant proteoglycan degrading molecule,

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2016204464 29 Jun 2016 for example a Chondroitinase ABCI deletion mutant or a fusion proteins including a deletion and or substitution mutant of a proteoglycan degrading polypeptide.

[0064] The production of reaction products from the catalytic activity of a proteoglycan degrading molecule or fusion protein can be determined by a measurement of the absorbance of the proteoglycan degradation product at a wavelength of 232 nm. A typical reaction mixture consisted of 120 μΐ of reaction mixture (40raM Tris, pH 8.0, 40mM NaAcetate, 0.002% casein) combined with a substrate (5 μΐ of 50 mM chondroitin C (MW

521), chondroitin 6 SO4, or dermatan sulfate) and 1.5 μΐ of chondroitinase ABCI (SEQ ID NO:1) or a mutant of chondroitinase like (SEQ ID NO:2). Reaction mixture aliquots of about 120 μΐ can be prepared at 30-37°C for 3 min or longer. The product formation is monitored as an increase in absorbance at 232 nm as a function of time at a wavelength of 232 nm using a spectrometer. The reaction may be stopped by addition of 0.1% SDS followed by boiling for 5 minutes. The observed activity may be converted to units (pmoles of product formed per minute) using the molar absorption coefficient for the C4-C5 double bond formed in the reaction (3800 cm'¹ min'¹).

[0065] Knowing the molar absorption coefficient for the reaction product, measuring the change in the absorbance of the reaction product at 232 nm reading over time upon addition of a known amount of the Chondroitinase ABCI (SEQ ID NO:1) or other other mutant proteoglycan degrading polypeptide to the 120 μΐ reaction mixture with 0002% casein and a chondroitin substrate added, the specific activity in umol/min/mg of the mutant proteoglycan degrading polypeptide can be determined. Seikagaku Chondroitinase ABC I has a specific activity under these assay conditions of about 450 umole/rnin/mg.

[0066] A proteoglycan degrading molecule like Chondroitinase ABC I (SEQ ID NO:37), digests axon growth inhibiting chondroitin present in CNS tissue and improves functional recovery in rats having contusion spinal cord injuries. It is reasonable to expect

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2016204464 29 Jun 2016 that mutants of proteoglycan degrading molecules, such as (SEQ ID NO: 11) NASO CA275 AC (T74-T426) polypeptide that show proteoglycan degrading activity may also show some regeneration of nerves, stimulate plasticity and be useful for diffusion of agents into tissues. The mode of administration, the timing of administration and the dosage are carried out such that the functional recovery from impairment of the CNS is enhanced by the promotion of neurite outgrowth and plasticity. It is reasonable to expect that once the deletion or substitution mutants of proteoglycan degrading molecules such as (SEQ ID NO: 11) NA50 CA275 AC (T74-T426) protein are administered, the degradation of CSPGs can remove the inhibitory molecules in tissue that block drag diffusion, block neurite outgrowth, and promote the regeneration of neurites or other therapeutics into the affected area. The regeneration and plasticity of the nerve cells into the affected CNS area may allow the return of motor and sensory function. Clinically relevant improvements will range from a detectable improvement to a complete restoration of an impaired or lost nervous function, varying with the individual patients and injuries.

EXAMPLE 3 (0067] This example shows that deletion mutants of chondroitinase are biologically active.

[0068] Recombinantly produced chondroitinases AC and B have shown efficacy in vitro by overcoming the barrier of an inhibitory substrate border, such as aggrecan and result in neurite extension for rat cortical neurons. To facilitate effective transport of the above enzymes to the injury site, deletion mutants of these chondroitinases were prepared to determine the minimally-sized polypeptides capable of degrading CSPGs. The cleavage activity of all these mutants have been screened in vitro by zymographic assay using aggrecan as substrate. A truncated polypeptide of chondroitinase AC (nA50-cA275) (SEQ

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ID NO:11) lacking 50 and 275 amino acids from the amino and carboxy termini respectively having a molecular weight of 38 ldDa compared to 75kDa of the full length protein was found to be about the minimal size mutant chondroitinase AC that retains activity as tested by zymography assay FIG. 4(B). However, an even smaller mutant, the deletion mutant of chondroitinase B (nA 120-cA 120) (SEQ ID NO:17) lacking 120 amino acids from each of the amino and carboxy termini, having a molecular weight of 26 kDa compared to 52 kDa of tire full length protein has also shown to retain activity as well in zymography assay FIG. 6(B). These and other even smaller deletion mutants could be used as potential therapeutics with lesser immunogenicity and similar or higher tissue penetration ability compared to the mature enzyme and may be used for treatment of spinal cord injury.

[0069] A series of chondroitinase AC and B deletion mutants were generated by PCR using the full-length cDNAs for chondroitinases AC and B as templates and cloned in the pETl5b expression vector at the Ndel and BamHI sites. Full length and deletion mutants were constructed with Histidine-tags for ease of detection and purification. Each of these cDNAs was induced by Isopropyl-fi-D-Tinogaiactopyranoside (IPTG,) and the expression was confirmed by Western blotting using anti-His antibody (Novagen). FIG. 3(A) show various non-limiting deletion mutants schematically, and FIG 3(B) shows confirmation of expression of these chondroitinase AC mutant polypeptides by anti-histidine tag Western blotting. Figures 5 and 6 show the same information for chondroitinase B deletions. Western blots demonstrate proteins of predicted size. Zymographic PAGE of deletion mutants show intense bands of substrate digestion (light) and negative carbohydrate staining.

[0070] Zymography assay. SDS-polyacrylamide gels were poured with aggrecan (85 pg/ml) polym erized into it. Crude extracts of deletion mutants of chondroitinases AC and B were run and renatured at 37 °C overnight. After separation the gel is incubated in 0.2% Cetylpyridinium for 90 minutes at room temperature. The digestion of the

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2016204464 29 Jun 2016 proteoglycans by the chondroitinases is visualized by staining the gel with 0.2% Toludene Blue in ethanol-H₂O-acetic acid (50:49:1 v/v/v) for 30 minutes and destained with ethanolHiO-acetlc acid (50:49:1 v/v/v). Following destaining the gel is incubated overnight in a 50 pg/ml solution of Stains-all in 50% ethanol in the dark and destained with H2O. Appearance of clear bands on the gel shows the digestion of carboyhydrates by the chondroitinases of the CSPG leaving the core protein which remains unstained (FIG 4.and FIG. 6).

EXAMPLE 4 [0071] This example describes the linking of a His tag to a mutant proteoglycan degrading polypeptide.

[0072] Deletion mutants of the chondroitinase ABC I enzyme where the mutant is missing a certain number of amino acids from the N-terminal and maintains proteoglycan degrading activity can be generated (SEQ ID NO:2-4). These N-terminal deletion maintain a histidine-tag that is attached to the N-terminus; however similarly tagged full length chondroitinase ABC I (SEQ ID NO :1) did not maintain the histidine-tag after experession.

[0073] Catalytically active deletion mutants of chondroitinase ABC I can be prepared for example but not limited to deleting 20, and 60 amino acids respectively from the N-terminus of the mature ABC I protein as shown in FIG. 7. A mutant polypeptide with both N and C terminal deletions such as chondroitinase ABC I-NA60-CA80 (SEQ ID NO:4) can also be made, [0074] These chondroitinase deletion mutants and mutants of other proteoglycan degrading molecules may used for construction of N-terminal fusion chimeric protein. Assay tests with these fusion polypeptides for chondroitin degradation and may be used to determine the efficacy of mature ABCI versus various deletion mutant in compositions and fusion proteins with respect to the substrate specificity, substrate binding and tissue

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2016204464 29 Jun 2016 penetration. Functional assay that can be performed to characterize the activity of mutant proteoglycan polypeptide or fusion polypeptides including them. In this functional assay, dorsal root ganglian (DRG) neurons can be plated on aggrecan or aggrecan treated with a mutant proteoglycan degrading polypeptide or a fusion polypeptide including the mutant. It is expected that neurons plated on aggrecan will failed to adhere to the plate and extend axons. In contrast, neurons plated on aggrecan treated with a mutant proteoglycan degrading polypeptide or a fusion polypeptide including the mutant in a composition or as part of a fusion polypeptide would be expected to adhere to the surface and extend axons. The extensive axon growth, which is observed for a chondroitinase like chondroitinase ABC I (SEQ ID NO: 1 or 37) treated aggrecan substrate is believed to be due to the digestion of the carbohydrates on the aggrecan core protein which creates a more permissive substrate for axon growth.

EXAMPLE 5 [0075] This phrophetic example describes a mutant of chondroitinase ABC I that has native protein structure, but lacks proteoglycan degrading catalytic activity.

[0076] This mutant may he prepared as a null or a negative control for bioassays and SCI studies. Based on the crystal structure of chondroitinase ABC I a site-specific mutant designated H501a and Y508a (SEQ ID NO: 36) to knock out catalytic activity in the putative active site can be prepared. Such mutants can be tested for inactivation of catalytic activity and SEC to compare to the wild-type enzyme. The null activity mutant can also be used to provide a negative control for the various proteoglycan degrading fusion proteins for use in bioassays and ultimately in SCI animal studies.

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EXAMPLE 6 [0077] This example illustrates examples of mutant proteoglycan degrading polypeptides that include both substitution and deletions from polypeptides of the present invention.

[0078] The chondroitinase ABC I sequence (SEQ ID NO: 37) is a published sequence for a mature chondroitinase ABC I peptide and includes the leader sequence. Chondroitinase ABC I sequence (SEQ ID NO: 37) is similar to (SEQ ID NO: 1 or 29), however (SEQ ID NO: 1) does not have the first 25 amino acids of (SEQ ID NO: 37), and amino acids at positions 154 and 195 of (SEQ ID NO: 37) differ from those (substitutions) found in similar positions when (SEQ ID NO: 1) and (SEQ ID NO: 37) are aligned.

[0079] (SEQ ID NO: 38-40) illustrate deletions from either the N or C terminal of the (SEQ ID NO: 37) polypeptide and substitutions relative to (SEQ ID NO: 1). These mutant polypeptides are NA20 (SEQ ID NO: 38), ΝΔ60 (SEQ ID NO: 39) and ΝΔ60 CA80 (SEQ ID NO: 40).

EXAMPLE 7 [0080] This example illustrates non-limiting illustrations of mutant polypeptides of the present invention fused with a membrane transduction polypeptide such as but not limited to a polypeptide portion of a HIV TAT protein. Full sequence listings for the mutants fusion polypeptides are provided in the Sequence listing included in the specification.

[0081] A nucleotide sequence for TAT-chondroitinase ΑΒΟ-ηΔ20 (SEQ ID NO. 41), a portion of which is illustrated below, shows the TAT sequence nucleotides highlighted by underlining linked to chondroitinase nucleotides.

ggte gtaaaaagcg tcgtcaacgt cgtcgtcctc ctcaatgcgc acaaaataac 61 ccattagcag acttctcatc agataaaaac tcaatactaa cgttatctga taaacgtagc

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2016204464 29Jun2016 [0082] The underlined nucleotides in this portion of the nucleic acid sequence denote a TAT sequence attached to the 5’ of chondroitinase ABC Ϊ-ΝΔ20 nucleic acid (SEQ ID NO. 47).

[0083] An amino acid sequence for TAT-chondroitinase ABCX-nA20 (SEQ ID NO. 42), a portion of which is shown below, illustrates the TAT sequence amino acids highlighted by underlining at the N-terminus of chondroitinase ABCI-NA20 (SEQ ID NO. 2).

grkkrrqrrrppqcaqnnpladfssdknsiltlsdkrsimgnosilwkwkggssfilhkklivptdkeaskawgrsstpvfsfwly nekpidgyltidfgeklistseaqagfkvkldftgwrtvgvslnndlenremtlnatntssdgtqdsigrslgakvdsirfkapsnvsq geiy [0084] A nucleotide sequence for TAT-ABCI-ΝΔόΟ (SEQ ID NO. 43), a portion of which is illustrated below, shows the N-terminal TAT (SEQ ID NO. 49) nucleotides highlighted by underlining.

ggtcgtaaaaagcgtogtcaacgtogtcgtcctectcaatgctttactttacataaaaaactgattgtccccaccgataaagaagcatcta aagcatggggacgctcatccacccccgttttctcattttggctttacaatgaaaaaccgattgatggttatcttactatcgatttcgg......

[0085] Amino acid sequence for TAT-ABCI-nA60 (SEQ ID NO. 44) a portion of which is shown below, illustrates the TAT sequence (SEQ ID NO. 50) highlighted by underlining at the N-terminus of chondroitinase ABC I-ΝΔ60 (SEQ ID NO. 3).

g^tegnrppqcftlhkklivptdkeaskawgrsstpvfsfwlynekpidgyltidfgeklistseaqagfkvkldftgwrtvgvsl nndlenremtlnatntssdgtqdsigrslgakvdsirfkapsnvsqgeiyidrimfsvddaryqwsdyqvktrlsepeiqf..., [0086] Nucleotide sequence for ABCI-TAT-C (SEQ ID NO. 45), a portion of which is illustrated below, shows the C-terminal TAT sequence nucleotides highlighted by underlining. The stop codon from chondroitinase ABC I (SEQ ID NO. 28) was replaced by the TAT sequence and was placed at the 3’end of the TAT sequence.

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... gattaatggcaaatggcaatctgctgataaaaatagtgaagtgaaatatcaggtttctggtgataacactgaactgacgtttacgagtt actttggtattccacaagaaatcaaactctcgccactccct ggtcgtaaaaagcgtcgteaacgtcgtcgtcctcctcaatgctag

2016204464 29 Jun 2016 [0087] Amino acid sequence for ABCI-TAT-C (SEQ ID NO. 46), a portion of which is shown below, illustrates the TAT sequence, highlighted by underlining, linked to the chondroitinase polypeptide at the C-terminus of the mature chondroitinase ABC I (SEQ ID NO. 1).

... aekvnvsrqhqvsaenknrqptegnfssawidhstrpkdasyeymvfldatpekmgemaqkirennglyqvlrkdkdvhi ildklsnvtgyafyqp^iedkwikkvnkpaivmthrqkdtlivsavtpdlnmtrqkaatpvtmvtmgkwqsadknsevkyq vsgdnteitftsvfgipqeiklsplpgrkkrrqrrrppqc

EXAMPLE 8 [0088] This example illustrates the sequence of chondroitinase nucleic acid and polypeptides which may be used for deletions or substitutions in mutants of the present invention. In these sequence, discrepancies from published sequences are highlighted in bold text at both the nucleotide level and at the amino acid level. These are illustrative of substitutions in the present invention.

SEQ ID NO: 26 Present invention Chondroitinase ABC II Nucleic acid >_ ABC II nature 2973 nt vs, > ABC II {present invention) 2974 nt scoring matrix: , gap penalties: -12/-2

99,0% identity; ' Global alignment score: 11684

20 30 40 50 60

806559 TTACCCACTCTGTCTCATGAAGCTTTCGGCGATATTTATCTTTTTGAAGGTGAATTACCC _ TTACCCACTCTGTCTCATGAAGCTTTCGGCGATATTTATCTTTTTGAAGGCGAATTACCC

20 30 40 50 SO

80 90 100 110 120

0 6 5 5 9 AATACCCTTACCACTTCAAATAATAATCAATTATCGCTAAGCAAACAGCATGCTAAAGAT

AATATCCTTACCACTTCAAATAATAATCAATTATCGCTAAGCAAACAGCATGCTAAAGAT 70 80 90 100 110 120

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130 140 150 160 170 180

GGTGAACAATCACTCAAATGGCAATATCAACCACAAGCAACATTAACACTAAATAATATT

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806559

806559

806559

806559

806S59

806559

806559

806559

806559

806559

GGTGAACAATCACTCAAATGGCAATATCAACCACAAGCAACATTAACACTAAATAATATT 130 140 150 160 170 180

190 200 210 220 230 240

GTTAATTACCAAGATGATAAAAATACAGCCACACCACTCACTTTTATGATGTGGATTTAT

GTTAATTACCAAGATGATAAAAATACAGCCACACCACTCACTTTTATGATGTGGATTTAT 190 200 210 220 230 240

250 260 270 280 290 300

AATGAAAAACCTCAATCTTCCCCATTAACGTTAGCATTTAAACAAAATAATAAAATTGCA

AATGAAAAACCTCAATCTTCCCCATTAACGTTAGCATTTAAACAAAATAATAAAATTGCA 250 260 270 280 290 300

310 320 330 340 350 360

CTAAGTTTTAATGCTGAACTTAATTTTACGQGGTGGCGAGGTATTGCTGTTCCTTTTCGT

CTAAGTTTTAATGCTGAACTTAATTTTACGGGGTGGCGAGGTATTGCTGTTCCTTTTCGT 310 320 330 340 350 360

370 380 390 400 410 420

GATATGCAAGGCTCTGTGACAGGTCAACTTGATCAATTAGTGATCACCGCTCCAAACCAA

GATATGCAAGGCTCTGCGACAGGTCAACTTGATCAATTAGTGATCACCGCTCCAAACCAA 370 380 390 400 410 420

430 440 450 460 470 480

GCCGGAACACTCTTTTTTGATCAAATCATCATGAGTGTACCGTTAGACAATCGTTGGGCA

GCCGGAACACTCTTTTTTGATCAAATCATCATGAGTGTACCGTTAGACAATCGTTGGGCA 430 440 450 460 470 480

490 500 510 520 530 540

GTACCTGACTATGz'V'ACACC'J/'rAGGTAAATAJYCGCAGTAAACACGATGGTTAGTAAAAAC

GTACCTGACTATCAAACACCTTACGTAAATAACGCAGTAAACACGATGGTTAGTAAAAAC 490 500 510 520 530 540

550 560 570 580 590 600

TGGAGTGCATTA'TTGATGTACGATCAGATGTTTCAAGCCCATTACCCTACTTTAAACTTC

TGGAGTGCATTATTGATGTACGATCAGATGTTTCAAGCCCATTACCCTACTTTAAACTTC 550 560 570 580 590 600

610 620 630 640 650 660

GATACTGAATTTCGCGATGACCAAACAGAAATGGCTTCGAGGTATCAGCGCTTTGAATAT

GATACTGAATTTCGCGATGACCAAACAGAAATGGCTTCGATTTATCAGCGCTTTGAATAT 610 620 630 640 650 660

670 680 690 700 710 720

TATCAAGGAATTCGTAGTGATAAAAAAATTACTCCAGATATGCTAGATAAACATTTAGCA

TATCAAGGAATTCGTAGTGATAAAAAAATTACTCCAGATATGCTAGATAAACATTTAGCG 670 680 690 700 710 720

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730 740 750 760 770 780

TTATGGGAAAAATTGGTGTTAACACAACACGCTGATGGTTCAATCACAGGAAAAGCCCTT

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806559

806559

806559

806559

806559

806559

806559

806559

806559

806559

TTATGGGAAAAATTGGGGTTAACACAACACGCTGATGGCTCAATCACAGGAAAAGCCCTT '730 740 750 760 770 780

790 800 810 820 830 840

GATCACCCTAACCGGCAACATTTTATGAAAGTCGAAGGTGTATTTAGTGAGGGGACTCAA

GATCACCCTAACCGGCAACATTTTATGAAAGTCGAAGGTGTATTTAGTGAGGGGACTCAA 790 800 810 820 830 840

850 860 870 880 890 900

AAAGCATTACTTGATGCCAATATGCTAAGAGATGTGGGCAAAACGCTTCTTCAAACTGCT

AAAGCATTACTTGATGCCAATATGCTAAGAGATGTGGGCAAAAGGCTTCTTCAAACTGCT 850 860 870 880 890 900

910 920 930 940 950 960

ATTTACTTGCGTAGCGATTCATTATCAGCAACTGATAGAAAAAAATTAGAAGAGCGCTAT

ATTTACTTGCGTAGCGATTCATTATCAGCAACTGGTAGAAAAAAATTAGAAGAGCGCTAT 910 920 930 940 950 960

970 980 990 1000 1010 2020

TTATTAGGTACTCGTTATGTCCTTGAACAAGGTTTTCACCGAGGAAGTGGTTATCAAATT

TTATTAGGTACTCGTTATGTCCTTGAACAAGGTTTTACACGAGGAAGTGGTTATCAAATT 970 980 990 1000 1010 1020

1030 1040 1050 1060 1070 1080

ATTAGCCATGTTGGTTACCAAACCAGAGAACTTTTTGATGCATGGTTTATTGGTCGTCAT

ATTACTCATGTTGGTTACCAAACCAGAGAACTTTTTGATGCATGGTTTATTGGCCGTCAT 1030 1040 1050 1060 1070 1080

1090 1100 1110 1120 1130 1140

GTTCTTGCAAAAAATAACCTTTTAGCCCCCACTCAACAAGCTATGATGTGGTAGAACGCC

GTTCTTGCAAAAAATAACCTTTTAGCCCCCACTCAACAAGCTATGATGTGGTACAACGCG 1090 1100 1110 1120 1130 1140

1150 1160 1170 1180 1190 1200

ACAGGACGTATTTTTGAAAAAAATAATGAAATTGTTGATGCAAATGTCGATATTCTCAAT

ACAGGACGTATTTTTGAAAAAGATAATGAAATTGTTGATGCAAATGTCGATATTCTCAAT 1150 1160 1170 1180 1190 1200

2210 1220 1230 1240 1250 1260

ACTCAATTGCAATGGATGATAAAAAGCTTATTGATGCTACCGGATTATCAACAACGTCAA

ACTCAATTGCAATGGATGATAAAAAGCTTATTGATGCTACCGGATTATCAACAACGTCAA 1220 1220 1230 1240 1250 1260

2270 1280 1290 1300 1310 1320

CAAGCCTTAGCGCAACTGCAACGTTGGCTAAATAAAACCATTCTAAGCTCAAAAGGTGTT

CAAGCCTTAGCGCAACTGCAAAGTTGGCTAAATAAAACCATTCTAAGCTCAAAAGGTGTT 1270 1280 1290 1300 1310 1320

1330 1340 2350 1360 1370 1380

WO 2004/110360

PCT/US2004/015662

GCTGGCGGTTTCAAATCTGATGGTTCTATTTTTCACCATTCACAACATTACCCCGCTTAT

2016204464 29 Jun 2016

806559

806559

806559

806559

806559

806559

806559

806559

806559

806559

GCTGGCGGTTTCAAATCTGATGGTTCTATTTTTCACCATTCACAACATTACCCCGCTTAT 1330 1340 1350 1360 1370 1380

1390 1400 1410 1420 1430 1440

GCTAAAGATGCATTTGGTGGTTTAGCACCCAGTGTTTATGCATTAAGTGATTCACCTTTT

GCTAAAGATGCATTTGGTGGTTTAGCACCCAGTGTTTATGCATTAAGTGATTCACCTTTT 1390 1400 1410 1420 1430 1440

1450 1460 1470 1480 1490 1500

CGCTTATCTACTTCAGCACATGAGCGTTTAAAAGATGTTTTGTTAAAAATGCGGATCTAC

CGCTTATCTACTTCAGCACATGAGCATTTAAAAGATGTTTTGTTAAAAATGCGGATCTAC 1450 1460 1470 1480 1490 1500

1510 1520 1530 1540 1550 1560

ACCAAAGAGACACAAATTCCTGCTGTATTAAGTGGTCGTCATCCAACTGGGTTGCATAAA

ACCAAAGAGACACAAATTCCTGTGGTATTAAGTGGTCGTCATCCAACTGGGTTGCATAAA 1510 1520 1530 1540 1550 1560

1570 1580 1590 1600 1610 1620

ATAGGGATCGCGCCATTTAAATGGATGGCATTAGCAGGAACCCCAGATGGCAAACAAAAG

ATAGGGATCGCGCCATTTAAATGGATGGCATTAGCAGGAACCCCAGATGGCAAACAAAAG 1570 1580 1590 1600 1610 1620

1630 1640 1650 1660 1670 1680

TTAGATACCACATTATCCGCCGCTTATGCAAAATTAGACAACAAAACGCATTTTGAAGGC

TTAGATACCACATTATCCGCCGCTTATGCAAACTTAGACAACAAAACGCATTTTGAAGGC 1630 1640 1650 1660 1670 1680

1690 1700 1710 1720 1730 1740

ATTAAGGCTGAAAGTGAGCCAGTCGGCGCATGGGCAATGAATTATGCATCAATGGCAATA

ATTAACGCTGAAAGTGAGGCAGTCGGCGCATGGGCAATGAATTATGCATCAATGGCAATA 1690 1700 1710 1720 1730 1740

1750 1760 1770 1780 1790 1800

CAACGAAGAGCATCGACCCAATCACCACAACAAAGCTGGCTCGCCATAGCGCGCGGTTTT

CAACGAAGAGCATCGACCCAATCACCACAACAAAGCTGGCTCGCCATAGCGCGCGGTTTT 1750 1760 1770 1780 1790 1800

1810 1820 1830 1840 1850 I860

AGCCGTTATCTTGTTGGTAATGAAAGCTATGAAAATAACAACCGTTATGGTCGTTATTTA

AGCCGTTATCTTGTTGGTAATGAAAGCTATGAAAATAACAACCGTTATGGTCGTTATTTA 1810 1820 1830 1840 1850 1860

1870 1880 1890 1900 1910 1920

CAATATGGACAATTGGAAATTATTCCAGCTGATTTAACTCAATCAGGGTTTAGCCATGGT

CAATATGGACAATTGGAAATTATTCCAGCTGATTTAACTCAATCAGGGTTTAGCCATGCT 1870 1880 1890 1900 1910 1920

1930 1940 1950 1960 1970 1980

GGATGGGATTGGAATAGATATCCAGGTACAACAACTATTCATCTTCCCTATAACGAACTT

806559

WO 2004/110360

PCT/US2004/015662

2016204464 29 Jun2016

806559

806559

806559

806559

806559

806559

806559

806559

806559

GGATGGGATTGGAATAGATA'JA/CAGGTAC/VACAAGTATTCATCTTCCCTATAACGAAGTT 1930 1940 1950 1960 1970' 1980

1990 2000 2010 2020 2030 2040

GAAGCAAAACTTAATCAATTACCTGCTGCAGGTATTGAAGAAATGTTGCTTTCAACAGAA

GAAGCAAAACTTAATCAATTACCTGCTGCAGGTATTGAAGAAATGTTGCTTTCAACAGAA 1990 2000 2010 2020 2030 2040

2050 2060 2070 2080 2090 2100

AGTTACTCTGGTGCAAATACCeTTAATAATAACAGTATOTTTGCCATGAAATTACACGGT

AGTTACTCTGGTGCAAATACCCTTAATAATAACAGTATGTTTGCCATGAAATTAGACGGT 2050 2060 2070 2080 2090 2100

2110 2120 2130 2140 2150 2160

CCAAGTAAATATCAACAACAAAGCTTAAGGGCAAATAAATCCTATTTCTTATTTGATAAT

CACAGTAAATATCAACAACAAAGCTTAAGGGCAAATAAATCCTATTTCTTATTTGATAAT 2110 2120 2130 2140 2150 2160

2170 2180 2190 2200 2210 2220

AGAGTTATTGCTTTAGGCTCAGGTATTGAAAATGATGATAAACAACATACGACCGAAACA

AGAGTTATTGCTTTAGGCTCAGGTATTGAAAATGATGATAAACAACATACGACCGAAACA 2170 2180 2190 2200 2210 2220

2230 2240 2250 2260 2270 2280

ACACTATTCCAGTTTGCCGTCCCTAAATTACAGTCAGTGATCATTAATGGCAAAAAGGTA

ACACTATTCCAGTTTGCCGTCCCTAAATTACAGTCAGTGATCATTAATGGCAAAAAGGTA 2230 2240 2250 2260 2270 2280

2290 2300 2310 2320 2330 2340

AATCAATTAGATACTCAATTAACTTTAAATAATGCAGATACATTAATTGATCCTGCCGGC aatcaattagatactcaattaactttaaataatgcagatacattaattgatcctgccggc

2290 2300 2310 2320 2330 2340

2350 2360 2370 2380 2390 2400

AATTTATATAAGCTCACTAAAGGACAAACTGTAAAATTTAGTTATCAAAAACAACATTCA

AATTTATATAAGCTCACTAAAGGACAAACTGTAAAATTTAGTTATCAAAAACAACATTCA 2350 2360 2370 2380 2390 2400

2410 2420 2430 2440 2450 2460

CTTGATGATAGAAATTCAAAACCAACAGAACAATTATTTGCAACAGCTGTTATTTCTCAT

CTTGATGATAGAAATTCAAAACCAACAGAACAATTATTTGCAACAGCTGTTATTTCTCAT 2410 2420 2430 2440 2450 2460

2470 2480 2490 2500 2510 2520

GGTAAGGCACGGAGTAATGAAAATTATGAATATGCAATAGCTATCGAAGCACAAAATAAT

GGTAAGGCACCGAGTAATGAAAATTATGAATATGCAATAGCTATCGAAGCACAAAATAAT 2470 2480 2490 2500 2510 2520

2530 2540 2550 2560 2570 2580

AAAGCTCCCGAATACACAGTATTACAACATAATGATCAGCCCCATGCGGTAAAAGATAAA

806559

WO 2004/110360

PCT/US2004/015662

AAAGCTCCCAAATACACAGTATTAGAACATAATGATCAGCTCCATGGGGTAAAAGATAAA

2016204464 29 Jun 2016

2530	2540	2550	2560	2570	2580
2590	2600	26X0	2620	2630

806559 ATAACCCAAGAAGAGGGAT ATGCTTTTTTTGAAGCCACTAAGTTAAAATCAGCGGATGC

ATAACCCAAGAAGAGGGATATGGTTTTTTTGAAGCCACTAAGTTA&AATCAGCGGATGC 2590 2600 2610 2620 2630 2640

2640 2650 2660 2670 2680 2690

0 6 S S 9 AACATTATTATCCAGTGATGCGCCGGTTATGGTCATGGCTAAAATACAAAATCAGCAATT

AACATTATTATCCAGTGATGCGCCGGTTATGGTCATGGCTAAAATACAAAATCAGCAATT

	2650	2660	2670	2680	2690	2700
2700	2710	2720	2730	2740	2750

06559 AACATTAAGTATTGTTAATCCTGATTTAAATTTATATC AAGGTAGAGAAAAAGATC AATT _ AACATTAAGTATTGTTAATCCTGATTTAAATTTATATCAAGGTAGAGAAAAAGATCAATT

2710 2720 2730 2740 2750 2760

2760 2770 2780 2790 2800 2810

806559 TGATGATAAAGGTAATCAAATCGAAGTTAGTGTTTATTCTCGTCATTGGCTTACAGCAGA __ TGATGATAAAGGTAATCAAATCGAAGTTAGTGTTTATTCTCGTCATTGGCTTACAGCAGA

2770 2780 2790 2800 2810 2820

2820 2830 2840 2850 2860 2870

806559 ATCGCAATCAACAAATAGTACTATTACCGTAAAAGGAATATGGAAATTAACGACACCTCA

ATCGCAATCAACAAATAGTACTATTACCGTAAAAGGAATATGGAAATTAACGACACCTCA

	2830	2840	2850	2860	2870	2880
2880	2890	2900	2910	2920	2930

806559 ACCCGGTGTTATTATTAAGCACCACAATAACAACACTCTTATTACGACAACAACCATACA

ACCCGGTGTTATTATTAAGCACCACAATAACAACACTCTTATTACGACAACAACCATACA 2890 2900 2910 2920 2930 2940

2940 2950 2960 2970

806559 GGCAACACCTACTGTTATTAATTTAGTTAAGTAA

GGCAACACCTACTGTTATTAATTTAGTTAAGTAA 2950 2960 2970

The above discrepancies, bold text, at the nucleotide level resulted in 98.3% identity at the amino acid level and the substituted residues are marked in bold text in the following.

SEQ ID NO: 27 Present Invention Chondroitinase ABC II protein >_ ABC {present invention) >_ ABC {mature) scoring matrix: , gap penalties: -12/-2

98.3% identity; Global alignment score: 6393

990 aa vs .

990 aa

WO 2004/110360

PCT/US2004/015662

20 30 40 50 60

457676 LPTIiSHEAF(^IYLFEGl^PNILTTSltI®QI>SLSKQHAICDGEQSIiKWQYQPQATItT]jNNI

2016204464 29 Jun 2016 _ LPTLSHEAEGDIYLFEGELPNTLTTSNWQLSLSKQHAroGSQSLK5iQYQPQATLTLHKri

20 30 40 50 60

80 90 100 110 120

5 7 6 7 6 VNYQDDKKTATPLTFMMWIYNEKPQSS PLTLAFKQKKKIALSFNAELNFTGWRGIAVPFR

VNYQDDKMTATPLTFPMWIYKEKPQSSPLTLAFKQNNKIALSFKAELNFTGWRGIAVPFR 70 80 90 x 100 110 120

130 140 150 160 170 180

5 7 6 7 6 DMQGSATGQLDQLVITAPNQAGTLFFDQIIMS VPLDNRWAVPD YQTP YVNNAVNTMVSKW

DMQGSVTGQLDQLVITAPMQAGTLFFDQI IMS VPLDKTRWAVPDYQTPYVKMAVNTMVSKN 130 140 150 160 170 180

190 200 210 220 230 240

5 7 6 7 6 WSALLMYDQMFQAHYPTLNFDTEFRDDQTBMASIYQRFEYYQGIRSDKKITPDMLDKHLA

WSALLMYDQMFQAHYPTLKFDTEFRDDQTEMASRYQRFEYYQGIRSDKKITPDMLDKHLA 190 200 210 220 230 240

250 260 270 280 290 300

457676 LWEKLGLTQHADG SITGKALDHPKRQHFMKVEGVFSEGTQKALLDAMMLRDVGKTLLQTA _ LV/EKLVLTQHADGS ITGKZiLDHPKRQHFMKVBGVFSEGTQKALLDANMLRDVGKTLLQTA

250 260 270 280 290 300

310 320 330 340 350 360

457676 IYLRSDSLSATGRKKLEERYLLGTRYVLEQGFTRGSGYQIITHVGYQTRELFDAWFIGRH

IYLRSDSLSATDRKKLEERYLLGTRYVLEQGFHRGSGYQIISHVGYQTRELFDAWFIGRH 310 320 330 340 350 360

370 380 390 400 410 420

457676 VLAKWLLAPTQQAMMWYNATGRIFEKDNEIVDANVDILNTQLQWMTKSLLMLPDYQQRQ _ VLAKNWLLAPTQQAMMMYNATGRIFEKKNEIVDAMVDILWTQLQWMIKSLLMLPDYQQRQ

370 380 ' 390 400 410 420

430 440 450 460 470 480

457676 QALAQLQSWLKKTILSSKGVAGGFKSDGSIFHHSQHYPAYAKDAFGGLAPSVYALSDSPF _ QALAQLQKWLNKTILSSKGVAGGFKSDGSIFHHSQHYPAYAKDAFGGLAPSVYALSDSPF

430 440 450 460 470 480

490 500 510 520 530 540

457676 RLSTSAHEHL KDVLLKMRIYTKETQIPWLSGRHPTGLHKIΘΙAPFKWMALAGTPDGKQK _ RLSTSAHERLKDVLLKMRIYTKETQIPAVLSGRHPTGLHKIGIAPFKWMALAGTPDGKQK

490 500 510 520 530 540

550 560 570 580 S90 600

457676 LDTTLSAAYAMLDNKTHFEGINAESEPVGAWAMHYASMAIQRRASTQSPQQSWLAIARGF

LDTTLSAAYAKLDEKTHFEGIKAESEPVGAWAMNYASMAIQRRASTQSPQQSWLAIARGF 550 560 570 580 590 600

610 620 630 640 650 660

WO 2004/110360

PCT/US2O04/O15662

457676 SRYL VGKTES YENNKRYGRYLQ YGQLE 11PADLTQSGFSHAGWDWNRYPGTTTIHLP YNEL

2016204464 29 Jun 2016 _ 3Ρ,νΐ·.:νσΝΕ3ΥΕΙ··ΤΚ14ι7ΥαΚΥη0Υ62ΒΕ;ΓΙΡΑϋΡΤ0βαΡεΗΆΟΚΠΜΜΡ.ΥΡαΤΤΤΙΗΜ’Ώ'7ΕΙι

610 620 630 640 650 660

670 680 690 700 710 720

457676 ΕΑΚχ,Νζ)ΒΡΑΑΟ1ΕΕΜΕη5ΤΒ3ΥΞσ7ΊΊΤΡΚΝΉ5ΜΕ71'·1Κ3ΗβΗ0ΚΥΟθς)εΐ,ΕΑΝΚ5ΥΕΡΡΌΚ

EAKLNQBPAAGIEEMI.LSTESYS<3ANTLNWSMFAMKLHGPSKYQQQSIjRA3SKSYETtFDM

670 680 690 700 710 720

730 740 750 760 770 780

457676 RVIALGSGIENDDKQHTTETTEFQFAVPKLQSVIIKGKKVNQLDTQLTLKNADTLIDP AG

ΡνίΑΡαθσίΕΝΠηΚΏΗΤΤΕΤΤΡΕΟΕΑνΡΚΡΟΕνίΙΝΟΚΚνίϊβΕΏΓΰΡΤΕΝϊίΑυΤΑΙΡΡΑΟ 730 740 750 760 770 780

790 800 810 820 830 840

457676 NLYKLTKGQTVKFSYQKQHSLDDRNSKPTEQLFATAVISHGKAPSNEMYEYAIAlEAQNN __ ΙΪΡΥΚΡΤΚΟΟΤνΚΡΕΥρΚΟΗδΡηηΚΝΞΚΡΤΕςΕΡΑΤΑνίδΗσΚΑΡδΚΓΕΝΥΕΥΑΙΑΙΕΑΟΝυ

790 800 810 820 830 840

850 860 870 880 890 900

457676 ΚΑΡΚΥΤνΕΟΗΜΕΟΡΗΑνκηΚΙΤΩΕΕΟΥΘΡΡΕΑΤΚΧιΚδΑΕΑΤΑηδΕΕΑΡνΜνΜΑΚΙ QNQQL

KAPEYTVLQHHDQPHAVKDKITQEEGYAFFEATKIiKSADATLLSSDAPVMVMAKIQN'QQL· •850 860 870 880 890 900

910 920 930 940 950 960

457676 ΤΡβίνΝΡϋΡΝΡΥΏσΕΕΚΟΟΕΡΟΚΟΚΟΙΕνβνΥΘΡΗΝΡΤΑΕδρβΤΝδΤίτνΚΘίνίΚΙιΤΤΡΰ _ TLSI νΝΡΟΡΝΙ,ΥΟΟΡΞΚΡςίΡϋϋΚβΚΟΙ EVSVYSRHWLTAES QSTKf STIT VKGIWKLTTPQ

910 920 930 940 950 960

970 980 990

457676 PGVIIKHHNWTLITTTTIQATPTVINLVK

PGVI1KHHNKWTLITTTTIQATPTVIWLVK 970 980 990

SEQ ID NO: 28 Present Invention Chondroitinase ABC I nucleic acid > ABC! present invention >_ ABCI mature scoring matrix: , gap penalties: -12/-2

99.7% identity; Global alignment score: 11909

2994 nt vs, 2994 nt

20 30 40 50 60

806559 GCCACCAGCAATCCTGCATTTGATCCTAAAAATCTGATGCAGTCAGAAATTTACCATTTT __ GCCACCAGCAATCCTGCATTTGATCCTAAAAATCTGATGCAGTCAGAAATTTACCATTTT

20 30 40 50 60

80 90 100 110 120

806559 GCACAAAATAACCCATTAGCAGACTTCTCATCAGATAAAAACTCAATACTAACGTTATCT

GCACAAAATAACCCATTAGCAGACTTCTCATCAGATAAAAACTCAATACTAACGTTATCT 70 80 90 100 110 120

WO 2004/110360

PCT/US2004/015662

130 140 150 160 170 180

GATAAACG’TAGCATTATGGGAAACCAATCTCTTTTATGGAAATGGAAAGGTGGTAGTAGC

2016204464 29 Jun 2016

806559

806559

806559

806559

806559

806559

906559

806559

806559

806559

GATAAACGTAGCATTATGGGAAACCAATCTCTTTTATGGAAATGGAAAGGTGGTAGTAGC '130 140 150 160 170 180

190 200 210 220 230 240

TTTACTTTACATAAAAAACTGATTGTCCCCACCGATAAAGAAGCATCTAAAGCATGGGGA

TTTACTTTACATAAAAAACTGATTGTCCCCACCGATAAAGAAGCATCTAAAGCATGGGGA 190 200 210 220 230 240

250 260 270 280 290 300

CGCTCATCCACCCCCGTTTTCTCATTTTGGCTTTACAATGAAAAACCGATTGATGG'rTAT

CGCTCATCTACCCCGGTTTTCTCATTTTGGCTTTACAATGAAAAACCGATTGATGGTTAT 250 260 270 280 290 300

310 320 330 340 350 360

CTTACTATCGATTTCGGAGAAAAACTCATTTCAACCAGTGAGGCTCAGGGAGGCTTTAAA

CTTACTATCGATTTCGGAGAAftAACTCATTTCAACCAGTGAGGCTCAGGCAGGCTTTAAA 310 320 330 340 350 360

370 380 390 400 410 420

GTAAAATTAGATTTCACTGGCTGGCGTACTGTGGGAGTCTCTTTAAATAACGATCTTGAA

GTAAAATTAGATTTCACTGGCTGGCGTGCTGTGGGAGTCTCTTTAAATAACGATCTTGAA 370 380 390 400 410 420

430 440 450 460 470 480

AATCGAGAGATGACCTTAAATGCAACCAATACCTCCTCTGATGGTACTCAAGACAGCATT

AATCGAGAGATGACCTTAAATGCAACCAATACCTCCTCTOATGGTACTCAAGACAGCATT -430 440 450 460 470 480

490 500 510 520 530 540

GGGCGTTCTTTAGGTGCTAAAGTCGATAGTATTCGTTTTAAAGCGCCTTCTAATGTGAGT

GGGCGTTCTTTAGGTGCTAAAGTCGATAGTATTCGTTTTAAAGCGGCTTCTAATGTGAGT 490 500 510 520 530 540

550 560 570 580 590 600

CAGGGTGAAATCTATATCGACCGTATTATGTTTTCTGTCGATGATGCTCGCTACCAATGG

CAGGGTGAAATCTATATCGACCGTATTATGTTTTCTGTCGATGATGCTCGCTACCAATGG 550 560 570 580 590 600

610 620 630 640 650 660

TCTGATTATGAAGTAAAAACTCGCTTATCAGAACCTGAAATTCAATTTCACAACGTAAAG

TCTGATTATCAAGTAAAAACTCGCTTATCAGAACCTGAAATTCAATTTCACAACGTAAAG 610 620 630 640 650 660

670 680 690 700 710 720

CCACAACTACCTGTAACACGTGAAAATTTAGCGGCCATTGATCTTATTCGCCAACGTCTA

CCACAACTACCTGTAACACCTGAAAATTTAGCGGCCATTGArCTTATTCGCCAACGrCTA 670 680 690 700 710 720

WO 2004/110360

PCT/US2004/015662

730 740 750 760 770 780

ATTAATGAATTTGTCGGAGGTGAAAAAGAGACAAACCTCGCATTAGAAGAGAATATCAGC

2016204464 29 Jun 2016

306559

806559

806559

806559

806559

806559

806559

806559

806559

806559

ATTAATGAATTTGTCGGAGGTGAAAAAGAGACAA&CCTCGCATTAGAAGAGAATATCAGC 730 740 750 760 770 780

790 800 810 820 830 840

AAATTAAAAAGTGATTTCGATGCTCTTAATACTCACACTTTAGCAAATGGTGGAACGCAA

AAATTAAAAAGTGATTTCGATGCTCTTAATATTCACACTTTAGCAAATGGTGGAACGCAA 790 800 810 820 830 840

850 860 870 880 890 900

GGCAGACATCTGATCACTGATAAACAAATCATTATTTATCAACCAGAGAATOTTAACTCT

GGCAGACATCTGATCACTGATAAACAAATCATTATTTATCAACCAGAGAATCTTAACTCC 850 860 ' 870 880 890 900

910 920 930 940 950 960

CAAGATAAACAACTATTTGATAATTATGTTATTTTAGGTAATTACACGACATTAATGTTT

CAAGATAAACAACTATTTGATAATTATGTTATTTTAGGTAATTACACGACATTAATGTTT '910 920 930 940 950 960

970 980 990 1000 1010 1020

AATATTAGCCGTGCTTATGTGCTGGAAAAAGATCCCACACAAAAGGCGCAACTAAAGCAG

AATATTAGCCGTGCTTATGTGCTGGAAAAAGATCCCACACAAAAGGCGCAACTAAAGCAG 970 980 990 1000 1010 1020

1030 1040 1050 1060 1070 1080

ATGTACTTATTAATGACAAAGCATTTATTAGATCAAGGCTTTGTTAAAGGGAGTGCTTTA

ATGTACTTATTAATGACAAAGCATTTATTAGATCAAGGCTTTGTTAAAGGGAGTGCTTTA 1030 1040 1050 1060 1070 1080

1090 1100 1110 1120 1130 1140

GTGACAACCCATCACTGGGGATACAGTTCTCGTTGGTGGTATATTTCCACGTTATTAATG

GTGACAA.CCCATCACTGGGGATACAGTTCTCGTTGGTGGTATATTTCCACGTTATTAATG 1090 1100 1110 1120 1130 1140

1150 1160 1170 1180 1190 1200

TCTGATGCACTAAAAGAAGCGAACCTACAAAGTCAAGTTTATGATTCATTACTGTGGTAT

TCTGATGCACTAAAAGAAGCGAACCTACAAACTCAAGTTTATGATTCATTACTGTGGTAT 1150 1160 1170 1180 1190 1200

1210 1220 1230 1240 1250 1260

TCACGTGAGTTTAAAAGTAGTTTTGATATGAAAGTAAGTGCTGATAGCTCTGATCTAGAT

TCACGTGAGTTTAAAAGTAGTTTTGATATGAAAGTAAGTGCTGATAGCTCTGATCTAGAT 1210 1220 1230 1240 1250 1260

1270 1280 1290 1300 1310 1320

TATTTCAATACCTTATCTCGCCAACATTTAGCCTTATTACTACTAGAGCCTGATGATCAA

TATTTCAATACCTTATCTCGCCAACATTTAGCCTTATTATTACTAGAGCCTGATGATCAA 1-270 1280 1290 1300 1310 1320

1330 1340 1350 1360 1370 1380

WO 2004/110360

PCT/US2004/015662

AAGCGTATCAACTTAGTTAATACTTTCAGCGATTATATCACTGGCGCATTAACGCAAGTG

2016204464 29 Jun 2016

806559

806559

806559

806559

806559

806559

806559

806559

806559

806559

AAGCGTATCAACTTAGTTAATACTTTCAGCCATTATATCACTGGCGCATTAACGCAAGTG 1330 1340 1350 1360 1370 1380

1390 1400 1410 1420 1430 1440

CCACCGGGTGGTAAAGATGGTTTACGCCCTGATGGTACAGCATGGCGACATGAAGGCAAC

CCACCGGGTGGTAAAGATGGTTTACGCCCTGATGGTACAGCATGGCGACATGAAGGCAAC 1390 1400 1410 1420 1430 1440

1450 1460 1470 1480 1490 1500

TATCCGGGCTACTCTTTCCCAGCCTTTAAAAATGCCTCTCAGCTTATTTATTTATTACGC

TATCCGGGCTACTCTTTCCCAGCCTTTAAAAATGCCTCTCAGCTTATTTATTTATTACGC 1450 1460 1470 1480 1490 1500

1510 1520 1530 1540 1550 1560

GATACACCATTTTCAGTGGGTGAAAGTGGTTGGAATAGCCTGAAAAAAGCGATGGTTTCA

GATACACCATTTTCAGTGGGTGAAAGTGGTTGGAATAACCTGAAAAAAGCGATGGTTTCA 1510 1520 1530 1540 1550 1560

1570 1580 1590 1600 1610 1620

GCGTGGATCTACAGTAATCCAGAAGTrGGATTACCGCTTGCAGGAAGACACCCTCTTAAC

GCGTGGATCTACAGTAATCCAGAAGTTGGATTACCGCTTGCAGGAAGACACCCTTTTAAC 1570 1580 1590 1600 1610 1620

1630 1640 1650 1660 1670 1680

TCACCTTCGTTAAAATCAGTCGCTCAAGGCTATTACTGGCTTGCCATGTCTGCAAAATCA

TCACCTTCGTTAAAATCAGTCGCTCAAGGCTATTACTGGCTTGCCATGTCTGCAAAATCA 1630 1640 1650 1660 1670 1680

1690 1700 1710 1720 1730 1740

TCGCCTGATAAAACACTTGCATCTATTTATCTTGCGATTAGTGATAAAACACAAAATGAA

TCGCCTGATAAAACACTTGCATCTATTrATCTTGCGATTAGTGATAAAACACAAAATGAA

1690 1700 1710 1720 1730 1740

1750 1760 1770 1780 1790 1800

TCAACTGCTATTTTTGGAGAAACTACTACACCAGCGTCTTTACGTCAAGGTTTCTATGCC

TCAACTGCTATTTTTGGAGAAACTATTACACCAGCGTCTTTACCTCAAGGTTTCTATGCC 1750 1760 1770 1780 1790 1800

1810 1820 1830 1840 1850 I860

TTTAATGGCGGTGCTTTTGGTATTCATCGTTGGCAAGATAAAATGGTGACACTGAAAGCT

TTTAATGGCGGTGCTTTTGGTATTCATCGTTGGCAAGATAAAATGGTGACACTGAAAGCT 1810 1820 1830 1840 1850 1860

1870 1880 1890 1900 1910 1920

TATAACACCAATGTTTGGTCATCTGAAATTTATAACAAAGATAACCGTTATGGCCGTTAC tataacaccaatgtttggtcatctgaaatttataacaaagataaccgttatggccgttac

1870 1880 1890 1900 1910 1920

1930 1940 1950 1960 1970 1980

CAAAGTCATGGTGTCGCTCAAATAGTGAGTAATGGCTCGCAGCTTTCACAGGGCTATCAG

806559

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806559

806559

806559

806559

806559

806559

806559

806559

806559

CAAAGTCATGGTGTCGCTCAAATAGTGAGTAATGGCTCGCAGCTTTCACAGGGCTATCAG 1930 1940 1950 1960 1970 1980

1990 2000 2010 2020 2030 2040

CAAGAAGGTTGGGATTGGAATAGAATGCCAGGGGCAACCACTATCCACCTTCCTCTTAAA

CAAGAAGGTTGGGATTGGAATAGAATGCAAGGGGCAACCACTATTCACCTTCCTCTTAAA 1990 2000 2010 2020 2030 2040

2050 2060 2070 2080 2090 2100

GACTTAGACAGTCCTAAACCTCATACCTTAATGCAACGTGGAGAGCGTGGATTTAGCGGA

GACTTAGACAGTCCTAAACCTCATACCTTAATGCAACGTGGAGAGCGTGGATTTAGCGGA 2050 2060 2070 2080 2090 2100

2110 2120 2130 2140 2150 2160

ACATCATCCCTTGAAGGTCAATATGGCATGATGGCATTCGATCTTATTTATCCCGCCAAT

ACATCATCCCTTGAAGGTCAATATGGCATGATGGCATTCGATCTTATTTATCCCGCCAAT 2110 2120 2130 2140 2150 2160

2170 2180 2190 2200 2210 2220

CTTGAGCGTTTTGATCCTAATTTCACTGCGAAAAAGAGTGTATTAGCCGCTGATAATCAC

CTTGAGCGTTTTGATCCTAATTTCACTGCGAAAAAGAGTGTATTAGCCGCTGATAATCAC 2170 2180 2190 2200 2210 2220

2230 2240 2250 2260 2270 2280

TTAATTTTTATTGGTAGCAATATAAATAGTAGTGATAAAAATAAAAATGTTGAAACGACC

TTAATTTTTATTGGTAGCAATATAAATAGTAGTGATAAAAATAAAAATGTTGAAACGACC 2230 2240 2250 2260 2270 2280

2290 2300 2310 2320 2330 2340

TTATTCCAACATGCCATTACTCCAACATTAAATACGCTTTGGATTAATGGACAAAAGATA

TTATTCCAACATGCCATTACTCCAAGATTAftATACCCTTTGGATTAATGGACAAAAGATA 2290 2300 2310 2320 2330 2340

2350 2360 2370 2380 2390 2400

GAAAACATGCCTTATCAAACAACACTTCAACAAGGTGATTGGTTAATTGATAGCAATGGC

GAAAACATGCCTTATCAAACAACACTTCAACAAGGTGATTGGTTAATTGATAOCAATGGC 2350 2360 2370 ' 2380 2390 2400

2410 2420 2430 2440 2450 2460

AATGGTTACTTAATTACTCAAGCAGAAAAAGTAAATGTAAGTCGCCAACATCAGGTTTCA

AATGGTTACTTAATTACTCAAGCAGAAAAAGTAAATGTAAGTCGCCAACATCAGGTTTCA 2410 2420 2430 2440 2450 2460

2470 2480 2490 2500 2510 2520 gcggaaaataaaaatcgccaaccgacagaaggaaactttagctcggcatggatcgatcac

GCGGAAAATAAAAATCGCCAACCGACAGAAGGAAACTTTAGCTCGGCATGGATCGATCAC 2470 2480 2490 2500 2510 2520

2530 2540 2550 2560 2570 2580

AGCACTCGCCCCAAAGATGCCAGTTATGAGTATATGGTCTTTTTAGATGCGACACCTGAA

806559

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2530 2540 2550 2560 2570 2580

2590 2600 2610 2620 2630 2640

806559 AAAATGGGAGAGATGGCACAAAAATTCCGTGAAAATAATGGGTTATATCAGGTTCTTCGT aaaatgggagagatggcacaaaaattccgtgaaaataatgggttatatcaggttcttcgt

2590 2600 2610 2620 2630 2640

2650 2660 2670 2680 2690 2700

806559 AAGGATAAAGACGTTCATATTATTCTCGATAAACTCAGCAATGTAACGGGATATGCCTTT

AAGGATAAAGACGTTCATATTATTCTCGATAAACTCAGCAATGTAACGGGATATGCCTTT 2650 2660 2670 2680 2690 2700

2710 2720 2730 2740 2750 2760

806559 TATCAGCCAGCATCAATTGAAGACAAATGGATCAAAAAGGTTAATAAACCTGCAATTGTG

TATCAGCCAGCATCAATTGAAGACAAATGGATCAAAAAGGTTAATAAACCTGCAATTGTG 2710 2720 2730 2740 2750 2760

2770 2780 2790 2800 2810 2820

806559 ATGACTCATCGACAAAAAGACACTCTTATTGTCAGTGCAGTTACACCTGATTTAAATATG __ ATGACTCATCGACAAAAAGACACTCTTATTGTCAGTGCAGTTACACCTGATTTAAATATG

2770 2780 2790 2800 2810 2820

2830 2840 2850 2860 2870 2880

806559 ACTCGCCAAAAAGCAGCAACTC CTGTCACCATCAATGTCACGATTAATGGCAAATGGCAA __ actcgccaaaaagcagcaactcctgtcaccatcaatgtcacgattaatggcaaatggcaa

2830 2840 2850 2860 2870 2880

2890 2900 2910 2920 2930 2940

806559 TCTGCTGATAAAAATAGTGAAGTGAAATATCAGGTTTCTGGTGATAACACTGAACTGACG _ TCTGCTGATAAAAATAGTGAAGTGAAATATCAGGTTTCTGGTGATAACACTGAACTGACG

2890 2900 2910 2920 2930 2940

2950 2960 2970 2980 2990

806559 TTTACGAGTTACTTTGGTATTCCACAAGAAATCAAACTCTCGCCACTCCCTTGA

TTTACGAGTTACTTTGGTATTCCACAAGAAATCAAACTCTCGCCACTCCCTTGA

2950 2960 2970 2980 2990

The sequence identity at the amino acid level is shown below:

SEQ ID NO: 29 Present Invention Chondroitinase ABC I protein >_ ABCI Present invention 997 aa vs.

>„ ABC! mature 997 aa scoring matrix:, gap penalties: -12/-2

99.5% identity; Global alignment score: 6595

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20 30 40 50 60

365019 ATSNPAFDPKNLMQSEIYHFAQNNPLADFSSDKNSILTLSDKRSIMGNQSLLWKWKGGSS

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ATSNPAFDPKNLMQSEIYHFAQNNPLADFSSDKNSILTLSDKRSIMGNQSLLWKWKGGSS 10 20 30 40 50 60

80 90 100 110 120

365019 FTLHKKLIVPTDKEASKAWGRSSTPVFSFWLYNEKPIDGYLTIDFGEKLISTSEAQAGFK

FTLHKKLIVPTDKEASKAWGRSSTPVFSFWLYNEKPIDGYLTiDFGEKLISTSEAQAGFK 70 80 90 100 110 120

130 140 150 160 170 180

365019 VKLDFTGWRTVGVSLNNDLENREMTLNATNTSSDGTQDSIGRSLGAKVDSIRFKAPSNVS

VKLDFTGWRAVGVSLNNDLENREMTLNATNTSSDGTQDSIGRSLGAKVDSSRFKAPSNVS 130 140 150 160 170 180

190 200 210 220 230 240

365019 GGEIYIDRIMFSVDDARYQWSDYGVKTRLSEPEIQFHNVKPQLPVTPENLAAIDLIRQRL

QGEIYIDRIMFSVDDARYQWSDYQVKTRLSEPEIQFHNVKPQLPVTPENLAAIDLIRQRL

190 200 210 220 230 240

250 260 270 280 290 300

365019 INEFVGGEKETNLALEENISKLKSDFDALNTHTLANGGTQGRHLITDKQIIIYQPENLNS

INEFVGGEKETNLALEENISKLKSDFDALNIHTLANGGTQGRHLITDKQIIIYQPENLNS

250 260 270 280 290 300

310 320 330 340 350 360

365019 QDKQLFDNYVILGNYTTLMFNiSRAYVLEKDPTQKAQLKQMYLLMTKHLLDQGFVKGSAL

QDKGLFDNYVILGNYTTLMFNISRAYVLEKDPTQKAQLKQMYILMTKHLLDQGFVKGSAL 310 320 330 340 350 360

370 380 390 400 410 420

365019 VTTHHWGYSSRWWYISTLLMSDALKEANLQTQVYDSLLWYSREFKSSFDMKVSADSSDLD

VTTHHWGYSSRWWYISTLLMSDALKEANLQTQVYDSLLWYSREFKSSFDMKVSADSSDLD

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370	380	390	400	410	420
430	440	450	460	470	480

365019 YFNTLSRQHLALLLLEPDDQKRINLVNTFSHYITGALTQVPPGGKDGLRPDGTAWRHEGN

YFNTLSRQHLALLLLEPDDQKRINLVNTFSHYITGALTQVPPGGKDGLRPDGTAWRHEGN 430 440 450 460 470 480

490 500 510 520 530 540

365019 YPGYSFPAFKNASGLIYLLRDTPFSVGESGWNSLKKAMVSAWIYSNPEVGLPLAGRHPLN

YPGYSFPAFKNASQLIYLLRDTPFSVGESGWNNLKKAMVSAWIYSNPEVGLPLAGRHPFN 490 500 510 520 530 540

550 560 570 580 590 600

365019 SPSLKSVAQGYYWLAMSAKSSPDKTLASlYLAiSDKTQNESTAlFGETITPASLPQGFYA

SPSLKSVAQGYYWLAMSAKSSPDKTLASIYLAISDKTGNESTAIFGETITPASLPQGFYA 550 560 570 580 590 600

610 620 630 640 650 660

365019 FNGGAFGIHRWQDKMVTLKAYNTNVWSSElYNKDNRYGRYQSHGVAQiVSNGSQLSGGYQ

FNGGAFGIHRWQDKMVTLKAYNTNVWSSEIYNKDNRYGRYQSHGVAGIVSNGSQLSQGYQ 610 620 630 640 650 660

670 680 690 700 710 720

365019 GEGWDWNRMPGATTIHLPLKDLDSPKPHTLMGRGERGFSGTSSLEGQYGMMAFDLIYPAN

GEGWDWNRMQGATTIHLPLKDLDSPKPHTLMQRGERGFSGTSSLEGQYGMMAFDLIYPAN 670 680 690 700 710 720

730 740 750 760 770 780

365019 LERFDPNFTAKKSVLAADNHUFIGSNINSSDKNKNVETTLFQHAITPTLNTLWINGQKI

LERFDPNFTAKKSVLAADNHLIFIGSNINSSDKNKNVETTLFQHAITPTLNTLWINGQKI 730 740 750 760 770 780

790 800 810 820 830 840

365019 ENMPYQTTLGQGDWLIDSNGNGYLITQAEKVNVSRQHQVSAENKNRGPTEGNFSSAWIDH

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ENMPYQTTLGQGDWLiDSNGNGYLlTQAEKVNVSRQHQVSAENKNRQPTEGNFSSAWIDH 790 800 810 820 830 840

850 860 870 880 890 900

365019 STRPKDASYEYMVFLDATPEKMGEMAGKFRENNGLYGVLRKDKDVHHLDKLSNVTGYAF

STRPKDASYEYMVFLDATPEKMGEMAQKFREIMNGLYQVLRKDKDVHIILDKLSNVTGYAF

850 860 870 880 890 900

910 920 930 940 950 960

365019 YQP AS IEDK Wi KKVN KPAl VMTH RQKDTLi VS AVTPDLN MTRQKAATPVT) N VT! N GKWQ

YQPASlEDKWIKKVNKPAlVMTHRQKDTLiVSAVTPDLNMTRQKAATPVTiNVTiNGKWQ 910 920 930 940 950 960

970 980 990

365019 S ADKNSEVKYQVSGDNTELTFTSYFGIPQEI KLSPLP

SADKNSEVKYQVSGDNTELTFTSYFGIPQEIKLSPLP 970 980 990

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REFERENCES

1. Fethiere J, Eggimann B, Cygler M (1999) Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes. J Mol Biol. 288:635-47,

2. Pojasek K, Shriver Z, Kiley, P Venkataraman G and Sasisekharan R. (2001) Biochem Biophys Res Commun. 286:343-51.

3. Huang W, Matte A, Li Y, Kim YS, Linhardt RJ, Su H, Cygler M. (1999) Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution. J Mol Biol, 294:1257-69,

4. Miura RO, Yamagata S, Miura Y, Harada T and Yamagata T. (1995) Anal Biochem. 225:333-40.

5. Yamagata T, Saito H, Habuchi O and Suzuki S. (1968) J Biol Chem.

243:1536-42.

[0089] Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore the spirit and scope of the appended claims should not be limited to the description and the preferred versions contain within this specification.

2016204464 08 Mar 2018

Claims (22)

1. What is claimed is:

   1. A purified chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the N-terminus when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.
2. 2. A purified chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the C-terminus when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.
3. 3. A purified chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the N-terminus and a deletion up to 120 amino acids at the C-terminus, when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.
4. 4. The purified chondroitinase B mutant polypeptide of claims 1-3 wherein the polypeptide is selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.
5. 5. A composition comprising chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the N-terminus when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.
6. 6. A composition comprising chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the C-terminus when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.

   2016204464 08 Mar 2018
7. 7. A composition comprising chondroitinase B mutant polypeptide having a deletion up to 120 amino acids at the N-terminus and a deletion up to 120 amino acids at the C-terminus, when compared to the mature chondroitinase B polypeptide of 481 amino acids, and wherein the chondroitinase B mutant polypeptide retains proteoglycan degrading activity.
8. 8. The composition of claims 5-7, wherein the polypeptide is selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.
9. 9. The composition of any of claims 5-8 further comprising a pharmaceutically acceptable excipient.
10. 10. The composition of any of claims 5-9 further comprising compounds that facilitate plasticity, neurite growth and regeneration, molecules that overcome neurite outgrowth inhibition or promote nerve growth, therapeutic molecules, diagnostic molecules or a combination of these.
11. 11. The composition of claim 10, wherein the compounds that facilitate plasticity and neurite growth and regeneration are selected from soluble NOGO antagonists, neural cell adhesion molecules, neurotrophic factors, growth factors, phosphodiesterase inhibitors, inhibitors of MAG or MOG, neuregulins and antibodies that promote remyelination.
12. 12. A method of treating a central nervous system injury or disorder comprising administering an effective amount of composition of any of claims 5-11.
13. 13. The method of claim 12, wherein the central nervous system injury or disorder is selected from a contusion injury, a traumatic brain injury, a stroke, multiple sclerosis, a brachial plexus injury, amblioplia, and a spinal cord injury.
14. 14. The method of claim 12, wherein the administration is by topical application, bolus injection, intravenous injection, intrathecal injection, intramuscular injection, peritoneal injection, subcutaneous injection, continuous infusion, sustained release from implants, sustained release pharmaceuticals, and oral delivery.

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    Anti-His-tag Western blot (top) and Zymogram (bottom) demonstrating Chondroitinase B deletion

    ΝΔ120 CA120 mutant expression and activity

    1/7

    2016204464 29 Jun2016 (A)

    Anti-His-tag immunoblot zymography

    Anti-His-tag Western blot (top) and Zymogram (bottom) demonstrating Chondroitinase AC deletion ΝΔ50 CA275 mutant expression and activity (B)

    -— Anti-His-tag immunoblot zymography

    FIG.l

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    2/7

    Ο (Μ §

    (Μ ^1^- ^ι^ιο (Μ

    Ο (Μ

    Relative Activities of AC deletions

    FIG. 2

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    2016204464 29 Jun2016

    23 100

    NH2-BBBBBiBfi * ί ........-.....—............

    ΝΔ200Δ200

    ΝΔ500Δ200 = ΝΔ1000Δ200

    200 300 400

    His225Tyr Arg288 GIU371

    234

    500 600 700 ^^^aaaa -COOH

    53CA200 (Q23^5oo) ΌΔ220 (Q23^->^48o) -^^(^-43^500) ^-3*(T₇₄-T₅₀₀) (S₁23^-T₅₀₀)

    3ΝΔ50ΟΔ275 (T₇₄-L₄₂₆) (A)

    Confirmation of Expression of Chondroitinase AC Deletion Mutants by Western Blotting (B)

    FIG. 3

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    2016204464 29 Jun 2016

    k. o 4-> o < O o o O O CM in o £ o o < b- § σ> c CM <1 CM <1 o CM <1 ω I O O o CM o o o o w O o o CM CM CM m T“ in ε □ < <1 <1 <1 < <3 LU LL o o z Z z Z

    (A) (B)

    37.5

    FIG. 4

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    2016204464 29 Jun 2016 i— ioo~

    200

    300

    400

    Ίϋο

    NH2*ΝΔ80 *ΝΔ120Ρ

    K250 H272 E333 R364

    -COOH (θ106“^50θ)

    0146^506)

    ΟΔ1 9(Q₂₆ ^_L₄₈₈)

    ΟΔ49 (Q26 ^-429) *1......-.....................- ......................................* ΟΔ120 (O₂₆-K₃₉₀)

    ........... - —ί ΝΔ120ΟΔ120(Ι₁₄₆-Κ₃₉₀)

    FIG. 5

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    2016204464 29 Jun 2016

    FIG. 6

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    2016204464 29 Jun 2016 (^85-^942)

    FIG.7

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    SEQUENCE LISTING

    2016204464 29 Jun 2016

    SEQ ID NO: 1 Chondroitinase ABC I protein

    ORIGIN atsnpa fdpknlmqse iyhfaqnnpl adfssdknsi 61 ltlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwrTvgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnThtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgfvk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 p '

    SEQ ID NO: 2 ΝΔ20 ABCI (A45-N1023), protein aqnnpl adfssdknsi

    61 ltlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwrtvgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnthtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgfvk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 p

    1/22

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    SEQ ID NO: 3 ΝΔ60 ABCI (F₈₅-Ni₀2₃) protein ftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwrtvgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnthtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgfvk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 p

    SEQ ID No. 4: ΝΔ60 CA80 ABCI (F₈₅-A₉₄2) protein ftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwrtvgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnthtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgfvk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk pa

    SEQ ID NO: 5 Chondroitinase AC protein Locus 1HMW_A

    ORIGIN

    1 mkklfvtciv ffsilspall iaqqtgtael imkrvmldlk kplmmdkva eknlntlqpd 61 gswkdvpykd damtnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist agktgrgkit tfkaqgqfwl lhdaigyyfp eganlslstq

    2/22

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    2016204464 29 Jun 2016

    541 sqkgnwfhin nshskdevsg dvfklwinhg arpenaqyay ivlpginkpe eikkyngtap 601 kvlantnqlq avyhqqldmv qaifytagkl svagieietd kpcavlikhi ngkqviwaad 661 plqkektavl sirdlktgkt nrvkidfpqq efagatvelk

    SEQ ID NO: 6 CA200 AC (Q23-T500) protein qqtgtael imkrvmldlk kplrnmdkva eknlntlqpd 61 gswkdvpykd damtnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

    SEQ ID NO: 7 CA220 AC (Q23-A480) protein qqtgtael imkrvmldlk kplrnmdkva eknlntlqpd 61 gswkdvpykd damtnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna

    SEQ ID NO: 8 ΝΔ20 CA200 AC (L43-T500) protein lmmdkva eknlntlqpd

    61 gswkdvpykd damtnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

    SEQ ID NO: 9 ΝΔ50 CA200 AC (T₇₄-T₅oo) protein tnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfhvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

    3/22

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    PCT/US2004/015662

    2016204464 29 Jun 2016

    SEQ ID NO: 10 ΝΔ100 CA200 AC (S123-T500) protein smwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltklwgeq gsndfaggvs dgvygasaya ldydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

    SEQ ID NO: 11 ΝΔ50 CA275 AC (T74-L426) protein tnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltkl

    SEQ ID NO: 12 Chondroitinase B Locus Q46079 protein

    ORIGIN

    1 mkmlnklagy llpimvllnv apclgqwas netlyqvvke vkpgglvqia dgtykdvqli 61 vsnsgksglp itikalnpgk vfftgdakve lrgehlileg iwfkdgnrai qawkshgpgl 121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq Imlkgnlffk dkpyvypffk ddyfiagkns wtgnvalgve 421 kgipvnisan rsaykpvkik diqpiegial dlnaliskgi tgkplswdev rpywlkempg 481 tyaltarlsa draakfkavi kmkeh

    SEQ ID NO: 13 NASO Chase B (G106-H506) protein gnrai qawkshgpgl

    121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq Imlkgnlffk dkpyvypffk ddyfiagkns wtgnvalgve 421 kgipvnisan rsaykpvkik diqpiegial dlnaliskgi tgkplswdev rpywlkempg 481 tyaltarlsa draakfkavi kmkeh

    SEQ ID NO: 14 ΝΔ120 Chase B (Ii₄₆-H₅06) protein ittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl

    4/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    361 derrkeycaa nrlkfetphq lmlkgnlffk dkpyvypffk ddyfiagkns wtgnvalgve

    421 kgipvnisan rsaykpvkik diqpiegial dlnaliskgi tgkplswdev rpywlkempg

    481 tyaltarlsa draakfkavi krnkeh

    SEQ ID NO: 15 CA19 Chase B (Q26-L488) protein qwas netlyqwke vkpgglvqia dgtykdvqli 61 vsnsgksglp itikalnpgk vfftgdakve lrgehlileg iwfkdgnrai qawkshgpgl 121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq lmlkgnlffk dkpyvypffk ddyfiagkns wtgnvalgve 421 kgipvnisan rsaykpvkik diqpiegial dlnaliskgi tgkplswdev rpywlkempg 481 tyaltarl

    SEQ ID NO: 16 ΟΔ120 Chase B (Q26-K390) protein qwas netlyqwke vkpgglvqia dgtykdvqli 61 vsnsgksglp itikalnpgk vfftgdakve lrgehlileg iwfkdgnrai qawkshgpgl 121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq lmlkgnlffk

    SEQ ID NO: 17 ΝΔ120 CA120 Chase Β (Ii₄6~K₃9o) protein ittsl tedgkvpqhc ridhcsftdk itfdqvinln

    181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyyrndigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq lmlkgnlffk

    SEQ ID NO: 18 Choindroitinase AC nucleotide locus CHU27583

    ORIGIN

    1 atgaagaaat tatttgtaac ctgtatagtc tttttctcta ttttaagtcc tgctctgctt 61 attgcacagc agaccggtac tgcagaactg attatgaagc gggtgatgct ggaccttaaa 121 aagcctttgc gcaatatgga taaggtggcg gaaaagaacc tgaatacgct gcagcctgac 181 ggtagctgga aggatgtgcc ttataaagat gatgccatga ccaattggtt gccaaacaac 241 cacctgctac aattggaaac tattatacag gcttatattg aaaaagatag tcactattat 301 ggcgacgata aagtgtttga ccagatttcc aaagctttta agtattggta tgacagcgac 361 ccgaaaagcc gcaactggtg gcacaatgaa attgccactc cgcaggccct tggtgaaatg 421 ctgatcctga tgcgttacgg taaaaagccg cttgatgaag cattggtgca taaattgacc 481 gaaagaatga agcggggcga accggagaag aaaacggggg ccaacaaaac agatatcgcc 541 ctgcattact tttatcgtgc tttgttaacg tctgatgagg ctttgctttc cttcgccgta 601 aaagaattgt tttatcccgt acagtttgta cactatgagg aaggcctgca atacgattat 661 tcctacctgc agcacggtcc gcaattacag atatcgagct acggtgccgt atttattacc 721 ggggtactga aacttgccaa ttacgttagg gatacccctt atgctttaag taccgagaaa 781 ctggctatat tttcaaagta ttaccgcgac agttatctga aagctatccg tggaagttat

    5/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun2016

    841 atggatttta acgtagaagg ccgcggagta agccggccag acattctaaa taaaaaggca 901 gaaaaaaaga ggttgctggt ggcgaagatg atcgatctta agcatactga agaatgggct 961 gatgcgatag ccaggacaga tagcacagtt gcggccggct ataagattga gccctatcac 1021 catcagttct ggaatggtga ttatgtgcaa catttaagac ctgcctattc ttttaatgtt 1081 cgtatggtga gtaagcggac ccgacgcagt gaatccggca ataaagaaaa cctgctgggc 1141 aggtatttat ctgatggggc tactaacata caattgcgcg gaccagaata ctataacatt 1201 atgccggtat gggaatggga caagattcct ggcataacca gccgtgatta tttaaccgac 1261 agacctttga cgaagctttg gggagagcag gggagcaatg actttgcagg aggggtgtct 1321 gatggtgtat acggggccag tgcctacgca ttggattacg atagcttaca ggcaaagaaa 1381 gcctggttct tttttgacaa agagattgta tgtcttggtg ccggtatcaa cagcaatgcc 1441 cctgaaaaca ttaccactac ccttaaccag agctggttaa atggcccggt tataagtact 1501 gcaggtaaaa ccggccgggg taaaataaca acgtttaaag cacagggaca gttctggttg 1561 ttgcacgatg cgattggtta ttactttcct gaaggggcca accttagtct gagtacccag 1621 tcgcaaaaag gcaattggtt ccacatcaac aattcacatt caaaagatga agtttctggt 1681 gatgtattta agctttggat caaccatggt gccaggccag aaaatgcgca gtatgcttat 1741 atcgttttgc cgggaataaa caagccggaa gaaattaaaa aatataatgg aacggcaccg 1801 aaagtccttg ccaataccaa ccagctgcag gcagtttatc atcagcagtt agatatggta 1861 caggctatct tctatacagc tggaaaatta agcgtagcgg gcatagaaat tgaaacagat 1921 aagccatgtg cagtgctgat caagcacatc aatggcaagc aggtaatttg ggctgccgat 1981 ccattgcaaa aagaaaagac tgcagtgttg agcatcaggg atttaaaaac aggaaaaaca 2041 aatcgggtaa aaattgattt tccgcaacag gaatttgcag gtgcaacggt tgaactgaaa 2101 tag //

    SEQ ID NO: 19 Chondriotinase AC nucleic acid deletion ΝΔ50 C Δ275 (a22o - ti27s) atgccatga ccaattggtt gccaaacaac

    241 cacctgctac aattggaaac tattatacag gcttatattg aaaaagatag tcactattat 301 ggcgacgata aagtgtttga ccagatttcc aaagctttta agtattggta tgacagcgac 361 ccgaaaagcc gcaactggtg gcacaatgaa attgccactc cgcaggccct tggtgaaatg 421 ctgatcctga tgcgttacgg taaaaagccg cttgatgaag cattggtgca taaattgacc 481 gaaagaatga agcggggcga accggagaag aaaacggggg ccaacaaaac agatatcgcc 541 ctgcattact tttatcgtgc tttgttaacg tctgatgagg ctttgctttc cttcgccgta 601 aaagaattgt tttatcccgt acagtttgta cactatgagg aaggcctgca atacgattat 661 tcctacctgc agcacggtcc gcaattacag atatcgagct acggtgccgt atttattacc 721 ggggtactga aacttgccaa ttacgttagg gatacccctt atgctttaag taccgagaaa 781 ctggctatat tttcaaagta ttaccgcgac agttatctga aagctatccg tggaagttat 841 atggatttta acgtagaagg ccgcggagta agccggccag acattctaaa taaaaaggca 901 gaaaaaaaga ggttgctggt ggcgaagatg atcgatctta agcatactga agaatgggct 961 gatgcgatag ccaggacaga tagcacagtt gcggccggct ataagattga gccctatcac 1021 catcagttct ggaatggtga ttatgtgcaa catttaagac ctgcctattc ttttaatgtt 1081 cgtatggtga gtaagcggac ccgacgcagt gaatccggca ataaagaaaa cctgctgggc 1141 aggtatttat ctgatggggc tactaacata caattgcgcg gaccagaata ctataacatt 1201 atgccggtat gggaatggga caagattcct ggcataacca gccgtgatta tttaaccgac 1261 agacctttga cgaagctt

    SEQ ID NO: 20 Chondroitinase B nuclei acid Locus CHU27584

    ORIGIN

    1 atgaagatgc tgaataaact agccggatac ttattgccga tcatggtgct gctgaatgtg 61 gcaccatgct taggtcaggt tgttgcttca aatgaaactt tataccaggt tgtaaaggag

    6/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    121 gtaaaacccg gtggtctggt acagattgcc gatgggactt ataaagatgt tcagctgatt 181 gtcagcaatt caggaaaatc tggtttgccc atcactatta aagccctgaa cccgggtaag 241 gtttttttta ccggagatgc taaagtagag ctgaggggcg agcacctgat actggaaggc 301 atctggttta aagacgggaa cagagctatt caggcatgga aatcacatgg acccggattg 361 gtggctatat atggtagcta taaccgcatt accgcatgtg tatttgattg ttttgatgaa 421 gccaattctg cttacattac tacttcgctt accgaagacg gaaaggtacc tcaacattgc 481 cgcatagacc attgcagttt taccgataag atcacttttg accaggtaat taacctgaac 541 aatacagcca gagctattaa agacggttcg gtgggaggac cggggatgta ccatcgtgtt 601 gatcactgtt ttttttccaa tccgcaaaaa ccgggtaatg ccggaggggg aatcaggatt 661 ggctattacc gtaatgatat aggccgttgt ctggtagact ctaacctgtt tatgcgtcag 721 gattcggaag cagagatcat caccagcaaa tcgcaggaaa atgtttatta tggtaatact 781 tacctgaatt gccagggcac catgaacttt cgtcacggtg atcatcaggt ggccattaac 841 aatttttata taggcaatga ccagcgattt ggatacgggg gaatgtttgt ttggggaagc 901 aggcatgtca tagcctgtaa ttattttgag ctgtccgaaa ccataaagtc gagggggaac 961 gccgcattgt atttaaaccc cggtgctatg gcttcggagc atgctcttgc tttcgatatg 1021 ttgatagcca acaacgcttt catcaatgta aatgggtatg ccatccattt taatccattg 1081 gatgagcgca gaaaagaata ttgtgcagcc aataggctta agttcgaaac cccgcaccag 1141 ctaatgttaa aaggcaatct tttctttaag gataaacctt atgtttaccc attttttaaa 1201 gatgattatt ttatagcagg gaaaaatagc tggactggta atgtagcctt aggtgtggaa 1261 aagggaatcc ctgttaacat ttcggccaat aggtctgcct ataagccggt aaaaattaaa 1321 gatatccagc ccatagaagg aatcgctctt gatctcaatg cgctgatcag caaaggcatt 1381 acaggaaagc cccttagctg ggatgaagta aggccctact ggttaaaaga aatgcccggg 1441 acgtatgctt taacggccag gctttctgca gatagggctg caaagtttaa agccgtaatt 1501 aaaagaaata aagagcactg a '

    SEQ ID NO: 21 Chondriotinase B nucleic acid deletion ΝΔ120 CA120 (a436 - gi 170) attac tacttcgctt accgaagacg gaaaggtacc tcaacattgc 481 cgcatagacc attgcagttt taccgataag atcacttttg accaggtaat taacctgaac 541 aatacagcca gagctattaa agacggttcg gtgggaggac cggggatgta ccatcgtgtt 601 gatcactgtt ttttttccaa tccgcaaaaa ccgggtaatg ccggaggggg aatcaggatt 661 ggctattacc gtaatgatat aggccgttgt ctggtagact ctaacctgtt tatgcgtcag 721 gattcggaag cagagatcat caccagcaaa tcgcaggaaa atgtttatta tggtaatact 781 tacctgaatt gccagggcac catgaacttt cgtcacggtg atcatcaggt ggccattaac 841 aatttttata taggcaatga ccagcgattt ggatacgggg gaatgtttgt ttggggaagc 901 aggcatgtca tagcctgtaa ttattttgag ctgtccgaaa ccataaagtc gagggggaac 961 gccgcattgt atttaaaccc cggtgctatg gcttcggagc atgctcttgc tttcgatatg 1021 ttgatagcca acaacgcttt catcaatgta aatgggtatg ccatccattt taatccattg 1081 gatgagcgca gaaaagaata ttgtgcagcc aataggctta agttcgaaac cccgcaccag 1141 ctaatgttaa aaggcaatct tttctttaag

    SEQ ID NO: 22 Chondroitinase ABCI nucleic acid Locus 129953

    ORIGIN

    1 ggaattccat cactcaatca ttaaatttag gcacaacgat gggctatcag cgttatgaca 61 aatttaatga aggacgcatt ggtttcactg ttagccagcg tttctaagga gaaaaataat 121 gccgatattt cgttttactg cacttgcaat gacattgggg ctattatcag cgccttataa 181 cgcgatggca gccaccagca atcctgcatt tgatcctaaa aatctgatgc agtcagaaat 241 ttaccatttt gcacaaaata acccattagc agacttctca tcagataaaa actcaatact 301 aacgttatct gataaacgta gcattatggg aaaccaatct cttttatgga aatggaaagg

    7/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    361 tggtagtagc tttactttac ataaaaaact gattgtcccc accgataaag aagcatctaa 421 agcatgggga cgctcatcta cccccgtttt ctcattttgg ctttacaatg aaaaaccgat 481 tgatggttat cttactatcg atttcggaga aaaactcatt tcaaccagtg aggctcaggc 541 aggctttaaa gtaaaattag atttcactgg ctggcgtgct gtgggagtct ctttaaataa 601 cgatcttgaa aatcgagaga tgaccttaaa tgcaaccaat acctcctctg atggtactca 661 agacagcatt gggcgttctt taggtgctaa agtcgatagt attcgtttta aagcgccttc 721 taatgtgagt cagggtgaaa tctatatcga ccgtattatg ttttctgtcg atgatgctcg 781 ctaccaatgg tctgattatc aagtaaaaac tcgcttatca gaacctgaaa ttcaatttca 841 caacgtaaag ccacaactac ctgtaacacc tgaaaattta gcggccattg atcttattcg 901 ccaacgtcta attaatgaat ttgtcggagg tgaaaaagag acaaacctcg cattagaaga 961 gaatatcagc aaattaaaaa gtgatttcga tgctcttaat attcacactt tagcaaatgg 1021 tggaacgcaa ggcagacatc tgatcactga taaacaaatc attatttatc aaccagagaa 1081 tcttaactcc caagataaac aactatttga taattatgtt attttaggta attacacgac 1141 attaatgttt aatattagcc gtgcttatgt gctggaaaaa gatcccacac aaaaggcgca 1201 actaaagcag atgtacttat taatgacaaa gcatttatta gatcaaggct ttgttaaagg 1261 gagtgcttta gtgacaaccc atcactgggg atacagttct cgttggtggt atatttccac 1321 gttattaatg tctgatgcac taaaagaagc gaacctacaa actcaagttt atgattcatt 1381 actgtggtat tcacgtgagt ttaaaagtag ttttgatatg aaagtaagtg ctgatagctc 1441 tgatctagat tatttcaata ccttatctcg ccaacattta gccttattat tactagagcc 1501 tgatgatcaa aagcgtatca acttagttaa tactttcagc cattatatca ctggcgcatt 1561 aacgcaagtg ccaccgggtg gtaaagatgg tttacgccct gatggtacag catggcgaca 1621 tgaaggcaac tatccgggct actctttccc agcctttaaa aatgcctctc agcttattta 1681 tttattacgc gatacaccat tttcagtggg tgaaagtggt tggaataacc tgaaaaaagc 1741 gatggtttca gcgtggatct acagtaatcc agaagttgga ttaccgcttg caggaagaca 1801 cccttttaac tcaccttcgt taaaatcagt cgctcaaggc tattactggc ttgccatgtc 1861 tgcaaaatca tcgcctgata aaacacttgc atctatttat cttgcgatta gtgataaaac 1921 acaaaatgaa tcaactgcta tttttggaga aactattaca ccagcgtctt tacctcaagg 1981 tttctatgcc tttaatggcg gtgcttttgg tattcatcgt tggcaagata aaatggtgac 2041 actgaaagct tataacacca atgtttggtc atctgaaatt tataacaaag ataaccgtta 2101 tggccgttac caaagtcatg gtgtcgctca aatagtgagt aatggctcgc agctttcaca 2161 gggctatcag caagaaggtt gggattggaa tagaatgcaa ggggcaacca ctattcacct 2221 tcctcttaaa gacttagaca gtcctaaacc tcatacctta atgcaacgtg gagagcgtgg 2281 atttagcgga acatcatccc ttgaaggtca atatggcatg atggcattcg atcttattta 2341 tcccgccaat cttgagcgtt ttgatcctaa tttcactgcg aaaaagagtg tattagccgc 2401 tgataatcac ttaattttta ttggtagcaa tataaatagt agtgataaaa ataaaaatgt 2461 tgaaacgacc ttattccaac atgccattac tccaacatta aatacccttt ggattaatgg 2521 acaaaagata gaaaacatgc cttatcaaac aacacttcaa caaggtgatt ggttaattga 2581 tagcaatggc aatggttact taattactca agcagaaaaa gtaaatgtaa gtcgccaaca 2641 tcaggtttca gcggaaaata aaaatcgcca accgacagaa ggaaacttta gctcggcatg 2701 gatcgatcac agcactcgcc ccaaagatgc cagttatgag tatatggtct ttttagatgc 2761 gacacctgaa aaaatgggag agatggcaca aaaattccgt gaaaataatg ggttatatca 2821 ggttcttcgt aaggataaag acgttcatat tattctcgat aaactcagca atgtaacggg 2881 atatgccttt tatcagccag catcaattga agacaaatgg atcaaaaagg ttaataaacc 2941 tgcaattgtg atgactcatc gacaaaaaga cactcttatt gtcagtgcag ttacacctga 3001 tttaaatatg actcgccaaa aagcagcaac tcctgtcacc atcaatgtca cgattaatgg 3061 caaatggcaa tctgctgata aaaatagtga agtgaaatat caggtttctg gtgataacac 3121 tgaactgacg tttacgagtt actttggtat tccacaagaa atcaaactct cgccactccc 3181 ttgatttaat caaaagaacg ctcttgcgtt ccttttttat ttgcaggaaa tctgattatg 3241 ctaataaaaa accctttagc ccacgcggtt acattaagcc tctgtttatc attacccgca 3301 caagcattac ccactctgtc tcatgaagct ttcggcgata tttatctttt tgaaggtgaa

    8/22

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    2016204464 29 Jun 2016

    3361 ttacccaata cccttaccac ttcaaataat aatcaattat cgctaagcaa acagcatgct 3421 aaagatggtg aacaatcact caaatggcaa tatcaaccac aagcaacatt aacactaaat 3481 aatattgtta attaccaaga tgataaaaat acagccacac cactcacttt tatgatgtgg 3541 atttataatg aaaaacctca atcttcccca ttaacgttag catttaaaca aaataataaa 3601 attgcactaa gttttaatgc tgaacttaat tttacggggt ggcgaggtat tgctgttcct 3661 tttcgtgata tgcaaggctc tgcgacaggt caacttgatc aattagtgat caccgctcca 3721 aaccaagccg gaacactctt ttttgatcaa atcatcatga gtgtaccgtt agacaatcgt 3781 tgggcagtac ctgactatca aacaccttac gtaaataacg cagtaaacac gatggttagt 3841 aaaaactgga gtgcattatt gatgtacgat cagatgtttc aagcccatta ccctacttta 3901 aacttcgata ctgaatttcg cgatgaccaa acagaaatgg cttcgattta tcagcgcttt 3961 gaatattatc aaggaattcc //

    SEQ ID NO: 23 TAT fusion chondroitinase ABCI nucleic acid ggtcgtaaaaagcgtcgtcaacgtcgtcg tggtggtggtggtggtgccaccagca atcctgcatt tgatcctaaa aatctgatgc agtcagaaat

    241 ttaccatttt gcacaaaata acccattagc agacttctca tcagataaaa actcaatact 301 aacgttatct gataaacgta gcattatggg aaaccaatct cttttatgga aatggaaagg 361 tggtagtagc tttactttac ataaaaaact gattgtcccc accgataaag aagcatctaa 421 agcatgggga cgctcatcta cccccgtttt ctcattttgg ctttacaatg aaaaaccgat 481 tgatggttat cttactatcg atttcggaga aaaactcatt tcaaccagtg aggctcaggc 541 aggctttaaa gtaaaattag atttcactgg ctggcgtgct gtgggagtct ctttaaataa 601 cgatcttgaa aatcgagaga tgaccttaaa tgcaaccaat acctcctctg atggtactca 661 agacagcatt gggcgttctt taggtgctaa agtcgatagt attcgtttta aagcgccttc 721 taatgtgagt cagggtgaaa tctatatcga ccgtattatg ttttctgtcg atgatgctcg 781 ctaccaatgg tctgattatc aagtaaaaac tcgcttatca gaacctgaaa ttcaatttca 841 caacgtaaag ccacaactac ctgtaacacc tgaaaattta gcggccattg atcttattcg 901 ccaacgtcta attaatgaat ttgtcggagg tgaaaaagag acaaacctcg cattagaaga 961 gaatatcagc aaattaaaaa gtgatttcga tgctcttaat attcacactt tagcaaatgg 1021 tggaacgcaa ggcagacatc tgatcactga taaacaaatc attatttatc aaccagagaa 1081 tcttaactcc caagataaac aactatttga taattatgtt attttaggta attacacgac 1141 attaatgttt aatattagcc gtgcttatgt gctggaaaaa gatcccacac aaaaggcgca 1201 actaaagcag atgtacttat taatgacaaa gcatttatta gatcaaggct ttgttaaagg 1261 gagtgcttta gtgacaaccc atcactgggg atacagttct cgttggtggt atatttccac 1321 gttattaatg tctgatgcac taaaagaagc gaacctacaa actcaagttt atgattcatt 1381 actgtggtat tcacgtgagt ttaaaagtag ttttgatatg aaagtaagtg ctgatagctc 1441 tgatctagat tatttcaata ccttatctcg ccaacattta gccttattat tactagagcc 1501 tgatgatcaa aagcgtatca acttagttaa tactttcagc cattatatca ctggcgcatt 1561 aacgcaagtg ccaccgggtg gtaaagatgg tttacgccct gatggtacag catggcgaca 1621 tgaaggcaac tatccgggct actctttccc agcctttaaa aatgcctctc agcttattta 1681 tttattacgc gatacaccat tttcagtggg tgaaagtggt tggaataacc tgaaaaaagc 1741 gatggtttca gcgtggatct acagtaatcc agaagttgga ttaccgcttg caggaagaca 1801 cccttttaac tcaccttcgt taaaatcagt cgctcaaggc tattactggc ttgccatgtc 1861 tgcaaaatca tcgcctgata aaacacttgc atctatttat cttgcgatta gtgataaaac 1921 acaaaatgaa tcaactgcta tttttggaga aactattaca ccagcgtctt tacctcaagg 1981 tttctatgcc tttaatggcg gtgcttttgg tattcatcgt tggcaagata aaatggtgac 2041 actgaaagct tataacacca atgtttggtc atctgaaatt tataacaaag ataaccgtta 2101 tggccgttac caaagtcatg gtgtcgctca aatagtgagt aatggctcgc agctttcaca 2161 gggctatcag caagaaggtt gggattggaa tagaatgcaa ggggcaacca ctattcacct 2221 tcctcttaaa gacttagaca gtcctaaacc tcatacctta atgcaacgtg gagagcgtgg

    9/22

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    2016204464 29 Jun 2016

    2281 atttagcgga acatcatccc ttgaaggtca atatggcatg atggcattcg atcttattta 2341 tcccgccaat cttgagcgtt ttgatcctaa tttcactgcg aaaaagagtg tattagccgc 2401 tgataatcac ttaattttta ttggtagcaa tataaatagt agtgataaaa ataaaaatgt 2461 tgaaacgacc ttattccaac atgccattac tccaacatta aatacccttt ggattaatgg 2521 acaaaagata gaaaacatgc cttatcaaac aacacttcaa caaggtgatt ggttaattga 2581 tagcaatggc aatggttact taattactca agcagaaaaa gtaaatgtaa gtcgccaaca 2641 tcaggtttca gcggaaaata aaaatcgcca accgacagaa ggaaacttta gctcggcatg 2701 gatcgatcac agcactcgcc ccaaagatgc cagttatgag tatatggtct ttttagatgc 2761 gacacctgaa aaaatgggag agatggcaca aaaattccgt gaaaataatg ggttatatca 2821 ggttcttcgt aaggataaag acgttcatat tattctcgat aaactcagca atgtaacggg 2881 atatgccttt tatcagccag catcaattga agacaaatgg atcaaaaagg ttaataaacc 2941 tgcaattgtg atgactcatc gacaaaaaga cactcttatt gtcagtgcag ttacacctga 3001 tttaaatatg actcgccaaa aagcagcaac tcctgtcacc atcaatgtca cgattaatgg 3061 caaatggcaa tctgctgata aaaatagtga agtgaaatat caggtttctg gtgataacac 3121 tgaactgacg tttacgagtt actttggtat tccacaagaa atcaaactct cgccactccc 3181 ttgatttaat caaaagaacg ctcttgcgtt ccttttttat ttgcaggaaa tctgattatg 3241 ctaataaaaa accctttagc ccacgcggtt acattaagcc tctgtttatc attacccgca 3301 caagcattac ccactctgtc tcatgaagct ttcggcgata tttatctttt tgaaggtgaa 3361 ttacccaata cccttaccac ttcaaataat aatcaattat cgctaagcaa acagcatgct 3421 aaagatggtg aacaatcact caaatggcaa tatcaaccac aagcaacatt aacactaaat 3481 aatattgtta attaccaaga tgataaaaat acagccacac cactcacttt tatgatgtgg 3541 atttataatg aaaaacctca atcttcccca ttaacgttag catttaaaca aaataataaa 3601 attgcactaa gttttaatgc tgaacttaat tttacggggt ggcgaggtat tgctgttcct 3661 tttcgtgata tgcaaggctc tgcgacaggt caacttgatc aattagtgat caccgctcca 3721 aaccaagccg gaacactctt ttttgatcaa atcatcatga gtgtaccgtt agacaatcgt 3781 tgggcagtac ctgactatca aacaccttac gtaaataacg cagtaaacac gatggttagt 3841 aaaaactgga gtgcattatt gatgtacgat cagatgtttc aagcccatta ccctacttta 3901 aacttcgata ctgaatttcg cgatgaccaa acagaaatgg cttcgattta tcagcgcttt 3961 gaatattatc aaggaattcc

    SEQ ID NO: 24

    G R K K R

    SEQ ID NO: 25

    An HIV TAT sequence and Gly penta linker protein

    RQRRRGGGGG

    Tat sequence and Gly penta linker nucleic acid ggt cgt aaa aag cgt cgt caa cgt cgt cgt ggt ggt ggt ggt ggt

    SEQ ID NO: 26 Present Invention Chondroitinase ABC II Nucleic acid ttacccactctgtctcatgaagctttcggcgatatttatctttttgaaggcgaattacccaatatccttaccacttcaaataataatcaattatcg ctaagcaaacagcatgctaaagatggtgaacaatcactcaaatggcaatatcaaccacaagcaacattaacactaaataatattgttaatt accaagatgataaaaatacagccacaccactcacttttatgatgtggatttataatgaaaaacctcaatcttccccattaacgttagcattta aacaaaataataaaattgcactaagttttaatgctgaacttaattttacggggtggcgaggtattgctgttccttttcgtgatatgcaaggctc tgcgacaggtcaacttgatcaattagtgatcaccgctccaaaccaagccggaacactcttttttgatcaaatcatcatgagtgtaccgtta gacaatcgttgggcagtacctgactatcaaacaccttacgtaaataacgcagtaaacacgatggttagtaaaaactggagtgcattattg atgtacgatcagatgtttcaagcccattaccctactttaaacttcgatactgaatttcgcgatgaccaaacagaaatggcttcgatttatcag cgctttgaatattatcaaggaattcgtagtgataaaaaaattactccagatatgctagataaacatttagcgttatgggaaaaattggggtta acacaacacgctgatggctcaatcacaggaaaagcccttgatcaccctaaccggcaacattttatgaaagtcgaaggtgtatttagtga ggggactcaaaaagcattacttgatgccaatatgctaagagatgtgggcaaaacgcttcttcaaactgctatttacttgcgtagcgattca ttatcagcaactggtagaaaaaaattagaagagcgctatttattaggtactcgttatgtccttgaacaaggttttacacgaggaagtggtta

    10/22

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    PCT/US2004/015662

    2016204464 29 Jun 2016 tcaaattattactcatgttggttaccaaaccagagaactttttgatgcatggtttattggccgtcatgttcttgcaaaaaataaccttttagccc ccactcaacaagctatgatgtggtacaacgccacaggacgtatttttgaaaaagataatgaaattgttgatgcaaatgtcgatattctcaat actcaattgcaatggatgataaaaagcttattgatgctaccggattatcaacaacgtcaacaagccttagcgcaactgcaaagttggcta aataaaaccattctaagctcaaaaggtgttgctggcggtttcaaatctgatggttctatttttcaccattcacaacattaccccgcttatgcta aagatgcatttggtggtttagcacccagtgtttatgcattaagtgattcaccttttcgcttatctacttcagcacatgagcatttaaaagatgtt ttgttaaaaatgcggatctacaccaaagagacacaaattcctgtggtattaagtggtcgtcatccaactgggttgcataaaatagggatcg cgccatttaaatggatggcattagcaggaaccccagatggcaaacaaaagttagataccacattatccgccgcttatgcaaacttagac aacaaaacgcattttgaaggcattaacgctgaaagtgagccagtcggcgcatgggcaatgaattatgcatcaatggcaatacaacgaa gagcatcgacccaatcaccacaacaaagctggctcgccatagcgcgcggttttagccgttatcttgttggtaatgaaagctatgaaaata acaaccgttatggtcgttatttacaatatggacaattggaaattattccagctgatttaactcaatcagggtttagccatgctggatgggatt ggaatagatatccaggtacaacaactattcatcttccctataacgaacttgaagcaaaacttaatcaattacctgctgcaggtattgaaga aatgttgctttcaacagaaagttactctggtgcaaatacccttaataataacagtatgtttgccatgaaattacacggtcacagtaaatatca acaacaaagcttaagggcaaataaatcctatttcttatttgataatagagttattgctttaggctcaggtattgaaaatgatgataaacaacat acgaccgaaacaacactattccagtttgccgtccctaaattacagtcagtgatcattaatggcaaaaaggtaaatcaattagatactcaatt aactttaaataatgcagatacattaattgatcctgccggcaatttatataagctcactaaaggacaaactgtaaaatttagttatcaaaaaca acattcacttgatgatagaaattcaaaaccaacagaacaattatttgcaacagctgttatttctcatggtaaggcaccgagtaatgaaaatt atgaatatgcaatagctatcgaagcacaaaataataaagctcccaaatacacagtattacaacataatgatcagctccatgcggtaaaag ataaaataacccaagaagagggatatggtttttttgaagccactaagttaaaatcagcggatgcaacattattatccagtgatgcgccggt tatggtcatggctaaaatacaaaatcagcaattaacattaagtattgttaatcctgatttaaatttatatcaaggtagagaaaaagatcaattt gatgataaaggtaatcaaatcgaagttagtgtttattctcgtcattggcttacagcagaatcgcaatcaacaaatagtactattaccgtaaa aggaatatggaaattaacgacacctcaacccggtgttattattaagcaccacaataacaacactcttattacgacaacaaccatacaggc aacacctactgttattaatttagttaagtaa

    SEQ ID NO: 27 Present Invention Chondroitinase ABC II protein >_ ABC (present invention) >_ ABC (mature) scoring matrix: , gap penalties: -12/-2

    98.3% identity; Global alignment score: 6393

    990 aa vs. 990 aa

    457676 LPTLSHEAFGDIYLFEGELPNILTTSNNNQLSLSKQHAKDGEQSLKWQYQPQATLTLNNI

    LPTLSHEAFGDIYLFEGELPNTLTTSNNNQLSLSKQHAKDGEQSLKWQYQPQATLTLNNI 10 20 30 40 50 60

    70 80 90 100 110 120

    457676 VNYQDDKNTATPLTFMMWIYNEKPQSSPLTLAFKQNNKIALSFNAELNFTGWRGIAVPFR _ VNYQDDKNTATPLTFMMWIYNEKPQSSPLTLAFKQNNKIALSFNAELNFTGWRGIAVPFR

    70 80 90 100 110 120

    130 140 150 160 170 180

    457676 DMQGSATGQLDQLVITAPNQAGTLFFDQIIMSVPLDNRWAVPDYQTPYVNNAVNTMVSKN

    DMQGSVTGQLDQLVITAPNQAGTLFFDQIIMSVPLDNRWAVPDYQTPYVNNAVNTMVSKN 130 140 150 160 170 180

    190 200 210 220 230 240

    457676 WSALLMYDQMFQAHYPTLNFDTEFRDDQTEMASIYQRFEYYQGIRSDKKITPDMLDKHLA

    11/22

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    2016204464 29 Jun 2016

    WSALLMYDQMFQAHYPTLNFDTEFRDDQTEMASRYQRFEYYQGIRSDKKITPDMLDKHLA 190 200 210 220 230 240

    250 260 270 280 290 300

    457676 LWEKLGLTQHADGSITGKALDHPNRQHFMKVEGVFSEGTQKALLDANMLRDVGKTLLQTA

    LWEKLVLTQHADGSITGKALDHPNRQHFMKVEGVFSEGTQKALLDANMLRDVGKTLLQTA 250 260 270 280 290 300

    310 320 330 340 350 360

    457 67 6 IYLRSDSLSATGRKKLEERYLLGTRYVLEQGFTRGSGYQIITHVGYQTRELFDAWFIGRH

    IYLRSDSLSATDRKKLEERYLLGTRYVLEQGFHRGSGYQIISHVGYQTRELFDAWFIGRH 310 320 330 340 350 360

    370 380 390 400 410 420

    457676 VLAKNNLLAPTQQAMMWYNATGRIFEKDNEIVDANVDILNTQLQWMIKSLLMLPDYQQRQ

    VLAKNNLLAPTQQAMMWYNATGRIFEKNNEIVDANVDILNTQLQWMIKSLLMLPDYQQRQ 370 380 390 400 410 420

    430 440 450 460 470 480

    457676 QALAQLQSWLNKTILSSKGVAGGFKSDGSIFHHSQHYPAYAKDAFGGLAPSVYALSDSPF

    QALAQLQRWLNKTILSSKGVAGGFKSDGSIFHHSQHYPAYAKDAFGGLAPSVYALSDSPF 430 440 450 460 470 480

    490 500 510 520 530 540

    457676 RLSTSAHEHLKDVLLKMRIYTKETQIPVVLSGRHPTGLHKIGIAPFKWMALAGTPDGKQK

    RLSTSAHERLKDVLLKMRIYTKETQIPAVLSGRHPTGLHKIGIAPFKWMALAGTPDGKQK 490 500 510 520 530 540

    550 560 570 580 590 600

    457676 LDTTLSAAYANLDNKTHFEGINAESEPVGAWAMNYASMAIQRRASTQSPQQSWLAIARGF

    LDTTLSAAYAKLDNKTHFEGIKAESEPVGAWAMNYASMAIQRRASTQSPQQSWLAIARGF 550 560 570 580 590 600

    610 620 630 640 650 660

    457676 SRYLVGNESYENNNRYGRYLQYGQLEIIPADLTQSGFSHAGWDWNRYPGTTTIHLPYNEL

    SRYLVGNESYENNNRYGRYLQYGQLEIIPADLTQSGFSHAGWDWNRYPGTTTIHLPYNEL

    610 620 630 640 650 660

    670 680 690 700 710 720

    457676 EAKLNQLPAAGIEEMLLSTESYSGANTLNNNSMFAMKLHGHSKYQQQSLRANKSYFLFDN

    EAKLNQLPAAGIEEMLLSTESYSGANTLNNNSMFAMKLHGPSKYQQQSLRANKSYFLFDN 670 680 690 700 710 720

    730 740 750 760 770 780

    457676 RVIALGSGIENDDKQHTTETTLFQFAVPKLQSVIINGKKVNQLDTQLTLNNADTLIDPAG

    RVIALGSGIENDDKQHTTETTLFQFAVPKLQSVIINGKKVNQLDTQLTLNNADTLIDPAG 730 740 750 760 770 780

    790 800 810 820 830 840

    457676 NLYKLTKGQTVKFSYQKQHSLDDRNSKPTEQLFATAVISHGKAPSNENYEYAIAIEAQNN

    NLYKLTKGQTVKFSYQKQHSLDDRNSKPTEQLFATAVISHGKAPSNENYEYAIAIEAQNN

    12/22

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    2016204464 29 Jun 2016

    790 800 810 820 830 840

    850 860 870 880 890 900

    457676 KAPKYTVLQHNDQLHAVKDKITQEEGYGFFEATKLKSADATLLSSDAPVMVMAKIQNQQL _ KAPEYTVLQHNDQPHAVKDKITQEEGYAFFEATKLKSADATLLSSDAPVMVMAKIQNQQL

    850 860 870 880 890 900

    910 920 930 940 950 960

    457676 TLSIVNPDLNLYQGREKDQFDDKGNQIEVSVYSRHWLTAESQSTNSTITVKGIWKLTTPQ _ TLSIVNPDLNLYQGREKDQFDDKGNQIEVSVYSRHWLTAESQSTNSTITVKG1WKLTTPQ

    910 920 930 940 950 960

    970 980 990

    457676 PGVIIKHHNNNTLITTTTIQATPTVINLVK

    PGVIIKHHNNNTLITTTTIQATPTVINLVK 970 980 990

    SEQ ID NO: 28 Present Invention Chondroitinase ABC I nucleic acid gccaccagcaatcctgcatttgatcctaaaaatctgatgcagtcagaaatttaccattttgcacaaaataacccattagcagacttctcatc agataaaaactcaatactaacgttatctgataaacgtagcattatgggaaaccaatctcttttatggaaatggaaaggtggtagtagcttta ctttacataaaaaactgattgtccccaccgataaagaagcatctaaagcatggggacgctcatccacccccgttttctcattttggctttac aatgaaaaaccgattgatggttatcttactatcgatttcggagaaaaactcatttcaaccagtgaggctcaggcaggctttaaagtaaaatt agatttcactggctggcgtactgtgggagtctctttaaataacgatcttgaaaatcgagagatgaccttaaatgcaaccaatacctcctctg atggtactcaagacagcattgggcgttctttaggtgctaaagtcgatagtattcgttttaaagcgccttctaatgtgagtcagggtgaaatc tatatcgaccgtattatgttttctgtcgatgatgctcgctaccaatggtctgattatcaagtaaaaactcgcttatcagaacctgaaattcaatt tcacaacgtaaagccacaactacctgtaacacctgaaaatttagcggccattgatcttattcgccaacgtctaattaatgaatttgtcggag gtgaaaaagagacaaacctcgcattagaagagaatatcagcaaattaaaaagtgatttcgatgctcttaatactcacactttagcaaatg gtggaacgcaaggcagacatctgatcactgataaacaaatcattatttatcaaccagagaatcttaactctcaagataaacaactatttgat aattatgttattttaggtaattacacgacattaatgtttaatattagccgtgcttatgtgctggaaaaagatcccacacaaaaggcgcaacta aagcagatgtacttattaatgacaaagcatttattagatcaaggctttgttaaagggagtgctttagtgacnacccatcactggggataca gttctcgttggtggtatatttccacgttattaatgtctgatgcactaaaagaagcgaacctacaaactcaagtttatgattcattactgtggtat tcacgtgagtttaaaagtagttttgatatgaaagtaagtgctgatagctctgatctagattatttcaataccttatctcgccaacatttagcctt attactactagagcctgatgatcaaaagcgtatcaacttagttaatactttcagccattatatcactggcgcattaacgcaagtgccaccgg gtggtaaagatggtttacgccctgatggtacagcatggcgacatgaaggcaactatccgggctactctttcccagcctttaaaaatgcct ctcagcttatttatttattacgcgatacaccattttcagtgggtgaaagtggttggaatagcctgaaaaaagcgatggtttcagcgtggatct acagtaatccagaagttggattaccgcttgcaggaagacaccctcttaactcaccttcgttaaaatcagtcgctcaaggctattactggct tgccatgtctgcaaaatcatcgcctgataaaacacttgcatctatttatcttgcgattagtgataaaacacaaaatgaatcaactgctattttt ggagaaactattacaccagcgtctttacctcaaggtttctatgcctttaatggcggtgcttttggtattcatcgttggcaagataaaatggtg acactgaaagcttataacaccaatgtttggtcatctgaaatttataacaaagataaccgttatggccgttaccaaagtcatggtgtcgctca aatagtgagtaatggctcgcagctttcacagggctatcagcaagaaggttgggattggaatagaatgccaggggcaaccactatccac cttcctcttaaagacttagacagtcctaaacctcataccttaatgcaacgtggagagcgtggatttagcggaacatcatcccttgaaggtc aatatggcatgatggcattcgatcttatttatcccgccaatcttgagcgttttgatcctaatttcactgcgaaaaagagtgtattagccgctg ataatcacttaatttttattggtagcaatataaatagtagtgataaaaataaaaatgttgaaacgaccttattccaacatgccattactccaac attaaataccctttggattaatggacaaaagatagaaaacatgccttatcaaacaacacttcaacaaggtgattggttaattgatagcaatg gcaatggttacttaattactcaagcagaaaaagtaaatgtaagtcgccaacatcaggtttcagcggaaaataaaaatcgccaaccgaca gaaggaaactttagctcggcatggatcgatcacagcactcgccccaaagatgccagttatgagtatatggtctttttagatgcgacacct gaaaaaatgggagagatggcacaaaaattccgtgaaaataatgggttatatcaggttcttcgtaaggataaagacgttcatattattctcg ataaactcagcaatgtaacgggatatgccttttatcagccagcatcaattgaagacaaatggatcaaaaaggttaataaacctgcaattgt gatgactcatcgacaaaaagacactcttattgtcagtgcagttacacctgatttaaatatgactcgccaaaaagcagcaactcctgtcacc

    13/22

    WO 2004/110360

    PCT/US2004/015662 atcaatgtcacgattaatggcaaatggcaatctgctgataaaaatagtgaagtgaaatatcaggtttctggtgataacactgaactgacgt ttacgagttactttggtattccacaagaaatcaaactctcgccactcccttga

    2016204464 29 Jun 2016

    SEQ ID NO: 29 Present Invention Chondroitinase ABC I protein

    365019 ATSNPAFDPKNLMQSEIYHFAQNNPLADFSSDKNSILTLSDKRSIMGNQSLLWKWKGGSS

    365019 FTLHKKLIVPTDKEASKAWGRSSTPVFSFWLYNEKPIDGYLTIDFGEKLISTSEAQAGFK 365019 VKLDFTGWRTVGVSLNNDLENREMTLNATNTSSDGTQDSIGRSLGAKVDSIRFKAPSNVS 365019 QGEIYIDRIMFSVDDARYQWSDYQVKTRLSEPEIQFHNVKPQLPVTPENLAAIDLIRQRL 365019 INEFVGGEKETNLALEENISKLKSDFDALNTHTLANGGTQGRHLITDKQIIIYQPENLNS 365019 QDKQLFDNYVILGNYTTLMFNISRAYVLEKDPTQKAQLKQMYLLMTKHLLDQGFVKGSAL 365019 VTTHHWGYSSRWWYISTLLMSDALKEANLQTQVYDSLLWYSREFKSSFDMKVSADSSDLD 365019 YFNTLSRQHLALLLLEPDDQKRINLVNTFSHYITGALTQVPPGGKDGLRPDGTAWRHEGN 365019 YPGYSFPAFKNASQLIYLLRDTPFSVGESGWNSLKKAMVSAWIYSNPEVGLPLAGRHPLN 365019 SPSLKSVAQGYYWLAMSAKSSPDKTLASIYLAISDKTQNESTAIFGETITPASLPQGFYA 365019 FNGGAFGIHRWQDKMVTLKAYNTNVWSSEIYNKDNRYGRYQSHGVAQIVSNGSQLSQGYQ 365019 QEGWDWNRMPGATTIHLPLKDLDSPKPHTLMQRGERGFSGTSSLEGQYGMMAFDLIYPAN 365019 LERFDPNFTAKKSVLAADNHLIFIGSNINSSDKNKNVETTLFQHAITPTLNTLWINGQKI 365019 ENMPYQTTLQQGDWLIDSNGNGYLITQAEKVNVSRQHQVSAENKNRQPTEGNFSSAWIDH 365019 STRPKDASYEYMVFLDATPEKMGEMAQKFRENNGLYQVLRKDKDVHIILDKLSNVTGYAF 365019 YQPASIEDKWIKKVNKPAIVMTHRQKDTLIVSAVTPDLNMTRQKAATPVTINVTINGKWQ 365019 SADKNSEVKYQVSGDNTELTFTSYFGIPQEIKLSPLP hyaluronidase 1 protein, SEQ ID NO: 30, Locus HSU96078 hyaluronidase 2 protein, SEQ ID NO: 31, hyaluronidase 3 protein, SEQ ID NO: 32, Locus BC012892 hyaluronidase 4 protein, SEQ ID NO: 33, AF009010

    PH-20 protein, SEQ ID NO: 34,

    Tat peptide SEQ ID NO: 35

    ABCI a site-specific mutant designated H501a and Y508a SEQ ID NO: 36

    SEQ ID NO: 37 Chondroitinase ABCI protein Locus P59807

    ORIGIN

    1 mpifrftala mtlgllsapy namaatsnpa fdpknlmqse iyhfaqnnpl adfssdknsi 61 ltlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwravgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf

    14/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnihtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgivk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 pXX

    SEQ ID NO: 38 ΝΔ20 ABCI (A45-N1023), protein aqnnpl adfssdknsi

    61 ltlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwravgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnihtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgivk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 pXX

    SEQ ID NO: 39 ΝΔ60 ABCI (F₈₅-Nio2₃) protein ftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldft gwravgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnihtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgivk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr
15. 15/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafhli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgenia qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl 1021 pXX

    SEQ ID NO. 40: ΝΔ60 CA80 ABCI (F₈₅-A₉₄2) protein ftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg eklistseaq agfkvkldfl gwravgvsln ndlenremtl natntssdgt 181 qdsigrslga kvdsirfkap snvsqgeiyi drimfsvdda ryqwsdyqvk trlsepeiqf 241 hnvkpqlpvt penlaaidli rqrlinefvg geketnlale enisklksdf dalnihtlan 301 ggtqgrhlit dkqiiiyqpe nlnsqdkqlf dnyvilgnyt tlmfnisray vlekdptqka 361 qlkqmyllmt khlldqgfvk gsalvtthhw gyssrwwyis tllmsdalke anlqtqvyds 421 llwysrefks sfdmkvsads sdldyfntls rqhlalllle pddqkrinlv ntfshyitga 481 ltqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih lplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkienmpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk pa

    SEQ ID NO. 41: I. Nucleotide sequence for TAT-ABCI-nA20:

    1 ggtc gtaaaaagcg tcgtcaacgt cgtcgtcctc ctcaatgcgc acaaaataac 61 ccattagcag acttctcatc agataaaaac tcaatactaa cgttatctga taaacgtagc 121 attatgggaa accaatctct tttatggaaa tggaaaggtg gtagtagctt tactttacat 181 aaaaaactga ttgtccccac cgataaagaa gcatctaaag catggggacg ctcatccacc 241 cccgttttct cattttggct ttacaatgaa aaaccgattg atggttatct tactatcgat 301 ttcggagaaa aactcatttc aaccagtgag gctcaggcag gctttaaagt aaaattagat 361 ttcactggct ggcgtactgt gggagtctct ttaaataacg atcttgaaaa tcgagagatg 421 accttaaatg caaccaatac ctcctctgat ggtactcaag acagcattgg gcgttcttta 481 ggtgctaaag tcgatagtat tcgttttaaa gcgccttcta atgtgagtca gggtgaaatc 541 tatatcgacc gtattatgtt ttctgtcgat gatgctcgct accaatggtc tgattatcaa 601 gtaaaaactc gcttatcaga acctgaaatt caatttcaca acgtaaagcc acaactacct 661 gtaacacctg aaaatttagc ggccattgat cttattcgcc aacgtctaat taatgaattt 721 gtcggaggtg aaaaagagac aaacctcgca ttagaagaga atatcagcaa attaaaaagt 781 gatttcgatg ctcttaatac tcacacttta gcaaatggtg gaacgcaagg cagacatctg 841 atcactgata aacaaatcat tatttatcaa ccagagaatc ttaactctca agataaacaa 901 ctatttgata attatgttat tttaggtaat tacacgacat taatgtttaa tattagccgt 961 gcttatgtgc tggaaaaaga tcccacacaa aaggcgcaac taaagcagat gtacttatta 1021 atgacaaagc atttattaga tcaaggcttt gttaaaggga gtgctttagt gacnacccat 1081 cactggggat acagttctcg ttggtggtat atttccacgt tattaatgtc tgatgcacta 1141 aaagaagcga acctacaaac tcaagtttat gattcattac tgtggtattc acgtgagttt 1201 aaaagtagtt ttgatatgaa agtaagtgct gatagctctg atctagatta tttcaatacc
16. 16/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    1261 ttatctcgcc aacatttagc cttattacta ctagagcctg atgatcaaaa gcgtatcaac 1321 ttagttaata ctttcagcca ttatatcact ggcgcattaa cgcaagtgcc accgggtggt 1381 aaagatggtt tacgccctga tggtacagca tggcgacatg aaggcaacta tccgggctac 1441 tctttcccag cctttaaaaa tgcctctcag cttatttatt tattacgcga tacaccattt 1501 tcagtgggtg aaagtggttg gaatagcctg aaaaaagcga tggtttcagc gtggatctac 1561 agtaatccag aagttggatt accgcttgca ggaagacacc ctcttaactc accttcgtta 1621 aaatcagtcg ctcaaggcta ttactggctt gccatgtctg caaaatcatc gcctgataaa 1681 acacttgcat ctatttatct tgcgattagt gataaaacac aaaatgaatc aactgctatt 1741 tttggagaaa ctattacacc agcgtcttta cctcaaggtt tctatgcctt taatggcggt 1801 gcttttggta ttcatcgttg gcaagataaa atggtgacac tgaaagctta taacaccaat 1861 gtttggtcat ctgaaattta taacaaagat aaccgttatg gccgttacca aagtcatggt 1921 gtcgctcaaa tagtgagtaa tggctcgcag ctttcacagg gctatcagca agaaggttgg 1981 gattggaata gaatgccagg ggcaaccact atccaccttc ctcttaaaga cttagacagt 2041 cctaaacctc ataccttaat gcaacgtgga gagcgtggat ttagcggaac atcatccctt 2101 gaaggtcaat atggcatgat ggcattcgat cttatttatc ccgccaatct tgagcgtttt 2161 gatcctaatt tcactgcgaa aaagagtgta ttagccgctg ataatcactt aatttttatt 2221 ggtagcaata taaatagtag tgataaaaat aaaaatgttg aaacgacctt attccaacat 2281 gccattactc caacattaaa taccctttgg attaatggac aaaagataga aaacatgcct 2341 tatcaaacaa cacttcaaca aggtgattgg ttaattgata gcaatggcaa tggttactta 2401 attactcaag cagaaaaagt aaatgtaagt cgccaacatc aggtttcagc ggaaaataaa 2461 aatcgccaac cgacagaagg aaactttagc tcggcatgga tcgatcacag cactcgcccc 2521 aaagatgcca gttatgagta tatggtcttt ttagatgcga cacctgaaaa aatgggagag 2581 atggcacaaa aattccgtga aaataatggg ttatatcagg ttcttcgtaa ggataaagac 2641 gttcatatta ttctcgataa actcagcaat gtaacgggat atgcctttta tcagccagca 2701 tcaattgaag acaaatggat caaaaaggtt aataaacctg caattgtgat gactcatcga 2761 caaaaagaca ctcttattgt cagtgcagtt acacctgatt taaatatgac tcgccaaaaa 2821 gcagcaactc ctgtcaccat caatgtcacg attaatggca aatggcaatc tgctgataaa 2881 aatagtgaag tgaaatatca ggtttctggt gataacactg aactgacgtt tacgagttac 2941 tttggtattc cacaagaaat caaactctcg ccactccctt ga

    SEQ ID NO. 42: II. Amino acid sequence for TAT-ABCI-nA20:

    grkkrrqrrrppqcaqnnpladfssdknsiltlsdkrsimgnqsllwkwkggssftlhkklivptdkeaskawgrsstpvfsfw lynekpidgyltidfgeklistseaqagflivkldftgwrtvgvslnndlenremtlnatntssdgtqdsigrslgakvdsirflcapsnv sqgeiyidrimfsvddaryqwsdyqvktrlsepeiqfhnvkpqlpvtpenlaaidlirqrlinefvggeketnlaleenisklksdfd alnthtlanggtqgrhlitdkqiiiyqpenlnsqdkqlfdnyvilgnyttlmfnisrayvlekdptqkaqlkqmyllmtkhlldqgfv kgsalvtthhwgyssrwwyistllmsdalkeanlqtqvydsllwysrefkssfdmkvsadssdldyfntlsrqhlallllepddqkr inlvntfshyitgaltqvppggkdglrpdgtawrhegnypgysfpafknasqliyllrdtpfsvgesgwnslkkamvsawiysnp evglplagrhplnspslksvaqgyywlamsaksspdktlasiylaisdktqnestaifgetitpaslpqgfyafnggafgihrwqdk mvtlkayntnvwsseiynkdnrygryqshgvaqivsngsqlsqgyqqegwdwnrmpgattihlplkdldspkphtlmqrger gfsgtsslegqygmmafdliypanlerfdpnftakksvlaadnhlifigsninssdknknvettlfqhaitptlntlwingqkienm pyqttlqqgdwlidsngngylitqaekvnvsrqhqvsaenknrqptegnfssawidhstrpkdasyeymvfldatpekmgema qkfrennglyqvlrkdkdvhiildklsnvtgyafyqpasiedkwikkvnkpaivmthrqkdtlivsavtpdlnintrqkaatpvtm vtingkwqsadknsevkyqvsgdnteltftsyfgipqeiklsplp

    SEQ ID NO. 43: II. Nucleotide sequence for ΤΑΤ-ΑΒΟ-11Δ6Ο:
17. 17/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016 ggtcgtaaaaagcgtcgtcaacgtcgtcgtcctcctcaatgctttactttacataaaaaactgattgtccccaccgataaagaagcatcta aagcatggggacgctcatccacccccgttttctcattttggctttacaatgaaaaaccgattgatggttatcttactatcgatttcggagaaa aactcatttcaaccagtgaggctcaggcaggctttaaagtaaaattagatttcactggctggcgtactgtgggagtctctttaaataacgat cttgaaaatcgagagatgaccttaaatgcaaccaatacctcctctgatggtactcaagacagcattgggcgttctttaggtgctaaagtcg atagtattcgttttaaagcgccttctaatgtgagtcagggtgaaatctatatcgaccgtattatgttttctgtcgatgatgctcgctaccaatg gtctgattatcaagtaaaaactcgcttatcagaacctgaaattcaatttcacaacgtaaagccacaactacctgtaacacctgaaaatttag cggccattgatcttattcgccaacgtctaattaatgaatttgtcggaggtgaaaaagagacaaacctcgcattagaagagaatatcagca aattaaaaagtgatttcgatgctcttaatactcacactttagcaaatggtggaacgcaaggcagacatctgatcactgataaacaaatcatt atttatcaaccagagaatcttaactctcaagataaacaactatttgataattatgttattttaggtaattacacgacattaatgtttaatattagc cgtgcttatgtgctggaaaaagatcccacacaaaaggcgcaactaaagcagatgtacttattaatgacaaagcatttattagatcaaggc tttgttaaagggagtgctttagtgacnacccatcactggggatacagttctcgttggtggtatatttccacgttattaatgtctgatgcactaa aagaagcgaacctacaaactcaagtttatgattcattactgtggtattcacgtgagtttaaaagtagttttgatatgaaagtaagtgctgata gctctgatctagattatttcaataccttatctcgccaacatttagccttattactactagagcctgatgatcaaaagcgtatcaacttagttaat actttcagccattatatcactggcgcattaacgcaagtgccaccgggtggtaaagatggtttacgccctgatggtacagcatggcgacat gaaggcaactatccgggctactctttcccagcctttaaaaatgcctctcagcttatttatttattacgcgatacaccattttcagtgggtgaa agtggttggaatagcctgaaaaaagcgatggtttcagcgtggatctacagtaatccagaagttggattaccgcttgcaggaagacaccc tcttaactcaccttcgttaaaatcagtcgctcaaggctattactggcttgccatgtctgcaaaatcatcgcctgataaaacacttgcatctatt tatcttgcgattagtgataaaacacaaaatgaatcaactgctatttttggagaaactattacaccagcgtctttacctcaaggtttctatgcct ttaatggcggtgcttttggtattcatcgttggcaagataaaatggtgacactgaaagcttataacaccaatgtttggtcatctgaaatttataa caaagataaccgttatggccgttaccaaagtcatggtgtcgctcaaatagtgagtaatggctcgcagctttcacagggctatcagcaag aaggttgggattggaatagaatgccaggggcaaccactatccaccttcctcttaaagacttagacagtcctaaacctcataccttaatgc aacgtggagagcgtggatttagcggaacatcatcccttgaaggtcaatatggcatgatggcattcgatcttatttatcccgccaatcttga gcgttttgatcctaatttcactgcgaaaaagagtgtattagccgctgataatcacttaatttttattggtagcaatataaatagtagtgataaa aataaaaatgttgaaacgaccttattccaacatgccattactccaacattaaataccctttggattaatggacaaaagatagaaaacatgc cttatcaaacaacacttcaacaaggtgattggttaattgatagcaatggcaatggttacttaattactcaagcagaaaaagtaaatgtaagt cgccaacatcaggtttcagcggaaaataaaaatcgccaaccgacagaaggaaactttagctcggcatggatcgatcacagcactcgc cccaaagatgccagttatgagtatatggtctttttagatgcgacacctgaaaaaatgggagagatggcacaaaaattccgtgaaaataat gggttatatcaggttcttcgtaaggataaagacgttcatattattctcgataaactcagcaatgtaacgggatatgccttttatcagccagca tcaattgaagacaaatggatcaaaaaggttaataaacctgcaattgtgatgactcatcgacaaaaagacactcttattgtcagtgcagtta cacctgatttaaatatgactcgccaaaaagcagcaactcctgtcaccatcaatgtcacgattaatggcaaatggcaatctgctgataaaa atagtgaagtgaaatatcaggtttctggtgataacactgaactgacgtttacgagttactttggtattccacaagaaatcaaactctcgcca ctcccttga

    SEQ ID NO. 44: IV. Amino acid sequence for ΤΑΤ-ΑΒΟ-ηΔ60 grkkrrqrrrppqcftlhkklivptdkeaskawgrsstpvfsfwlynekpidgyltidfgeklistseaqagfkvkldftgwrtvgvsl nndlenremtlnatntssdgtqdsigrslgakvdsirfkapsnvsqgeiyidrimfsvddaryqwsdyqvktrlsepeiqfhnvkp qlpvtpenlaaidlirqrlinefvggeketnlaleenisklksdfdalnthtlanggtqgrhlitdkqiiiyqpenlnsqdkqlfdnyvil gnyttlmfnisrayvlekdptqkaqlkqmyllmtkhlldqgfvkgsalvtthhwgyssrwwyistllmsdalkeanlqtqvydsll wysrefkssfdmkvsadssdldyfntlsrqhlallllepddqkrinlvntfshyitgaltqvppggkdglrpdgtawrhegnypgys fpafknasqliyllrdtpfsvgesgwnslkkamvsawiysnpevglplagrhplnspslksvaqgyywlamsaksspdktlasiy laisdktqnestaifgetitpaslpqgfyafnggafgihrwqdkmvtlkayntnvwsseiynkdnrygryqshgvaqivsngsqls qgyqqegwdwnrmpgattihlplkdldspkphtlmqrgergfsgtsslegqygmmafdliypanlerfdpnftakksvlaadn hlifigsninssdknknvettlfqhaitptlntlwingqkienmpyqttlqqgdwlidsngngylitqaekvnvsrqhqvsaenknr qptegnfssawidhstrpkdasyeymvfldatpekmgemaqkfrennglyqvlrkdkdvhiildklsnvtgyafyqpasiedk wikkvnkpaivmthrqkdtlivsavtpdlnmtrqkaatpvtinvtingkwqsadknsevkyqvsgdnteltftsyfgipqeiklsp lp
18. 18/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun 2016

    SEQ ID NO. 45: V. Nucleotide sequence for ABCI-TAT-C:

    gccaccagcaatcctgcatttgatcctaaaaatctgatgcagtcagaaatttaccattttgcacaaaataacccattagcagacttctcatc agataaaaactcaatactaacgttatctgataaacgtagcattatgggaaaccaatctcttttatggaaatggaaaggtggtagtagcttta ctttacataaaaaactgattgtccccaccgataaagaagcatctaaagcatggggacgctcatccacccccgttttctcattttggctttac aatgaaaaaccgattgatggttatcttactatcgatttcggagaaaaactcatttcaaccagtgaggctcaggcaggctttaaagtaaaatt agatttcactggctggcgtactgtgggagtctctttaaataacgatcttgaaaatcgagagatgaccttaaatgcaaccaatacctcctctg atggtactcaagacagcattgggcgttctttaggtgctaaagtcgatagtattcgttttaaagcgccttctaatgtgagtcagggtgaaatc tatatcgaccgtattatgttttctgtcgatgatgctcgctaccaatggtctgattatcaagtaaaaactcgcttatcagaacctgaaattcaatt tcacaacgtaaagccacaactacctgtaacacctgaaaatttagcggccattgatcttattcgccaacgtctaattaatgaatttgtcggag gtgaaaaagagacaaacctcgcattagaagagaatatcagcaaattaaaaagtgatttcgatgctcttaatactcacactttagcaaatg gtggaacgcaaggcagacatctgatcactgataaacaaatcattatttatcaaccagagaatcttaactctcaagataaacaactatttgat aattatgttattttaggtaattacacgacattaatgtttaatattagccgtgcttatgtgctggaaaaagatcccacacaaaaggcgcaacta aagcagatgtacttattaatgacaaagcatttattagatcaaggctttgttaaagggagtgctttagtgacnacccatcactggggataca gttctcgttggtggtatatttccacgttattaatgtctgatgcactaaaagaagcgaacctacaaactcaagtttatgattcattactgtggtat tcacgtgagtttaaaagtagttttgatatgaaagtaagtgctgatagctctgatctagattatttcaataccttatctcgccaacatttagcctt attactactagagcctgatgatcaaaagcgtatcaacttagttaatactttcagccattatatcactggcgcattaacgcaagtgccaccgg gtggtaaagatggtttacgccctgatggtacagcatggcgacatgaaggcaactatccgggctactctttcccagcctttaaaaatgcct ctcagcttatttatttattacgcgatacaccattttcagtgggtgaaagtggttggaatagcctgaaaaaagcgatggtttcagcgtggatct acagtaatccagaagttggattaccgcttgcaggaagacaccctcttaactcaccttcgttaaaatcagtcgctcaaggctattactggct tgccatgtctgcaaaatcatcgcctgataaaacacttgcatctatttatcttgcgattagtgataaaacacaaaatgaatcaactgctattttt ggagaaactattacaccagcgtctttacctcaaggtttctatgcctttaatggcggtgcttttggtattcatcgttggcaagataaaatggtg acactgaaagcttataacaccaatgtttggtcatctgaaatttataacaaagataaccgttatggccgttaccaaagtcatggtgtcgctca aatagtgagtaatggctcgcagctttcacagggctatcagcaagaaggttgggattggaatagaatgccaggggcaaccactatccac cttcctcttaaagacttagacagtcctaaacctcataccttaatgcaacgtggagagcgtggatttagcggaacatcatcccttgaaggtc aatatggcatgatggcattcgatcttatttatcccgccaatcttgagcgttttgatcctaatttcactgcgaaaaagagtgtattagccgctg ataatcacttaatttttattggtagcaatataaatagtagtgataaaaataaaaatgttgaaacgaccttattccaacatgccattactccaac attaaataccctttggattaatggacaaaagatagaaaacatgccttatcaaacaacacttcaacaaggtgattggttaattgatagcaatg gcaatggttacttaattactcaagcagaaaaagtaaatgtaagtcgccaacatcaggtttcagcggaaaataaaaatcgccaaccgaca gaaggaaactttagctcggcatggatcgatcacagcactcgccccaaagatgccagttatgagtatatggtctttttagatgcgacacct gaaaaaatgggagagatggcacaaaaattccgtgaaaataatgggttatatcaggttcttcgtaaggataaagacgttcatattattctcg ataaactcagcaatgtaacgggatatgccttttatcagccagcatcaattgaagacaaatggatcaaaaaggttaataaacctgcaattgt gatgactcatcgacaaaaagacactcttattgtcagtgcagttacacctgatttaaatatgactcgccaaaaagcagcaactcctgtcacc atcaatgtcacgattaatggcaaatggcaatctgctgataaaaatagtgaagtgaaatatcaggtttctggtgataacactgaactgacgt ttacgagttactttggtattccacaagaaatcaaactctcgccactccct ggtcgtaaaaagcgtcgtcaacgtcgtcgtcctcctcaatgctag

    SEQ ID NO. 46: V. Amino acid sequence for ABCI-TAT-C:

    atsnpafdpknlmqseiyhfaqnnpladfssdknsiltlsdkrsimgnqsllwkwkggssftlhkklivptdkeaskawgrsstpv fsfwlynekpidgyltidfgeklistseaqagfkvkldftgwrtvgvslnndlenremtlnatntssdgtqdsigrslgakvdsirfka psnvsqgeiyidrimfsvddaryqwsdyqvktrlsepeiqfhnvkpqlpvtpenlaaidlirqrlinefvggeketnlaleenisklk sdfdalnthtlanggtqgrhlitdkqiiiyqpenlnsqdkqlfdnyvilgnyttlmfnisrayvlekdptqkaqlkqmyllmtkhlld qgfvkgsalvtthhwgyssrwwyistllmsdalkeanlqtqvydsllwysrefkssfdmkvsadssdldyfntlsrqhlallllepd dqkrinlvntfshyitgaltqvppggkdglrpdgtawrhegnypgysfpafknasqliyllrdtpfsvgesgwnslkkanrvsawi ysnpevglplagrhplnspslksvaqgyywlamsaksspdktlasiylaisdktqnestaifgetitpaslpqgfyafhggafgihr wqdkmvtlkayntnvwsseiynkdnrygryqshgvaqivsngsqlsqgyqqegwdwnrmpgattihlplkdldspkphtlm qrgergfsgtsslegqygmmafdliypanlerfdpnftakksvlaadnhlifigsninssdknknvettlfqhaitptlntlwingqki
19. 19/22

    WO 2004/110360

    PCT/US2004/015662 enmpyqttlqqgdwlidsngngylitqaekvnvsrqhqvsaenknrqptegnfssawidhstrpkdasyeymvfldatpekm gemaqkffennglyqvlrkdkdvhiildklsnvtgyafyqpasiedkwikkvnkpaivmthrqkdtlivsavtpdlnmtrqkaa tpvtinvtingkwqsadknsevkyqvsgdnteltftsyfgipqeiklsplpgrkkrrqrrrppqc

    2016204464 29 Jun 2016

    SEQ ID NO. 47 Nucleotide sequence for chondroitinase ΑΒΟ-ηΔ20 gc acaaaataac

    61 ccattagcag acttctcatc agataaaaac tcaatactaa cgttatctga taaacgtagc 121 attatgggaa accaatctct tttatggaaa tggaaaggtg gtagtagctt tactttacat 181 aaaaaactga ttgtccccac cgataaagaa gcatctaaag catggggacg ctcatccacc 241 cccgttttct cattttggct ttacaatgaa aaaccgattg atggttatct tactatcgat 301 ttcggagaaa aactcatttc aaccagtgag gctcaggcag gctttaaagt aaaattagat 361 ttcactggct ggcgtactgt gggagtctct ttaaataacg atcttgaaaa tcgagagatg 421 accttaaatg caaccaatac ctcctctgat ggtactcaag acagcattgg gcgttcttta 481 ggtgctaaag tcgatagtat tcgttttaaa gcgccttcta atgtgagtca gggtgaaatc 541 tatatcgacc gtattatgtt ttctgtcgat gatgctcgct accaatggtc tgattatcaa 601 gtaaaaactc gcttatcaga acctgaaatt caatttcaca acgtaaagcc acaactacct 661 gtaacacctg aaaatttagc ggccattgat cttattcgcc aacgtctaat taatgaattt 721 gtcggaggtg aaaaagagac aaacctcgca ttagaagaga atatcagcaa attaaaaagt 781 gatttcgatg ctcttaatac tcacacttta gcaaatggtg gaacgcaagg cagacatctg 841 atcactgata aacaaatcat tatttatcaa ccagagaatc ttaactctca agataaacaa 901 ctatttgata attatgttat tttaggtaat tacacgacat taatgtttaa tattagccgt 961 gcttatgtgc tggaaaaaga tcccacacaa aaggcgcaac taaagcagat gtacttatta 1021 atgacaaagc atttattaga tcaaggcttt gttaaaggga gtgctttagt gacnacccat 1081 cactggggat acagttctcg ttggtggtat atttccacgt tattaatgtc tgatgcacta 1141 aaagaagcga acctacaaac tcaagtttat gattcattac tgtggtattc acgtgagttt 1201 aaaagtagtt ttgatatgaa agtaagtgct gatagctctg atctagatta tttcaatacc 1261 ttatctcgcc aacatttagc cttattacta ctagagcctg atgatcaaaa gcgtatcaac 1321 ttagttaata ctttcagcca ttatatcact ggcgcattaa cgcaagtgcc accgggtggt 1381 aaagatggtt tacgccctga tggtacagca tggcgacatg aaggcaacta tccgggctac 1441 tctttcccag cctttaaaaa tgcctctcag cttatttatt tattacgcga tacaccattt 1501 tcagtgggtg aaagtggttg gaatagcctg aaaaaagcga tggtttcagc gtggatctac 1561 agtaatccag aagttggatt accgcttgca ggaagacacc ctcttaactc accttcgtta 1621 aaatcagtcg ctcaaggcta ttactggctt gccatgtctg caaaatcatc gcctgataaa 1681 acacttgcat ctatttatct tgcgattagt gataaaacac aaaatgaatc aactgctatt 1741 tttggagaaa ctattacacc agcgtcttta cctcaaggtt tctatgcctt taatggcggt 1801 gcttttggta ttcatcgttg gcaagataaa atggtgacac tgaaagctta taacaccaat 1861 gtttggtcat ctgaaattta taacaaagat aaccgttatg gccgttacca aagtcatggt 1921 gtcgctcaaa tagtgagtaa tggctcgcag ctttcacagg gctatcagca agaaggttgg 1981 gattggaata gaatgccagg ggcaaccact atccaccttc ctcttaaaga cttagacagt 2041 cctaaacctc ataccttaat gcaacgtgga gagcgtggat ttagcggaac atcatccctt 2101 gaaggtcaat atggcatgat ggcattcgat cttatttatc ccgccaatct tgagcgtttt 2161 gatcctaatt tcactgcgaa aaagagtgta ttagccgctg ataatcactt aatttttatt 2221 ggtagcaata taaatagtag tgataaaaat aaaaatgttg aaacgacctt attccaacat 2281 gccattactc caacattaaa taccctttgg attaatggac aaaagataga aaacatgcct 2341 tatcaaacaa cacttcaaca aggtgattgg ttaattgata gcaatggcaa tggttactta 2401 attactcaag cagaaaaagt aaatgtaagt cgccaacatc aggtttcagc ggaaaataaa 2461 aatcgccaac cgacagaagg aaactttagc tcggcatgga tcgatcacag cactcgcccc 2521 aaagatgcca gttatgagta tatggtcttt ttagatgcga cacctgaaaa aatgggagag 2581 atggcacaaa aattccgtga aaataatggg ttatatcagg ttcttcgtaa ggataaagac
20. 20/22

    WO 2004/110360

    PCT/US2004/015662

    2016204464 29 Jun2016

    2641 gttcatatta ttctcgataa actcagcaat gtaacgggat atgcctttta tcagccagca 2701 tcaattgaag acaaatggat caaaaaggtt aataaacctg caattgtgat gactcatcga 2761 caaaaagaca ctcttattgt cagtgcagtt acacctgatt taaatatgac tcgccaaaaa 2821 gcagcaactc ctgtcaccat caatgtcacg attaatggca aatggcaatc tgctgataaa 2881 aatagtgaag tgaaatatca ggtttctggt gataacactg aactgacgtt tacgagttac 2941 tttggtattc cacaagaaat caaactctcg ccactccctt ga

    SEQ ID NO. 48: II. Nucleotide sequence for ΑΒΟ-ηΔόΟ:

    tttactttacataaaaaactgattgtccccaccgataaagaagcatctaaagcatggggacgctcatccacccccgttttctcattttggctt tacaatgaaaaaccgattgatggttatcttactatcgatttcggagaaaaactcatttcaaccagtgaggctcaggcaggctttaaagtaa aattagatttcactggctggcgtactgtgggagtctctttaaataacgatcttgaaaatcgagagatgaccttaaatgcaaccaatacctcc tctgatggtactcaagacagcattgggcgttctttaggtgctaaagtcgatagtattcgttttaaagcgccttctaatgtgagtcagggtga aatctatatcgaccgtattatgttttctgtcgatgatgctcgctaccaatggtctgattatcaagtaaaaaetcgcttatcagaacctgaaatt caatttcacaacgtaaagccacaactacctgtaacacctgaaaatttagcggccattgatcttattcgccaacgtctaattaatgaatttgtc ggaggtgaaaaagagacaaacctcgcattagaagagaatatcagcaaattaaaaagtgatttcgatgctcttaatactcacactttagca aatggtggaacgcaaggcagacatctgatcactgataaacaaatcattatttatcaaccagagaatcttaactctcaagataaacaactat ttgataattatgttattttaggtaattacacgacattaatgtttaatattagccgtgcttatgtgctggaaaaagatcccacacaaaaggcgca actaaagcagatgtacttattaatgacaaagcatttattagatcaaggctttgttaaagggagtgctttagtgacnacccatcactggggat acagttctcgttggtggtatatttccacgttattaatgtctgatgcactaaaagaagcgaacctacaaactcaagtttatgattcattactgtg gtattcacgtgagtttaaaagtagttttgatatgaaagtaagtgctgatagctctgatctagattatttcaataccttatctcgccaacatttag ccttattactactagagcctgatgatcaaaagcgtatcaacttagttaatactttcagccattatatcactggcgcattaacgcaagtgccac cgggtggtaaagatggtttacgccctgatggtacagcatggcgacatgaaggcaactatccgggctactctttcccagcctttaaaaat gcctctcagcttatttatttattacgcgatacaccattttcagtgggtgaaagtggttggaatagcctgaaaaaagcgatggtttcagcgtg gatctacagtaatccagaagttggattaccgcttgcaggaagacaccctcttaactcaccttcgttaaaatcagtcgctcaaggctattact ggcttgccatgtctgcaaaatcatcgcctgataaaacacttgcatctatttatcttgcgattagtgataaaacacaaaatgaatcaactgcta tttttggagaaactattacaccagcgtctttacctcaaggtttctatgcctttaatggcggtgcttttggtattcatcgttggcaagataaaatg gtgacactgaaagcttataacaccaatgtttggtcatctgaaatttataacaaagataaccgttatggccgttaceaaagtcatggtgtcgc tcaaatagtgagtaatggctcgcagctttcacagggctatcagcaagaaggttgggattggaatagaatgccaggggcaaccactatc caccttcctcttaaagacttagacagtcctaaacctcataccttaatgcaacgtggagagcgtggatttagcggaacatcatcccttgaag gtcaatatggcatgatggcattcgatcttatttatcccgccaatcttgagcgttttgatcctaatttcactgcgaaaaagagtgtattagccg ctgataatcacttaatttttattggtagcaatataaatagtagtgataaaaataaaaatgttgaaacgaccttattccaacatgccattactcc aacattaaataccctttggattaatggacaaaagatagaaaacatgccttatcaaacaacacttcaacaaggtgattggttaattgatagc aatggcaatggttacttaattactcaagcagaaaaagtaaatgtaagtcgccaacatcaggtttcagcggaaaataaaaatcgccaacc gacagaaggaaactttagctcggcatggatcgatcacagcactcgccccaaagatgccagttatgagtatatggtctttttagatgcgac acctgaaaaaatgggagagatggcacaaaaattccgtgaaaataatgggttatatcaggttcttcgtaaggataaagacgttcatattatt ctcgataaactcagcaatgtaacgggatatgccttttatcagccagcatcaattgaagacaaatggatcaaaaaggttaataaacctgca attgtgatgactcatcgacaaaaagacactcttattgtcagtgcagttacacctgatttaaatatgactcgccaaaaagcagcaactcctgt caccatcaatgtcacgattaatggcaaatggcaatctgctgataaaaatagtgaagtgaaatatcaggtttctggtgataacactgaactg acgtttacgagttactttggtattccacaagaaatcaaactctcgccactcccttga

    SEQ ID NO. 49: Nucleotide sequence for TAT ggtcgtaaaaagcgtcgtcaacgtcgtcgtcctcctcaatgc
21. 21/22

    WO 2004/110360

    PCT/US2004/015662 (SEQ ID NO. 50) Amino acid sequence for a TAT peptide grkkrrqrrrppqc

    2016204464 29 Jun 2016
22. 22/22