ES2638819T3 - Fusion proteins for CNS treatment - Google Patents

Abstract

A composition comprising: a polypeptide comprising a TAT protein transduction domain and a mutant by chondroitinase ABC I deletion, wherein the TAT protein transduction domain is anchored to the N-terminus of the mutant by ABC I chondroitinase deletion and the ABC I chondroitinase deletion mutant is selected from SEQ ID NO: 2 and SEQ ID NO: 3.

Description

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DESCRIPTION

Fusion proteins for the treatment of CNS Background and compendium

Spinal cord injury (SCI) inflicts trauma to the cells and tissues of the central nervous system (CNS) and causes a serious and debilitating condition in the individual. Following SCI, the limited regeneration of injured neurons results in permanent disability characterized by some loss of sensitivity, paralysis and autonomic dysfunction. One of the reasons why neurons do not regenerate is their inability to pass through the glial scar that develops after SCI. This glial scar contains extracellular matrix molecules that include chondroitin sulfate proteoglycans (PGSC). In vitro studies show that neurons fail to extend processes along PGSC-coated surfaces, while in vivo data correlates regeneration failure with PGSC expression zones. In myelin of the adult central nervous system (CNS), several axon growth inhibitor compounds have also been identified as myelin-associated glycoprotem (MAG), OMgp, and Reticulon 4 or Nogo that have been shown to be growth inhibitors of neurons.

Proteoglycan degradation enzyme, chondroitinase ABC type I (SEQ ID NO: X) has been used to enhance neuronal growth in a spinal cord damage model of the spinal cord. It has also been reported that treating a spinal cord injury with a NOGO receptor antagonist promotes some limited degree of neuronal regeneration. It has been further reported that the creation of a mouse with the deactivated NOGO gene resulted in certain limited and incoherent degrees of neuronal regeneration after dorsal hemisection of the spinal cord.

Experimental treatments for CNS injury have used the application of chondroitinase to the extracellular space, however the enzyme digests the PGSC in the extracellular matrix and not intracellular reserves. In addition, the diffusion of chondroitinase within the parenchyma, the essential and distinctive tissue of an organ or an abnormal growth that differs from its support framework) or between the anatomical compartments is limited. The limited access of drugs, imaging agents and anesthetics, etc., to the cells and / or tissues of the Central Nervous System (CNS) can reduce the usefulness or efficacy of any of these substances.

The supply of therapeutic and diagnostic molecules to cells and tissues depends, in part, on extracellular matrices, as well as carbohydrates and proteins bound to cell membranes. The extracellular matrix is partly composed of proteoglycans, including chondroitin sulfate proteoglycans (PGSC). PGSCs are a family of proteoglycans composed of a nucleus protein and covalently linked sulfated glycosaminoglycans. Each proteoglycan is determined by the glycosaminoglycan side chains. For the PGSCs, these side chains are composed of approximately 40 to 100 sulfated disaccharides composed of chondroitin sulfates 4, 6 and dermatan. The PGSC protein component is synthesized ribosomically and glycosylation occurs in the endoplasmic reticulum and in the Golgi apparatus. The sugar chains are then sulfated at positions 4 or 6 by means of various glycosaminoglycan sulfotransferases.

Transduction proteins can be used to transport loads of polypeptides and polynucleotides across anatomical barriers and into the cells. For example, the TAT protein of the human immunodeficiency virus (HIV) contains a protein transduction domain ("PTD") that is involved in the transduction of HIV into cells. PTD contains an 11 amino acid domain (TAT Peptide) that is responsible for PTD activity. The TAT Peptide can bind to proteins and facilitate the transduction of proteins to cells. The mechanism of transduction is independent of the molecular weight or chemical properties of the proteins that are linked to the TAT Peptide. In vivo studies show that if a fusion protein consisting of the 120 kd enzyme-linked TAT Peptide, beta-galactosidase (p-Gal), is injected into mice, a robust supply of p-Gal to a Wide variety of cells. When the TAT transduction peptide is urn, p-Gal activity was observed in the brain; without the TAT peptide, p-Gal was not observed in the brain. Transport through the blood-brain barrier is normally restricted to certain small hydrophobic molecules and specific low molecular weight lipophilic peptides. The transport of proteins as large as p-Gal to the brain is usually not possible without a substantial disruption of the blood-brain barrier, but the TAT Peptide facilitates transport while leaving the blood-brain barrier intact.

Chimeric proteins, also called fusion proteins, are hybrid proteins that combine at least parts of two or more precursor proteins or polypeptides. Chimeric proteins can be produced by recombinant technology, that is, by fusing at least a part of the coding sequence of one gene with at least a part of the coding sequence of another gene. The fused gene can then be used to transform a suitable organism that expresses the fusion protein in that case.

In Tat Peptide Complexes, Frankel et al. (U.S. Patents 5,804,604; 5,747,641; 5,674,980; 5,670,617; 5,652,122) describe the use of Tat peptides to transport biologically active loading molecules covalently linked to the cytoplasm and cell nuclei. Frankel only describes bound cargo molecules

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covalently they are (therapeutic, diagnostic or prophylactic), and does not illustrate or suggest the anchoring of molecules that facilitate diffusion, plasticity, neurite growth and axon regeneration. These molecules may include, but are not limited to, molecules that overcome neurite growth inhibition or promote nerve growth such as soluble NOGO antagonists such as NgR27-3ii, neural cell adhesion molecules such as Li, neurotrophic factors, factors growth, phosphodiesterase inhibitors and MAG or MOG inhibitors. Additionally, mutants can be combined by deletion with other compounds that promote remyelination such as neurregulins (GGF2) and antibodies that promote remyelination, or molecules that degrade proteoglycans to Tat peptides.

Regeneration after SCI is limited due to a variety of obstacles that include the deposition of PGSC in the glial scar, demyelination of axons, lack of trophic support and lack of axonal orientation. A single therapy directed against one aspect of SCI may not be as effective as a combinatorial approach. Fusion proteins with chondroitinase will allow combinatorial therapy with a single protein. The fusion partners for chondroitinase to be constructed in this proposal were chosen among the proteins that have evidence of efficacy in SCI.

The use of a molecule that has the ability to both degrade extracellular matrix glycoprotems and to block or overcome the inhibitory nature of myelin components can be used to improve the ability of damaged neurons to grow or regenerate compared to any of the treatments alone. Proteoglycan degradation molecules can also be advantageously used to provide a method for facilitating access and diffusion of substances to cells or tissues by using at least one enzyme capable of cleaving proteoglycans and preferably degrading chondroitin sulfate proteoglycans. (PGSC).

In accordance with the first aspect of the present invention, compositions are provided comprising a polypeptide comprising a TAT protein transduction domain and a mutant by deletion of chondroitinase ABC 1, wherein the TAT protein transduction domain is anchored to the N-terminus of the mutant by deletion of chondroitinase ABC 1 and the mutant by deletion of chondroitinase ABC 1 is selected from SEQ ID NO: 2 and SEQ ID NO: 3. Embodiments of the present invention therefore include compositions comprising polypeptides cleaving proteoglycans, optionally the compositions may additionally comprise polypeptides that block and / or exceed the activity of neuronal growth inhibitor molecules. The compositions also include molecules for the transduction of polypeptides through cell membranes and the blood-brain barrier. The compositions can be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The compositions can be used in the regeneration of damaged neurological tissue and facilitate the diffusion and transport of therapeutic molecules capable of blocking and / or overcoming the activity of the neuronal growth inhibitory molecule in diseased or damaged tissue.

The embodiments of the present invention include compositions that facilitate the delivery and diffusion of therapeutic or diagnostic agents, and preferably agents that promote the regeneration of nerves and axons, in cells or tissues. The composition includes the use of an enzyme capable of cleaving chondroitin sulfate proteoglycans (PGSC) to increase the diffusion of these agents to cells or tissues of the central nervous system.

The compositions of the present invention comprise chimeric or fusion proteins that may be susceptible of systematic use in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS), and in particular, fusion proteins capable of crossing the blood brain barrier. The fusion protein includes a polypeptide transduction domain, a polypeptide domain capable of cleaving a chondroitin sulfate proteoglycan (PGSC), and optionally a polypeptide domain that blocks and / or exceeds the activity of neuronal growth inhibitor molecules , all of which can be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The various domains of polypeptides can be linked or chemically linked to each other by polypeptide linkers.

Also contemplated are polynucleotides that encode chimeric or fusion proteins susceptible of systematic use in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS) and, in particular, encode fusion proteins capable of crossing the barrier Hematoencephalic The polynucleotides encoding these chimeric or fusion proteins may include a polynucleotide domain that encodes a polypeptide transduction domain, a polynucleotide domain that encodes a polypeptide domain capable of degrading a proteoglycan, preferably cleaving a chondroitin sulfate proteoglycan (PGSC) , a polynucleotide domain that encodes a polypeptide domain that blocks and / or exceeds the activity of neuronal growth inhibitor molecules, or any combination of these domains that can be used in the treatment of spinal cord injuries and related disorders of the central nervous system (CNS). The polynucleotide also includes one or more polynucleotide domains encoding polypeptides that connect the domains of the polypeptide to each other to form the fusion protein.

An embodiment of the present invention is a composition according to the first aspect that facilitates the access and distribution of the therapeutic and diagnostic agent of the composition to the cells, through membranes or through

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the tissues through the use of the enzyme capable of cleaving the PGSCs. The molecules or agents of the composition may include one or more growth factors, including brain-derived neurotrophic factor, insulin-like growth factor, fibroblast growth factor, ciliary neurotrophic factor, glia-derived neurotrophic factor, transforming growth factor , Glial growth factor 2, L1, GM1, Vascular endothelial growth factor, Nerve growth factor, Immunophilins. The molecules of the composition may include fluorescent or contrast agents for image formation. Agents may include cells for transplant stem cells, neurons, others, cells as delivery agents, chemotherapeutic agents, antibiotics, antibody therapies, Nogo receptor antagonists, other chondroitinase enzymes. The fusion protein may facilitate transport or modify the transport of such agents to cells, tissues, and / or other inaccessible locations; and / or enhance penetration rates, penetration distance; or provide a more uniform distribution of the concentration. Modified transport occurs through the use of at least one enzyme capable of cleaving PGSC. The compositions can be used to treat a CNS lesion, preferably the composition is used in the treatment of neuronal damage by a contusion injury.

Chimeric proteins of a proteoglycan degradation domain linked to a polypeptide that block the action of neuronal growth inhibitors such as, but not limited to, a Nogo receptor antagonist domain (NgR27-3ii) or a variant linked to a chondroitinase such as chondroitinase ABC 1 or a chondroitinase variant that has one or more N-terminal amino acids suppressed. The compound may include chimeric proteins of a proteoglycan degradation domain bound to a polypeptide that is a neural cell adhesion promoter such as a neural cell adhesion promoter domain Li or a variant linked to chondroitinase ABC 1 or a variant of Chondroitinase that has one or more N-terminal amino acids deleted. Chimeric proteins may include chimeric proteins of a proteoglycan degradation domain linked to a polypeptide that is a glial cell stimulator, such as, but not limited to, a GGF2 glial cell stimulator or a variant linked to chondroitinase ABC 1 or a chondroitinase variant that has one or more N-terminal amino acids deleted.

A recombinant E. coli expression system and a purification process can be used to produce essentially pure and catalytically active ABCI chondroitinase. These methods can be modified to produce chimeras of degradation molecules of proteoglycans and other agents.

The chimera can be analyzed to determine the chondroitinase enzymatic activity and the specific biological activity of each fusion partner. The methods for measuring chimera activities can be modified for those used to measure chondroitinase activity, including a spectrophotometric analysis, zymography, an HPLC analysis to detect PGSC disaccharide digestion products and an in vitro neurite growth analysis. A neuron growth cone collapse analysis can be used to evaluate NOGO receptor antagonists and a neurite growth analysis can be used to measure Li activity. GGF2 activity can be measured using a Schwann cell proliferation analysis.

The compositions of the present invention can be used for the treatment of spinal cord injuries and in the promotion of axon regeneration. The compositions of the present invention can also be used to promote plasticity, re-growth, repair and / or regeneration of dysfunctional neurons in the CNS that have been damaged as a result of a disease, such as degenerative diseases including Alzheimer's and Parkinson's disease. Advantageously, the use of proteoglycan degradation polypeptides and membrane transducer polypeptides in the compositions of the present invention also promotes the diffusion and access of damaged or diseased tissue to other therapeutic agents that promote the regeneration of neurons.

Description of the drawings

The file of this patent contains at least one drawing / photograph executed in color. Copies of this patent with drawings / color photographs will be provided by the Office upon request and payment of the necessary fee.

In part, other aspects, features, benefits and advantages of the embodiments of the present invention will be apparent with respect to the following description, appended claims and accompanying drawings in which:

FIG. 1 is an illustrative example of an ABCI TAT-chondroitinase construct; the DNA sequence for the entire gene fragment fused with the Tat sequence followed by the 5-glycine linker; the effector and antisense oligonucleotides that have the sequences such as 5'-tatgtatggtc gtaaaaagcgtc gtcaacgtcgtcgtgg tggtggtggtca-3 'and 5'-tatgaccaccaccaccaccacgac gacgttgacgac gcttttt acgaccataca-3 at the end of which are linked at the end of the end of the site ABCI gene cloned into pET15b (Novagen) at the NdeI and BamHI sites. The Tat48 s7 peptide sequence (GRKKRRQRR) is linked to the ABCI sequence by a pentaglycine connector.

FIG. 2 are images of brain sections; (I) illustrating adult rat lobes that were incubated in beta-galactodidase alone (B&D), or with the addition of ABCI Coindroitinase (A, 0.5 U / ml or C, 0.005 U / ml); (II) Eosin Y penetration through the cortex of a cerebral hemisphere treated with Coindroitinase and control

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which shows approximately the same penetration, Eosin Y is a zwiterion, which has a global negative charge at the low pH at which it was used, and is 692 kDa; (III) A saturated solution of Congo Red shows greater penetration through the cortex of a cerebral hemisphere treated with chondroitinase compared to the untreated brain, Congo Red is a negatively charged dye of 697 kDa;

FIG. 3 (A) is a diagram representing the complete GGF protein (GGF2-M1-E422) and the three GGF2 fragments containing the Ig and EGF domains alone and in combination; (B) a schematic presentation of chimeric proteins of a proteoglycan degradation molecule such as ABCI chondroitinase and a gGF2 isoform of the neurregulin gene 1;

FIG. 4 illustrates the structure of (A) an N-terminal ABG NgR27-3ii-chondroitinase chimeric protein with and without a peptide linker or spacer group; (B) an ABG C-terminal NgR27-3ii-chondroitinase chimeric protein with and without a peptide spacer or linker group; (C) an extracellular chimeric protein Li and N-terminal ABCI chondroitinase with and if, a spacer or linker peptide; (D) an extracellular domain chimeric protein Li and C-terminal ABCI chondroitinase with and without a spacer or linker peptide.

FIG. 5 (A) BBB scores of animals with spinal cord injuries treated with chondroitinase (Chondroitinase ABC I), penicillinase or artificial cerebrospinal fluid (aFCE); (B) Parasagittal sections of the spinal cord of injured animals treated with chondroitinase (left column) or penicillinase (right column). Sections were cut at 30 microns. Each set of images contains four parasagittal sections realigned with the rostral medulla on the left and the caudal medulla on the right. The most central section of each set has been removed and replaced below to facilitate visualization. The pairs of images of neuronal degeneration are taken with Weil, anti-GFAP and an aminocoupric dye (from top to bottom). (C) and (D) Distribution of individual BBB scores according to the treatment group. The scores are BBB scores at ten weeks after surgery. Group averages are shown below the data points.

Detailed description

Before describing the present compositions and methods, it should be understood that this invention is not limited to the compositions and associated molecules, methodologies or particular protocols described, as these may vary. It should also be understood that the terminology used in the description is intended to describe only the particular versions or embodiments and is not intended to limit the scope of the present invention that will be limited only by the appended claims.

It should also be noted that, as used herein and in the appended claims, the singular forms "a", "a", "one" and "the" and "the" include the plural reference unless the context state clearly otherwise. Thus, for example, the reference to a "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so on. Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those skilled in the art. Although any method and material similar or equivalent to those described herein may be used in the practice or testing of the embodiments of the present invention, the preferred methods, devices and materials are described below.

"Optional" or "optionally" means that the event or circumstance described below may or may not occur, and that the description includes cases in which the event occurs and cases in which it does not occur.

Enzymes that cleave PGSCs include ABC chondroitinase type I, chondroitinase ABC type II, chondroitinase AC and chondroitinase B, or mammalian enzymes with chondroitinase-like activity, such as hyaluronidase i, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, cathepsins, ADAMT and PH-20, or mixtures thereof.

PGSCs are a family of proteoglycans composed of a nucleus protein and covalently linked sulfated glycosaminoglycans. Polysaccharide chains are cleaved by several enzymes, including a family of chondroitinases. To date, this family of enzymes contains at least four members, chondroitinase ABC I, ABC II, AC and B. Chondroitinase ABC I is an exo-lyase that cleaves both chondroitin and dermatan sulfates. It has been widely used in the study of neuronal regeneration in vitro on PGSC-loaded substrates and more recently in in vivo studies after CNS injury.

The proteins and polypeptides that can be used in the compositions and fusion proteins of the present invention are those that promote plasticity as well as the regeneration of injured or diseased neurons or axons. These regenerating proteins and polypeptides may include cell adhesion proteins, those that stimulate glial cells, and polypeptides that block the inhibitory effect of the proteins that act as axonal growth inhibitors.

The plasticity of the nervous system refers to any type of functional reorganization. This reorganization occurs with development, learning and memory and brain repair. Structural changes that occur with plasticity can include synapse formation, synapse elimination, germination of

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Neurites and may even include strengthening or weakening of existing synapses. Regeneration is generally distinguished from plasticity by the long-term growth of axons in disorganized tracts that is characteristic of regeneration.

Proteins and polypeptides that are capable of blocking the activity of neuronal growth inhibitory molecules may include peptides and polypeptides that block peptide inhibitory properties such as, but not limited to Nogo, MAG, OMgp. Suitable compositions that exceed the activity of neuronal growth inhibitory molecules include, but are not limited to, inhibitors of the Protein Kinase C family, inhibitors and agents of the Rho Kinase family, such as inhibitors of phosphodiesterase, which increase intracellular cyclic AMP and L1. It has been shown that the peptide (NgR27-3ii) inhibits the binding of Nogo66, OMgp, MAG and MOG to membrane bound NogoR and overcomes the inhibitory effects of Nogo on the regeneration of nerve processes.

Proteins and polypeptides that affect cell adhesion or stimulate cells may include, but are not limited to, polypeptides such as Li and GGF2. Li is a neural cell adhesion protein that is a potent stimulator of in vitro neurite growth for which it has been found that the treatment of acute LME in rodents using a soluble form of Li leads to an increase in function recovery neurological GGF2 stimulates glial cells and has been shown to improve clinical outcome measures in a murine experimental allergic encephalomyelitis (EAE) model probably as a result of oligodendrocyte stimulation to promote remyelination.

Peptide sequences capable of crossing the cell membrane useful in the present invention can be obtained from the Tat protein of human immunodeficiency virus type i (HIV-i) (Fawell et al., Proc. Nat. Acad. Sci., 9i: 664-68, 1994). In particular, the Tat peptide may comprise any sequential residue from the motif of the alkaline peptide of the Tat 37-72 protein (Vives et al., 1997) (37-

CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72 (SEQ ID NO: 56)). The minimum number of amino acid residues may be in the range of about three to about six, preferably about three to about five and most preferably about four, that is, the minimum requirement for an alpha helical turn. A preferred embodiment comprises residues of the Tat 4857 protein (GRKKRRQRRR) (SEQ ID NO: 57).

Protemes and Peptides Tat. Tat is an 86 amino acid protein involved in the replication of the human immunodeficiency virus type i (HIV-i). The Tat transactivation protein of HIV-i is effectively absorbed by the cells (Mann and Frankel 1997; Vives et al., 1994), and low concentrations (nM) are sufficient to transactivate an reporter gene expressed from the HIV promoter. i (Mann and Frankel i99i). The exogenous Tat protein is capable of translocating through the plasma membrane and reaching the nucleus to transactivate the viral genome.

It is believed that a region of the Tat protein centered on a group of alkaline amino acids is responsible for this translocation activity (Vives et al., 1997). Cellular absorption and nuclear translocation mediated by Tat peptides have been demonstrated in several systems (Vives, et al., J. Biol. Chem. 272: i60i0-i60i7, 1997; Jones, Genes Dev ii: 2593-2599, 1997) . The chemical coupling of a peptide derived from Tat (residues 37-72) to various proteins results in its internalization in several cells or cell tissues (Fawell et al., Proc Natl Acad Sci USA 9i: 664-668, 1996, Anderson, et al., Biochem Biophys Res Commun i94: 876-8884, 1993, Fahraeus et al., Curr Biol 6: 84-9i, 1996, Nagahara et al., Nat Med 4: i449-i452, 1998). A synthetic peptide consisting of Tat alkaline amino acids 48-60 with a cystem residue coupled to the C-terminal to fluorescema maleimide is translocated to the cell nucleus as determined by fluorescence microscopy (Vives et al., 1997). In addition, a fusion protein (Tat-NLS-p-Gal) consisting of tat amino acids 48-59 fused by their amino terminal end to amino acids 9-i23 of p-galactosidase translocates to the cell nucleus of a independent of cytosolic factors, dependent on ATP (Efthymiadis et al., 1998).

Chimeric proteins, also referred to in the art as fusion proteins, are tubular proteins that combine parts of at least two or more precursor proteins or peptides. Chimeric proteins can be produced by recombinant technology, that is, by fusing at least a part of the coding sequence of one gene to at least a part of the coding sequence of another gene. When desirable, one or more genes for the linker peptides can be fused with the gene coding sequence for the other polypeptide domains of the fusion protein. The fused gene can then be used to transform a suitable organism such as, but not limited to, E. coli or CHO cells that then express the fusion protein.

The genes encoding mutants by N-terminal or C-terminal deletion of polypeptide domains of fusion proteins can be used in constructs for the expression of fusion proteins. Preferably, the deletion mutants generated maintain their proteoglycan catalytic degradation activity, blocking activity, growth activity or transduction activity. Deletion mutants generated from proteoglycan degradation molecules such as the ABCI chondroitinase enzyme in which the mutant does not have a certain number of amino acids from the N- and / or C-terminal end are those that retain some proteoglycan degradation activity. N-terminal chondroitinase deletions such as chondroitinase ABC I maintain a

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histidine tag that is anchored to the N-terminal end. It is expected that a TAT-mutant fusion DNA construct by chondroitinase ABC I deletion can be expressed without the removal of the TAT polypeptide during expression. For example, a TAT peptide fused at the N-terminus of the mutant by ABCI-NA20 or ABCI-A60 deletion. Fragments of polypeptides, such as those shown in FIG. 3 for GGF2 in the construction of chimeric fusion proteins.

Catalytically active deletion mutants of ABCI chondroitinase can be prepared by suppressing 20 or 60 amino acids respectively from the N-terminus of the mature ABCI protein. These deletion mutants can be used for the construction of the N-terminal fusion chimeric protein. Detailed comparative biochemical studies can be performed to determine the efficacy of mature ABCI chondroitinase against various mutants by deletion in fusion compositions and proteins with respect to substrate specificity, substrate binding and tissue penetration.

An ABCI chondroitinase mutant can be prepared that has a native protein structure, but lacks catalytic activity as a null or negative control for bioanalysis and LME studies. Based on the crystalline structure of ABCI chondroitinase, a site-specific mutant called H501a and Y508a can be prepared to inactivate the catalytic activity at the supposed active site. Such mutants can be tested for inactivation of catalytic activity and SEC for comparison with the wild-type enzyme. If the null activity mutant is created successfully it will provide a negative control for the various fusion proteins for use in bioanalysis and, ultimately, in animal studies with SCI.

An E. coli expression system can be used to make chondroitinase using PET expression vectors (Novagen). The ABG GGF2-chondroitinase fusion protein can be expressed in E. coli. Constructs for Tat-mutant fusion proteins by chondroitinase deletion, Tat-GGF2 fusion proteins or Tat-chondroitinase-GGF2 fusion proteins can be expressed from E. coli. Other fusion proteins can be expressed in CHO cell lines.

Table 1 illustrates several non-limiting components that can be used in compositions described herein in the form of a fusion or chimeric molecule. The chimeric composition or molecule described may include one or more of the molecules of Table 1 and all the features of claim 1. In the case of chimeric molecules, one or more connecting segments, preferably polypeptides, are used.

Table 1 Components of the compositions

 Transduction Molecules

 Proteoglycan degradation molecules Therapeutic, diagnostic molecules, receptor antagonists

 Residues 48-57 of the Tat protein Alkaline motif of the prote Rev of HIV-1 Alkaline motif of the prote Rev of HIV-1 Protema VP22 of the Herpes simplex virus

 Any agent that degrades extracellular matrix glycoprotems including: Crondroitinase ABC I, Chondroitinase ABC II, Chondroitinase AC, Chondroitinase B, Hyaluronidase 1, Hyaluronidase 2, Hyaluronidase 3, Hyaluronidase 4, deletion mutants and / or PH20 molecule replacement listed above that maintain enzymatic activity. Any peptide that blocks the inhibitory properties of Nogo, MAG, OMgp including: Nogo peptide 1-40 * Any component of Nogo peptide 1-40 that maintains the ability to block Nogo's inhibitory properties Other peptides that block Nogo Antibodies that recognize Nogo Peptides that block MAG Antibodies that recognize MAG Peptides that block OMgp Antibodies that recognize OMgp Antibodies that recognize the Nogo-66 receptor Peptides that block the P75 receptor Antibodies that recognize the neurotrophin receptor p75 Peptides or antibodies that block other Nogo receptors , MAG and / or oMgp Peptide that exceeds the inhibitory properties of Myelin, Nogo, MaG, OMgp including: Inhibitors of the protein kinase C family * Inhibitors of the Rho kinase family Agents that increase the concentration of intracellular cAMP L1

* Peptide 1-40 of Nogo: NEP-1-40 of 40 aa human (Acetyl-RIY KGV IQA IQK SDE GHP FRA YLE SEV AIS EEL VQK YSN S-amide corresponding to aa 1-40 of Nogo-66

In FIG. 4A and FIG. 4B, a schematic illustration of the chimeric fusion proteins is set forth.

5 A peptide component of any column of Table 1 that may be connected by means of an oligopeptide connector well known in the art such as a glycine-rich peptide, for example Gly-Gly-Gly-Gly-Gly, or connectors prepared including any of the natural amino acids as well as substituted amino acids or beta or gamma such as 4-aminobutmco acid or 6-aminocaproic acid. Other connectors may also be used, including but not limited to, alkyl diamines, amino, or alkyl diols. Preferably, the Transduction component 10 of the fusion protein is in a terminal position in the chimeric protein. Other examples of common connectors may include, but are not limited to Gly-Gly-Ala-Gly-Gly, connectors rich in Gly / Ser (eg Gly4Ser3), or connectors rich in Gly / Ala. Additionally, the connectors may have any length and design to promote or restrict the mobility of the components of the fusion shield.

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The incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids or one or more D-amino acids to the peptides (or other components of the complexes) described herein (referred to hereinafter as " D-peptides ") is advantageous in many different ways. Peptides containing D-amino acids show increased stability in vitro or in vivo compared to homologs containing L-amino acids. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing a better oral, transepithelial and transdermal supply of drugs and linked conjugates, better bioavailability of complexes that can penetrate the membrane, and intravascular lives. Prolonged interstitials when such properties are desirable. The use of D-peptides can also enhance the transdermal, oral and transepithelial supply of connected drugs and other loading molecules.

In a spinal cord injury, the axons of the ascending sensory neurons and descending motor neurons rupture, which can lead to loss of sensation and paralysis. These axons do not regenerate satisfactorily leading to permanent disability. A scar envelops the site of the injury that is believed to encapsulate the area of fragile tissue, stabilizes the blood-brain barrier and avoids an overwhelming cascade of uncontrolled tissue damage. This scar is made up of hypertrophic glial cells and an extracellular matrix (ECM). Chondroitin sulfate proteoglycans (PGSC) are an important component of the scar. They are expressed by glial cells and deposited in the MEC in regions of rupture of the blood-brain barrier. In vitro evidence demonstrates that these PGSCs are potent inhibitors for axon growth and without wishing to be limited by theone, they are believed to contribute to the failure of the spinal cord axons to regenerate and reform functional synapses. In vivo studies have shown that regenerating axons are able to grow inward, and even outward, from the scar.

PGSCs and white matter components are generally accepted as molecular barriers that neurons must overcome in order to regenerate and restore functional connections after injury. Transplanted adult sensory neurons placed distally with respect to a scar in formation can be robustly regenerated even along the pathways of white matter degeneration, however, regeneration abruptly ceases when the axons internalize the proteoglycan it contains. the glial scar Treatment of the CNS white matter with chondroitinase increases the ability of neurons to grow in these substrates.

The tissues of the central nervous system are strongly compacted with cells and have a limited extracellular space. The extracellular matrix proteins and carbohydrates provide osmotic and loading forces as well as specific and non-specific binding sites that can prevent the penetration of therapeutic agents. Enzymatic cleavage of these matrix and cell components can later facilitate the access of compounds or cells through tissues. A proteoglycan degradation molecule such as Chondroitinase ABC I can be used which is an enzyme that digests chondroitin sulfate proteoglycans to promote the diffusion of therapeutic molecules to the CNS. Tat peptides transport biologically active loading molecules covalently bound to the cytoplasm and cell nuclei. In the case of a fusion protein that has a polypeptide domain of protein transduction such as the HIV protection protein, therapeutic molecules for axon regeneration through the blood brain barrier can be supplied.

The treatment of lesions of the LME model, preferably the injury of the contusion model, can be used to determine the degree of regeneration and functional recovery obtained by the compositions and the method described herein. The degree of functional recovery can be demonstrated by a better conduction of the corticospinal tract, a better removal of the tape, a walk by a bar, a walk by a grating and placement of the legs after treatment with chondroitinase of a spinal cord injury. The improvement of the motor ability as well as the autonomic functions can also be used: bowel, bladder, sensory and sexual function as measures of improvement of function and can be related to the molecular structure and components of the compositions of The present invention.

In addition to the ability of chondroitinase to improve regeneration, chondroitinase digests components of the perineuronal network (RPN). The density of RPN is variable within the CNS and is particularly dense in the somatosensory, auditory, visual and hippocampal cortex. It has also been shown that RPN is dense around spinal motor neurons. The authors of the present invention have discovered dense RPN within the spinal horn. Digestion of RPN can increase plasticity within the hippocampus and visual cortex. Plasticity within intact systems in incomplete SCI, especially in the region of central pattern generators or in the reticular nucleus, can support the function of damaged or destroyed systems. The promotion of plasticity in these systems may be a different or additional mechanism to regeneration through which chondroitinase can improve function after CNS injury. In addition, regeneration and plasticity can work in concert to affect recovery after the injury; In fact, it has been shown that the corticospinal tract is critical for the modulation of spinal cord plasticity.

The recovery of neurological function after a contusion injury in the CNS or a disease state can be promoted by administering fusion proteins to cells, a tissue, or a subject that have

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damaged or diseased neurons, whether the injury or illness is immediate or if it is long lasting.

The fusion proteins herein are administered in an amount effective to degrade PGSCs and thereby promote the recovery of neurological function. Once the proteins or polypeptides of the compositions have been purified to the desired degree, they can be suspended or diluted in a physiological carrier or excipient suitable for the treatment of SCI. In models of SCI, effective intrathecal doses in rats have been approximately 0.06 units at alternate days for 14 days. A dose for a 70 kilogram human being can be approximately 17 units. At approximately 100 units / milligram, this will be equivalent to approximately 170 micrograms. Doses of up to 20 units appear safe in rat. Compositions that include a proteoglycan degradation molecule can be injected as part of a fusion protein diluted in a pharmaceutically acceptable carrier or excipient, generally at concentrations in the range of 1 µg to 500 mg / kg of host. The administration of the agent can be carried out by injection in stroke, intravenous administration, continuous infusion, sustained release of implants or sustained release drugs. Administration may be by injection, for example intramuscular, peritoneal, subcutaneous, intravenous. Oral administration may include tablets or capsules, preferably oral dosing is a sustained release formulation for administration once or twice a day. Percutaneous administration may be once a day, and preferably less than once a day. Administration to the human patient or other mairnfero subject may continue until a measurable improvement in autonomic or motor function is achieved in the patient.

PTD chondroitinase fusion proteins can be administered with a suitable pharmaceutical carrier. The administration of the chimeric protein compositions of the present invention may be topical, local or systematic. The chimeric fusion proteins can also be secreted by genetically modified cells, preferably a chimeric fusion protein can be secreted by genetically modified cells that are implanted, free or in capsules, at or near the site of CNS injury.

Once the compositions are administered, the degradation of the PGSCs eliminates the inhibitory molecules that block the growth of neurites, and allows the regeneration of neurites in the affected area. Chondroitinase ABC I is an endo and exo lyase that cleaves both CS and DS. The removal of CS and DS from the glial scar allows the regeneration of neurite growths in the damaged area.

Mixtures of any of these fusion polypeptides may be used to provide therapeutic treatment for CNS lesions and disorders that may include but are not limited to contusion injury, traumatic brain injury, stroke, multiple sclerosis, brachial plexus injury, amblyophage spinal cord injuries. Spinal cord injuries include diseases and traumatic injuries, such as the crushing of neurons caused by a car accident, coffee, bruise or gunshot wound, as well as other injuries. The practice of the present methods can confer weather benefits to the treated mammal, providing clinically relevant improvements in at least one of the subject's motor coordination functions and sensory perception. Clmically relevant improvements can range from a detectable improvement to a complete restoration of a damaged or lost CNS function.

The regeneration of nerve cells in the affected area of the CNS allows the return of motor and sensory function. The clinically relevant mussel ranges from a detectable improvement to a complete restoration of altered or lost nerve function, varying with individual patients and lesions.

A variant of a protein or fragments thereof refers to a molecule substantially similar to the complete protein or to a fragment, which possesses a biological activity that is substantially similar to a biological activity of the protein or complement fragments. A molecule is substantially similar to another molecule if both molecules have substantially similar structures or if both molecules have a similar biological activity.

Variants of complement proteins or their fragments are produced by chemical or recombinant means. Variants of polynucleotides constructed to express fusion proteins can also be prepared. Variants may include, for example, deletions, or insertions or substitutions, of amino acid residues within the amino acid sequence, or deletion, substitution or insertion of nucleic acids of a sequence encoding a fusion protein or a polypeptide domain of the concrete fusion protection. For example, in some cases the removal of one or more amino acid residues from a chondroitinase polypeptide can be performed without significant change in its PGSC degradation activity. Substantial changes in functional properties such as proteoglycan degradation or blocking activity against axon growth inhibitors are made by selecting substitutions that are less conservative, that is, that differ more significantly in their effect on the maintenance of the structure, the load or hydrophobic character of the main chain of the peptide in the substitution zone.

It is not expected that the majority of deletions, insertions and substitutions will produce radical changes in the characteristics of the protein molecule; However, when it is difficult to predict the exact effect of the substitution, deletion or insertion before performing them, a person skilled in the art will appreciate that the effect will be evaluated by routine scrutiny analysis. For example, a change in the regeneration character of the axon of the molecule

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Polypeptide, by means of a greater or lesser degradation of the proteoglycan, can be measured with functional recovery assays as well as HPLC analysis to detect disaccharides from PGSC digestion.

The use of a proteoglycan degradation molecule and an HIV tat protein, or a tat derived polypeptide, in the form of a fusion protein can supply a molecule of interest to the CNS or a site in the CNS where the damage has occurred of the neuronal tissue due to disease or trauma. In particular, the site of damage to the CNS is where scarring has occurred as a result of a contusion injury. The molecule of interest, optionally referred to as a loading agent or molecule, may be a therapeutic molecule that promotes plasticity, axon growth, a diagnostic molecule or a proteoglycan degradation molecule. The fusion protein that has an HIV protein transduction protein allows the therapeutic molecules for axon regeneration to be delivered through the blood-brain barrier or the proteoglycan degradation molecule can be supplied to the cells to degrade the Proteoglycan cell stores and promote axon regeneration.

The transport polypeptides of this invention may be advantageously anchored to loading molecules by means of chemical cross-linking or by means of genetic fusion. A single terminal system residue is a preferred means of chemical crosslinking. According to some preferred embodiments of this invention, the carboxy end of the transport radical is genetically fused with the amino end of the charge radical. The embodiment of the present invention consists of an amino-terminal methionine followed by tat 47-58 residues, followed by a chondroitinase polypeptide.

It will be appreciated that the 86 complete amino acids that constitute the tat protein may not be necessary for the absorption activity of tat. For example, a protein fragment or a peptide can be used that has less than 86 amino acids, but shows absorption in cells and / or can cross the blood-brain barrier, (a functionally effective fragment or portion of tat). For example, the tat protein that contains residues 1-72 may be sufficient for absorption activity and residues of tat 1-67 may mediate the entry of a heterologous protein into the cells. The synthetic peptide containing residues of tat 1-58 may have absorption activity.

The tat peptide may be a unique (ie, continuous) amino acid sequence present in the tat protein or it may consist of two or more amino acid sequences that are present in the tat protein, but in the natural protein they are separated by other sequences of amino acids. As used herein, the tat protein includes a sequence of naturally occurring amino acids that is the same as that of the natural tat protein, its functional equivalents or functionally equivalent fragments thereof (peptides). Such functional equivalents or functionally equivalent fragments may possess absorption activity into the cell or through the blood-brain barrier that is substantially similar to that of the natural tat protein. The tat protein can be obtained from natural sources or can be produced using genetic engineering techniques or chemical synthesis.

The amino acid sequence of the naturally occurring HIV protease tat can be modified, by addition, deletion and / or substitution of at least one amino acid present in the natural tat protein, to produce the modified tat protein (also referred to herein) protema or polypeptide of tat). Therefore, tat protein analogs or modified tat peptides with greater stability can be produced using known techniques. Therefore, tat proteins or peptides may have amino acid sequences that are substantially similar, but not identical, to that of the naturally occurring tat protein or portions thereof. In addition, cholesterol or other lipid derivatives can be added to the tat protein to produce a modified tat that has a greater membrane solubility.

The natural HIV-1 tat protein has a region (amino acids 22-37) where 7 of the 16 amino acids are a system. These cystem residues are capable of forming disulfide bonds with each other, with cystem residues of the region rich in cystem of other proteinaceous molecules and with cystem residues of a loading protein or the loading radical of a conjugate product. Such a formation of disulfide bonds can cause the loss of the biological activity of the charge. In addition, even if there is no possibility of binding disulfide to the loading radical (for example, when the loading protein does not have systemic residues), the formation of disulfide bonds between transport polypeptides can lead to the aggregation and insolubility of the polypeptide of transport, the conjugate product of transport-loading polypeptide, or both. The tat system-rich region can be suppressed to prevent the formation of disulfide bonds and prevent the aggregation and insolubility of the transport polypeptide, the transport-load polypeptide conjugate product, or both.

Chondroitinase is also able to promote plasticity in the CNS regions with a significantly dense RPN, including cortex, tectum, hippocampus and spinal cord. It is reasonable that a combination of effects, including regeneration, outbreak and plasticity, is responsible for the improvement of function after SCI with treatment with chondroitinase or treatment with fusion molecules that include chondroitinase or other proteoglycan degradation molecule. .

NbR27 -311: Nogo is a high molecular weight myelin component that inhibits the growth of neurites. The amino terminal region (Nogo66) is the part of the molecule that is specifically associated with the inhibition of

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neurite growth Expression cloning methods revealed that the Nogo66 (NgR) receptor is a GPI-anchored glycoprotem expressed primarily in neurons. NgR interacts not only with Nogo, but also with other myelin-associated inhibitors such as MAG and MOG. Because of its central role in the inhibitory properties of myelin in neurons, NgR has been the target of approaches to elicit antagonism about its interactions with its ligands. The soluble segment of NgR interacts with Nogo66, MAG and MOG. The region encompasses residues 27-311 and this NgR fragment can therefore act as a single receptor that will interfere with the inhibition of myelin-associated neurons. If NgR27-3ii is linked to a proteoglycan degradation molecule such as ABCI chondroitinase to form a chimeric fusion protein, it is expected that the domain of the NgR27-3ii polypeptide of the fusion protein will limit the inhibition of neurite growth associated with Myelin and promote axonal regeneration in the regions digested with chondroitinase from a spinal cord injury.

For the cloned product (NgR27-3ii), the precise region of interest, or the fragments may be derived from the initial clone. The biological activity of NgR27-3ii, its fragments and fusion polypeptides that include NgR27-3ii can be confirmed using an in vitro analysis for the collapse of growth cones in neurons. The analysis of the growth cone collapse has been established, and the addition of MAG or Nogo66 causes the collapse of the growth cones in a dose-dependent manner. If NgR27-3ii, its fragments and fusion polypeptides including NgR27-3ii are active, it is biologically active, in that case they must inhibit the collapse of the growth cone mediated by MAG and Nogo66. Growth cone collapse data can be collected for inspection of GRD neuron photomicrograffas in response to Nogo66 and MAG.

Polypeptide Li is a member of the immunoglobulin superfamily of cell adhesion molecules and is expressed in growing axons, glial progenitor cells and Schwann cells throughout life, but only has a limited CNS expression. Li interacts with itself and with other extracellular molecules, such as the FGF receptor to promote fasciculation and neurite growth. The expression of Li is generally associated with a permissive environment for axonal regeneration, the precise region of interest, or Li fragments may derive from the initial clone. For example, Schwann cells expressing Li support peripheral nerve regeneration and axonal growth is observed in the optic nerves of transgenic mice expressing Li in astrocytes but not in wild-type optic nerves. Fibroblasts designed to express Li support axonal growth when they are transplanted to the spinal cord. Finally, a soluble form of Li linked to Fc promoted functional recovery after an acute SCI. If Li is attached to a molecule of degradation of proteoglycans such as ABCI chondroitinase in a fusion protein, it is reasonable to expect that the fusion protein promotes axonal regeneration in the chondroitinase digested regions of a spinal cord injury.

Neurregulins and their receptors comprise a diverse system of growth factors and tyrosine kinase receptors that have been shown to be essential for organogenesis in the CNS, muscle epithelium and other tissues. GGF2 is a soluble isoform of the neurregulin i gene. Initially it was characterized as a Schwann 32 cell mitogen, but subsequent studies have shown direct actions on oligodendrocytes, neurons and other types of cells. GGF2 decreases demyelination and inflammation and improves remyelination in a mouse model for multiple sclerosis. Based on these results, it is reasonable to expect a recombinant human GGF2 to be a potential treatment for demyelination associated with SCI. When GGF2 is linked to a proteoglycan degradation molecule such as ABCI chondroitinase, it is reasonable to expect axon remyelination to be promoted in chondroitinase digested regions of an SCI.

Table 2. Examples of non-limiting GGF2 fragments for expression in E. coli for use in compositions and compositions of fusion polypeptides

 Construct

 Domain Sec Nucleotides Sec amino acids Molecular Weight

 GGF2-FL

 Full GGF2 Mi-E422 46 kDa

 GGF2 / i

 Ig domain + EGF domain 748-i206 L250-C402 i6.8 kDa

 GGF2 / 2

 Ig domain to end C 748-i266 L250-E422 i9 kDa

 GGF2 / 3

 Domain EGF i048-i206 T350-C402 5.7 kDa

The efficacy of a composition of the present invention can be evaluated using a rat LME model validated at three different levels of LME severity.

A molecule of proteoglycan degradation such as ABCI chondroitinase in small quantities is available commercially as a naturally occurring enzyme (Seikagaku Corporation) and can be manufactured

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by a recombinant production system so that it has essentially the same activity as the enzyme purified from Proteus vulgaris. Genomic DNA can be isolated from Proteus vulgaris using the DNeasy Tissue kit (Qiagen). PCR primers can be synthesized with a Ndel restriction site at the 5 'end and a BamHI site at the 3' end having the sequences 5'-CAT ATG GCC ACC AGC MT CCT GCA TTT G-3 '(F2) and 5'-GGA TCC TCA AGG GAG TGG CGA GAG-3 '(R), respectively, to synthesize the mature protein. The 3.0 kb PCR products can be ligated to the pCR 2.1 vector (TOPO cloning kit, Invitrogen) and transformed into competent DH5a cells (Invitrogen). Plasmid DNA can be isolated from numerous clones screened by digestion with the restriction enzyme EcoRI. The integrity of a gene prepared in this way can be confirmed by repeated DNA sequencing.

The ABCI chondroitinase sequence can be cloned into a PET vector (Novogen) for E. coli expression. After induction of IPTG gene expression, bacteria can be lysed by sonication with concomitant ABCI chondroitinase extraction with Triton. X-114 / PBS. The authors of the present invention discovered that the recombinant ABCI chondroitinase mayone was in the cytosolic fraction of the bacterial cell lysate product, which allowed the development of an ABCI chondroitinase purification protocol that produces an enzyme with high activity at high yields The protocol includes cation exchange chromatography as a capture stage and gel filtration as a refining stage. After these stages, ABCI chondroitinase reaches a purity of ~ 95%. Anion exchange membrane filtration (Intercept Q, Millipore) can be used for the removal of endotoxin and host DNA. This stage is expected to eliminate approximately 75% of the endotoxin. After filtration, ABCI chondroitinase can be dialyzed in a volatile buffer, pH 8.0 and lyophilized to dryness. The final product is stable at -70 ° C for long-term storage. The purified cABCI is a highly alkaline protein with pI ~ 9.5 as determined by IEF-PAGE analysis of the samples of the crude cell lysate product.

A variety of analytical methods can be used to compare the enzymatic activity of the recombinant version of ABCI chondroitinase with that of a commercially available form of the enzyme (Seikagaku Corporation) purified from Proteus vulgaris. The methods can be adapted to evaluate the activity of fusion proteins including proteoglycan degradation polypeptides such as chondroitinase. Specific measurements of activity were obtained using an accepted spectrophotometric analysis that measures the change in absorbance due to the production of reaction products from proteoglycan degradation. The recombinant form of ABCI chondroitinase has approximately 25% more specific activity than Seikagaku ABCI chondroitinase. Exclusion chromatography by size can be used to compare the hydrodynamic properties of enzymes. Elution profiles for the recombinant enzyme were identical to that of the natural enzyme.

A form of zymography can be used to further characterize the enzyme and can be adapted for the characterization of fusion proteins. Polyacrylamide gels can be polymerized in the presence of aggrecan, a substrate for ABCI chondroitinase. Enzymatic samples can be resolved on gels impregnated with aggrecan by electrophoresis in the presence of SDS. The gels can then be subjected to a renaturation stage where the SDS is extracted and the enzymes allowed to replicate. The enzyme is retracted and the activity is recovered, then the aggrecan in the gel is digested and the resulting carbohydrate loss in that region of the gel can be visualized by a specific carbohydrate stain. A similar loss of carbohydrate in the gel is expected for active forms of a fusion protein that will include a portion of proteoglycan degradation polypeptide. In the case of recombinant ABCI Chondroitinase, its activity can be visualized as a clear point on the zymogram. The results of the zymography were compatible with the spectrophotometric analysis that shows that the recombinant form of chondroitinase ABCI has the same or greater specific activity than the natural form.

HPLC methods can be used to detect disacarids with four and six sulfates (A4DS and A6DS, respectively) released as a result of digestion with ABCI chondroitinase of PGSC. The two disacarids can be effectively resolved by anion exchange chromatography. The HPLC analysis has been validated demonstrating that the quantification of A4DS and A6DS of the chromatograms provides a linear relationship with the amounts injected into the HPLC. The production of A4DS and A6DS of PGSC digestion is directly related to the amount of chondroitinase specific activity determined by the spectrophotometric analysis described above. This analysis can be used as a sensitive and accurate method to independently quantify the A4DS and A6DS released by digestion with chondroitinase of a variety of substrates and can also be used to determine the activity of chondroitinase polypeptides in a fusion protein.

Another functional analysis that can be performed to characterize the activity of the proteoglycan polypeptide is that in which dorsal root ganglion neurons (GRD) are grown on aggrecan or treated with aggrecan with a proteoglycan such as ABCI chondroitinase. It is expected that aggrecan-grown neurons fail to adhere to plaque or extend axons. In contrast, neurons cultured on aggrecan treated with a proteoglycan degradation polypeptide such as ABCI chondroitinase in a composition or as part of a fusion polypeptide were expected to adhere to the surface and extend the axons. It is believed that the intense growth of the axon, which is observed for chondroitinase ABCi is due to the digestion of carbohydrates in the aggrecan nucleus protein that creates a more permissive substrate for the growth of the

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axon

The LME rat contusion model is a clinically relevant model and can be used to evaluate the efficacy of fusion proteins and other compositions of the present invention to promote axon regeneration. With an SML due to bruising, the cells are destroyed, hemorrhage occurs and inflammation begins. The destroyed cells are eliminated by macrophages and a reactive gliosis begins. A chemical cavity is formed and the gliosis matures to a glial scar. The myelin in the area is destroyed and many local neurons that are not destroyed remain in a demyelinated state.

The LME fOrceps compression model is a developed and characterized contusion model. This model has been validated and results in injuries that are very similar to those of the most widely used impactor models. The model may involve compression with fOrceps at the vertebral level T9 / T10. The fOrceps compresses the medulla to a width of 0.9, 1.3 or 1.7 mm for 15 seconds. These three levels of compression allow serious, mild or moderate injury. This model has been validated using the open field locomotion test and the scoring system of Basso, Bresnahan and Beattie (BBB). It was also characterized histologically. Behavioral tests and BBB scoring demonstrated that the fOrceps produces a highly reproducible lesion, with a recovery similar to that observed with impactor models. These tests and punctuation data demonstrate that the compression model with fOrceps is comparable to other contusion models and reproducible enough to be used as an experimental LME model and can be used for the evaluation of compositions of the present invention.

The tissue of animals injured by compression with fOrceps can be processed histologically to examine the preservation of white matter, the glial scar and the formation of cysts. As with other models of LME by contusion, a central cyst is formed after the injury, with a size that increases with the increase in the severity of the injury (decrease in the space of the fcepceps). Around the cyst a glial scar is formed that is characterized by astrogliosis (GFAP), macrophage activation and myelin loss.

Several experimental procedures will be illustrated with reference to the following non-limiting examples.

Example 1

This example illustrates how a proteoglycan degradation molecule can be administered and studied and demonstrated that it shows a functional improvement in animals that have a bruised model lesion.

The LME fOrceps compression model is a developed and characterized contusion model. This model has been validated and results in injuries that are very similar to the most widely used impactor models. The model may involve compression with fOrceps at the vertebral level T9 / T10. The fOrceps compresses the medulla to a width of 0.9, 1.3 or 1.7 mm for 15 seconds. These three levels of compression allow serious, mild or moderate injury.

The rats were injured with the compression model with fOrceps in the T9 / T10 vertebra. At the time of surgery, an intrathecal catheter was placed for chondroitinase delivery. The animals were treated every day for a week and then on alternate days for a week with 0.06 U / dose of chondroitinase ABC I (Seikagaku), penicillinase or artificial cerebrospinal fluid (aFCE). Doses and controls were obtained from Bradbury et al., 2002. The behavior was assessed by open-field locomotive tests and the BBB scoring system on Day 2 and then weekly after the injury for ten weeks.

FIG. 5A shown are the average BBB scores for animals treated with chondroitinase ABC I, penicillinase and aFCE. Rats treated with aFCE or penicillinase were recovered to an average BBB score of approximately 4. Rats treated with chondroitinase were recovered to an average BBB score of approximately 8. Several animals recovered scores greater than 10, indicating a supraspinal entry. Chondroitinase scores were significantly different from those of the two control groups by ANOVA and post hoc Tukey.

The tissue of these animals was immunohistochemically processed to determine the glial fibrillar acid protein (GFAP) to evaluate the overall scar architecture. The tissue was also stained with a Weil dye and a silver degeneration dye to assess the degeneration of myelin and neurons, respectively. Interestingly, no obvious differences in these parameters were observed between experimentally treated and control tissues.

In FIG. 5B the large lesion comprises several segments and the preservation of the ventral medulla. Weil's staining revealed an intense demyelination in treated and untreated animals. The GFAP images (middle set) demonstrate the extent of the scar that forms after the injury with the fOrceps. The amino-cupric silver degeneration staining of the lower part shows the vast neural degeneration that extends rostrally and caudally after the injury. Again, no obvious differences were observed between the chondroitinase treated and control tissues.

Additional preliminary experiments have been performed at moderate injury levels and a

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significant improvement in locomotive activity in the open field with chondroitinase treatment. Animals that received ABCI chondroitinase recovered to an average BBB score of 9.1 at ten weeks after the injury, compared with 7.1 for penicillinase controls. The consistency of the data (ETM of 0.6 and 0.3, respectively) and the region of the scores on the BBB scale make this 2-point change not only statistically significant, but also clinically significant. A score of 9 indicates plantar placement with weight support or frequent weight support consistent with dorsal staggering, while a score of 7 indicates the movement of each joint in the hind limb but without weight support and without consistent limb scanning . An examination of the individual animal scores at ten weeks shows that 6 of 12 animals in the chondroitinase group recovered to scores of 9 or more, while only one of 12 animals in the penicillinase group recovered to a score of 9. A animal from each group was removed from the analysis because its score never exceeded more than 2.5, indicating a severity of the injury outside the model standards. FIG. 5C and FIG. 5D include a scatter plot of scores at 10 weeks for each animal in the penicillinase and chondroitinase treatment groups for moderate injury. Group averages are shown below.

The results show in this well-controlled study that chondroitinase improves locomotive function in the open field in rats that were injured with the compression model with forceps in the T9 / T10 vertebrae. Animals were recovered to average BBB scores of 9.7 and 9.9 for the penicillinase and chondroitinase groups, respectively. This study demonstrates a significant effect on the levels of severe and moderate injuries, but not on the levels of minor injuries. No significant differences were observed between the groups in the study of mild injury (forceps at 1.3 mm). It is not clear if chondroitinase is not effective with mild lesions, if chondroitinase makes changes that are not adequately analyzed by means of the open field locomotive test, such as stride length, leg placement, functions sensory or autonomous. The preliminary analysis of animal histology in this study confirmed the placement of catheters and lesions in each animal. Ongoing experiments that sacrifice animals at time points after injury with and without chondroitinase treatment will characterize the PGSC content of the scar and the effects of chondroitinase digestion on the expression of the munon antigen. Future experiments will expand the behavioral testing platform and examine the spinal cord to determine the evidence of regeneration with tracing of the tract.

Two acute toxicity studies were conducted in rats. The first was an intravenous (IV) study in which rats 0, 0.2, 0.775 or 7.775 mg / kg of ABCI chondroitinase were injected. In the second study, intrathecal catheters (IT) were placed on the spinal cord using the same methods used in the studies of SCI in animals and 0.06, 0.6 and 6.0 ABCI chondroitinase units were delivered through the IT catheters. These doses were 1, 10 and 100 times higher than the estimated local ABCI chondroitinase concentrations achieved with IT catheters in the LME experiments. Animals were monitored for pain and stress and body weights were obtained daily. No apparent reactions were observed during or immediately after administration of ABCI chondroitinase. No swelling, inflammation, bruising or necrosis was observed at the injection site for experiment IV. No changes in body temperature were observed in animals treated via IT. No alterations in feeding, grooming or vocalization were observed. Animals were evaluated to determine motor behavior in an open pool. No abnormalities were observed by animal care staff or behavioral specialists. The animals showed no signs of joint tenderness or swelling. There were no significant differences in weight change between treatment groups. The results demonstrate that treatment with ABCI chondroitinase is not associated with acute toxicity using doses IV and IT substantially greater than effective IT doses.

Example 2

This example describes the preparation of the Nogo receptor agonist domains, the L1 neural cell adhesion protein and the GGF2 polypeptide, which can be used for the compositions and fusion proteins described herein.

A soluble portion of the human Nogo receptor comprising amino acids 27 to 311 (NgR27-311) was selected, as it has been shown to inhibit the binding of Nogo66, MAG and MOG to membrane bound NgR. Primers that flanked this region were designed, and RT-PCR was performed using human hippocampus RNA (BD Biosciences). The 1.05 kb region was amplified and purified successfully.

L1 was prepared through a CHO cell line from Dr. Melitta Schachner's laboratory that secretes human L1 as a fusion protein with human Fc (L1-Fc). The cells were grown in rolling bottles and then L1-Fc was purified from the conditioned medium using affinity column chromatography with protein A. The purity of L1-Fc was evaluated by SDS-PAGE and a single band was observed. with the appropriate molecular weight. The biological activity of L1-Fc was confirmed using a neutic growth analysis. Tissue culture plates were coated with poly-L-lysine or L1-Fc and then cerebellar granular cells were isolated from 10-day-old rats and placed in culture on the substrates. It was observed that plaque-grown neurons on the L1-Fc substrate exhibited a substantial number of long neurites compared to the controls of the polylysine substrate. These results demonstrate that the L1-Fc produced is biologically active in promoting the growth of neurites. This neural growth analysis will be used to evaluate the biological activity related to the L1 portion of the ABCI chondroitinase fusion protein.

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A CHO cell line was obtained that secreted a soluble form of complete GGF2 glycoprotem from CeNes. Comprehensive optimization of means and methods of purification to obtain essentially pure and biologically active GGF2 was completed. Several analytical methods were developed to characterize GGF2 including SDS-PAGe, isoelectric focusing, peptide mapping and carbohydrate analysis. For example, a reduced and non-reduced SDG-PAGE gel of GGF2; The isolated product is essentially free of contaminating proteins and shows the expected molecular weight and monomeric structure. The biological activity of GGF2 was analyzed using a rat primary Schwann cell proliferation method and the expected effect was reproducibly obtained with four independent batches of GGF2. Another functional analysis was developed that mediates the phosphorylation of the Akt kinase, a component of cellular signaling downstream of the erbB receptor pathway. It was observed that there was a dose-dependent phosphorylation of the Akt kinase.

Example 3

This example illustrates the handling of ABCI chondroitinase for the construction of chimeras.

ABCI chondroitinase was cloned from P. vulgaris and expressed in E. coli. Surprisingly, repeated attempts to create N-terminal fusion proteins were unsuccessful because the N-terminal fusion portion was cleaved from ABCI chondroitinase during the synthesis. However, the catalytically active deletion mutants with His N-terminal His-Fusion tags called ABC I-NA2O, ABC I-NA4O and ABCI-NA6O were prepared by suppressing 20, 40 and 60 amino acids respectively from the N-terminal end of the mature ABCI protein. Unlike complete ABCI chondroitinase, N-terminal deletion mutants are capable of synthesizing a 6xHis tag as an N-terminal fusion protein. It was also observed that the deletion of 80 amino acids from the C-terminus formed a mutant with the proteoglycan degradation activity of ABCI chondroitinase as verified in a zymography analysis.

Fusion proteins with NgR27-3ii, or L1 with a proteoglycan degradation molecule such as ABCI chondroitinase will use the expression of mairnfero and can be made in CHO cells. The cDNA for ABCI chondroitinase has been cloned into the pSECTag vector (Invitrogen) in the appropriate reading frame. A CHO cell line having secretory ABCI chondroitinase can be developed and the conditioned medium can be tested for catalytic activity by zymography analysis to confirm that ABCI chondroitinase expressed in mammalian cells is functional. A version of ABCI chondroitinase optimized for mammalian cell codons can be synthesized by methods known in the art for use in the expression of CHO cells.

GGF2 is a splice variant of the NRG1 gene expressed in the brain and spinal cord of adult humans. It is a glycosylated protein of molecular mass between 66-90 kDa. The authors of the present invention have discovered that the recombinant GGF2 expressed in CHO cells is highly glycosylated and promotes the proliferation of Schwann cells in vitro, and additionally that an NRGi EGF-like domain expressed in E. coli is fully functional to promote the proliferation and survival of myocytes.

The authors of the present invention have expressed fragments of GGF2 in E. coli as described in the preliminary data section. The specific domain of GGF2 responsible for the proliferation of Schwann cells and, therefore, for remyelination can be determined. If the Ig and EGF domains together or separately show in vitro biological activity, they can be used to form chimeric fusion proteins.

Cloning and expression of NgR27-3ii in CHO cells. An NgR fragment corresponding to residues 1359 was isolated by RT-PCR from human hippocampal poly K RNA (BD Biosciences) and its structure can be confirmed by DNA sequencing. The genetic fragment corresponding to residues 27-311 can be cloned from the larger fragment and then subcloned into the pSECTag vector (Invitrogen) in the appropriate reading frame to express the fragment as a secretory protein in CHO cells. Plasmid DNA containing the NgR27-3ii gene can be transfected into CHO cells and the cell line that produces NgR27-3ii can be selected under selection pressure with hygromycin B. The chimera expression plasmid of NgR27-3ii-ABCI It can be constructed for expression in a CHO cell expression system using methods known in the art.

Example 4

This example illustrates methods that can be used to purify and isolate expressed domains of chondroitinase ABCI and GGF2 expressed in E. coli. These methods can be applied to the purification of chimeric fusion proteins of the present invention.

The authors of the present invention have developed an efficient E. coli recombinant expression system and a purification process for ABCI chondroitinase that can be applied for the purification of chimeric chondroitinase ABCI derivatives.

For example, the expression of various domains of GGF2 in the chondroitinase expression host ABCI, E. coli, has been carried out and the following peptides have been tested: aa250-402, aa250-422 and aa350-402 These expressed peptides were found in the soluble fraction of the lysate products of E. coli. It is reasonable to expect that the final chimeric products of these ABCI chondroitinase peptides in E. coli also

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be soluble. In addition, the charge characteristics of the chimeric products compared to the recombinant ABCI chondroitinase are expected to be similar. Therefore, the values of the theoretical isoelectric point (p1) for the GGF2 peptides are 9.3 for aa250-402, 9.18 for aa250-422 and 7.55 for aa350-402. The fusion of the first two peptides with ABCI chondroitinase is expected to result in chimeric protections with pi values still above 9. In this case, SP chromatography is expected to perform well as a capture stage. The smallest GGF2 peptide, aa 350-402, will reduce the pi of the final ABCI chondroitinase chimera to approximately 8.4. This change may require the optimization of the conditions of the capture stage.

ABCI chondroitinase chimeric products with peptides NgR27-3ii and Li can be expressed in a CHO cell system. Prior to purification of the expressed chimeric proteins, the growth conditions for the cell line that produces the NgR27-3ii or Li chimeric proteins will be optimized in serum-free medium. A detailed study of media optimization can be carried out to determine the highest production conditions. The volume of scale increase can be decided based on the production rate of the chimeric proteins (pg / cellMa). The conditioned media of the various chimera producing cell lines can be collected and subjected to tangential flow filtration. The ion exchange chromatography can be used to capture the secreted proteins from the conditioned media, the gel filtration chromatography can be used as a refining purification step and then the anion exchange membrane filtration can be used for the removal of endotoxins and DNA. In each stage the efficacy of the purification by SDS-PAGE, and the spectrophotometric quantification of the protein concentration will be analyzed.

Example 5

This prophetic example describes the in vitro evaluation of the biological activity of the chimera: Each chimera can be analyzed to determine the enzymatic activity of the chondroitinase and the specific biological activity of each fusion partner.

The first stage in the analysis can employ conventional biochemical methodologies of proteins to confirm the fidelity of the gene expression. These include SDSPAGE, IEE, mass spectrometry and handheld exclusion chromatography.

The specific activity of chondroitinase chimera can be determined using a conventional and uniformly accepted spectrophotometric analysis. The production of reaction products from the catalytic activity of a chimeric polypeptide with chondroitinase can be determined by measuring the absorbance of the product at a wavelength of 232 nm. A typical reaction mixture consists of i20 microliters of reaction mixture (40 mM Tris, pH 8.0, 40 mM Na acetate, 0.002% casema) combined with substrate (5 microliters of 50 mM C chondroitin) yi, 5 microliters of chimeric fusion polypeptide with ABCI chondroitinase or test sample. The change in absorbance units is the initial velocity that can be converted into a measure of unit activity.

HPLC analysis of disacarides: The catalytic activity of chimeric polypeptide with chondroitinase on a PGSC substrate is expected to release two species of sulfated disaccharides that include aA AUA- [i ^ -3] -GaINAc- 4S (A 4DS) and aA AUA - [i ^ -3] -GaINAc-6S (A 6DS). These species can be resolved by HPLC and the quantification of the resulting chromatograms is a sensitive and accurate measure of chondroitinase activity. To perform the analysis, samples of chondroitinase digestion reactions carried out with wild-type chondroitinase and chondroitinase fusion proteins can be clarified by centrifugation and then subjected to an validated anion exchange HPLC method as follows. . A Dionex CarboPac PA-i0 (4 x 250 mm) analytical column equipped with a Dionex CarboPac PA-i0 (4 x 50 mm) protective column with a mobile phase consisting of a water gradient at pH 3.5 (can be used) buffer A) and 2M NaCl, at pH 3.5 (buffer B). The detection can be set at a wavelength of 232 nm. A flow rate of i ml / minute and a continuous 45-minute gradient of 100% A to 100% B provides an acceptable resolution of A4Ds and A6DS. Standard curves can be generated using known amounts of A4DS and A6DS.

The zymography allows the resolution of proteins by their molecular weight with a concomitant evaluation of chondroitinase activity. A 0% polyacrylamide gel can be polymerized in the presence of 85 pg / ml of aggrecan. The samples can be boiled in an SDS-loading buffer and then the analytes can be resolved by electrophoresis. After separation, the gel can be incubated for 2.5 hours at room temperature in 2.5% Triton Xi00 and then at 6 ° C at 37 ° C in 2.5% Triton X-i00 again. During these incubations, the SDS can be extracted from the gel and the chondroitinase is retracted and digests the aggrecan in its immediate proximity. After the refolding process, the gel can be colored to determine carbohydrates. The gel can be incubated first in 0.2% Cetylpyridinium for 90 minutes at room temperature and then transferred to 0.2% Toluidine Blue in H2O 49: 50: i, Ethanol, and Acetic Acid for 30 minutes. The gel may be completely detached at that time. After detaching the gel, it can be incubated overnight in a solution of 50 micrograms / ml of Stains-All (Sigma) in 50% ethanol. Chondroitinase activity can be detected as a clear spot on the gel that matches the molecular weight of the enzyme. It has been shown that the size of the clearance is almost linearly related to the activity of a Unit.

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The chimera of NgR27-3ii-chondroitinase can be evaluated to determine the activity of the Nogo senuelo receptor. The analysis can be used to measure the collapse of the growth cones: Dorsal root ganglia (GRD) are dissected from Sprague Dawley rat cnas 1 day after birth (Pi) and dissociated into 200 U / ml collagenase I (Worthington) and 2.5 U / ml dispasa (Boehringer / Roche) 2 times for 30 min. at 37 ° C. Enzymes are removed and DNase (0.5 mg / ml) can be added to the ganglia. Crushing can be done with a pipette tip attached to a 1000 pl Pipetman. The resulting cell suspension can be filtered through a 40 micron cell filter and centrifuged at 70 xg for 5 min. The cells can be resuspended in DMEM / 10% FBS and pre-cultured on a plate for 2 hours on a non-coated tissue culture plate (100 mm in diameter). Non-adherent neurons are removed and plated at 10,000 cells / well in a 24-well plate coated with poly-lysine / laminin in serum-free Neurobasal / B27 with 50 ng / ml of NGF. After 20 to 24 hours, MAG or Nogo66 can be added at varying concentrations for 1 hour at 37 ° C to induce growth cone collapse. The chimera of NgR 27-311-chondroitinase can be added at various concentrations to compete with MAG and Nogo66 and thus protect the neurons from the collapse of the growth cone. The cultures can be fixed by adding an equal volume of preheated 8% paraformaldehyde / 0.6 M sucrose to the medium for 20 min. while the cells are kept on a hot plate at 37 ° C. Growth cones can be labeled with AlexaS68 phalloidin (Molecular Probes). Briefly, the cells can be permeabilized with 0.1% Triton-X 100 for 5 min. at room temperature, they are blocked for 20 min. in 1% BSA in PBS and incubated in phalloidin, dilute 1:40 in 1% BSA, in PBS for 20 min at RT. The cells can be washed in PBS and can be mounted in fluorescent mounting medium (DAKO). The percentage of collapsed growth cones is determined by analyzing a minimum of 100 growth cones per well under a 40x objective.

The biological activity of fusion proteins with L1 can be determined using a standardized analysis of neurite growth. After recording a 25 mm circle in the center of a 35 mm plate treated with tissue culture, 1 ml of 10 pg / ml poly-lysine can be added to each recorded circle and incubated at 37 ° C for 60 minutes Poly-lysine provides a negative control for the growth of neurites. The recorded circles can be washed twice with Hank's balanced saline solution plus calcium and magnesium (HBSS ++) after the last rinse; 1.2 pl of L1-Fc (0.6 pM, 0.3 pM, 0.15 pM, 0.075 pM) are applied on the petri dishes to serve as a positive control and various concentrations of the protein can be applied of fusion of L1-chondroitinase to the plates as test samples. The plates can be incubated for 1 hour at room temperature. The applications are aspirated slightly, so as not to dry them and immediately washed twice with 1 ml of HBSS ++ and then 1 ml of 1% BSA in PBS is added to each circle. After approximately 15 minutes at room temperature, the recorded drums can be washed twice with HBSS ++ and once with bioanalysis medium (NeuralBasal (Gibco) + B27 supplements (Gibco) + L-glutamine + L-glutamic acid, penicillin and streptomycin 100 U / ml) + 1% fetal bovine serum that will remain in the petri dishes until analysis. To provide neurons to the plaque on the substrates, cerebellar granule cells from days 9 or 10 after birth (PND) are collected. The brain is separated from the 1 cna skull and the cerebellum is separated from the rest of the tissue, the meninges are removed and the cerebellum is placed in ice-cold HBSS ++. The tissue is crushed and treated with trypsin using 0.25% trypsin for 15 minutes at 37 ° C. The trypsin action is inhibited by adding 0.5 mg / ml of soy trypsin inhibitor (Gibco). The tissue is rinsed with HBSS ++ and crushed with an EBS-coated flame pasteur pipette. The dissociated cells settle at 500 xg for 3 minutes, the supernatant is decanted and the cells are resuspended in 2 ml of growth medium. After lightly crushing, the cell suspension is carefully applied as a coating on the top of a 3.5% BSA pad (in PBS), and centrifuged at 500 xg for 3 minutes. The supernatant is aspirated and the sediment is resuspended in 2 ml of growth medium. A cell count is performed and the cells are diluted to a final working concentration of 1.5 x 105 cells / ml. A 300 µl alfcuot of diluted cells is added to the center of each plate, resulting in the uniform distribution of 6 x 104 cells throughout the entire surface area of the engraved circle. The cells are allowed to grow for 16 hours at 37 ° C in a humidified environment with a 5% CO2 supplement. The next day the plates are removed from the incubator at 37 ° C, the cells are fixed with 4% paraformaldetndo and the growth of neurites is recorded by photomicroscopy.

Biological activity analyzes for GGF2 can be performed using Schwann cell proliferation. The sciatic nerves of three-day-old Sprague Dawley rat rats are dissected and dissociated in L-15 medium (Invitrogen) containing 0.25% trypsin and 0.03% type I collagenase (SIGMA) for 15 minutes at 37 ° C. The nerves are centrifuged at 400 xg for 5 minutes and the dissociation medium is replaced by DMEM / 10% FBS. The nerves are crushed using a 10 ml syringe with a 21 g needle and then a 23 g needle. The cell suspension is filtered through a 40 pm mesh placed over the opening of a 50 ml conical tube. The cells can be centrifuged at 400 xg for 5 min, plated in a T-75 flask coated with poly-D-lysine (PDL) at a density of approximately 5 million cells in 15 ml of DMEM / 10% FBS / PenStrep and incubated in a 37 ° C incubator regulated with 10% CO2. After a 24-hour incubation, the cells can be washed twice with DMEM / 10% FBS and re-fed with fibroblast inhibition medium consisting of 10% DMEM / FBS and 10 pl / ml cytosine arabinoside 1 mM (Ara-C). After an incubation time of 2 to 3 days, the fibroblast inhibition medium is replaced by Schwann cell growth (DMEM / 10% FBS / 150 ng / ml GGF2 / 5 pM forskolin). Cells can be expanded and 2 x 10 6 cells / ml alfcuotas are frozen in DMEM / 10% DMSO, 54% FBS in liquid nitrogen. At the time of use, Schwann cells are thawed and plated at a density of approximately 16,000

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cells / well in a 96-well tissue culture plate coated with PDL in DMEM / 5% FBS. After approximately 24 hours, gGF2 is added in a serial dilution ranging from 100 ng / ml to 0.78 ng / ml containing 5 pM forskolin to establish a standard curve. Samples of GGF2 fusion proteins can also be added in serial dilution together with 5 pM Forskolin. BrdU 10 pM is added. The cells are incubated for 48 hours in a 10% CO2 incubator. A BrdU ELISA kit from Roche Applied Science (Catalog No. 1 647 229) can be used to detect the proliferation of Schwann cells. The medium is poured from the plate and the plate is covered with a paper handkerchief. A 200 µl / well almuota of Fix / Denat is added and incubated for 30 min at 15-25 ° C. A 100 µl / well almuot of anti-BrdU-POD working solution is dissolved (the lyophilized antibody is dissolved in 1.1 ml of doubly distilled water and diluted 1: 100 with antibody dilution solution) and incubated for 90 minutes at room temperature. The wells can be washed 3 times with 200300 pl / well wash solution. A 100 µl almuota of substrate solution is added and incubated for 15 minutes or until the color development is sufficient for photometric detection. The plates are read on a SpectraMax plate reader at 450 nm well before the addition of the stop solution at 370 nm well after the addition of 50 ml of 1N sulfuric acid to each well.

Akt ELISA [pS473]: C6 glioma cells, obtained from ATCC, are grown in DMEM / 10% FBS in a T-75 flask until confluence. After trypsin treatment, the cells are grown in a 24-well plate at a density of 500,000 cells / well in 0.5 ml of medium. One day after plating, the cells are treated with GGF2 (lot: Glu from Lonza Biologics) at serial dilutions ranging from 0.78 to 100 ng / ml for 30 minutes at 37 ° C to establish a standard curve. As a negative control, wortmanin, a PI3 kinase inhibitor, is added to the cells at 10 nnM for 30 minutes before the addition of GGF2. Samples of GGF2 fusion proteins will also be added in a serial dilution. The cells are washed with PBS and then extracted with 100 µl of cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EgTa, 1 mM NaF, 20 mM Na4P2O7, Na3VO4 2 mM, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, protease inhibitor cocktail (Pierce)). Cell extracts are kept at -80 ° C until later use. A Biosource ELlSA kit (Catalog Number KHOO1 II) can be used to measure phosphorylated Akt kinase levels. In summary, 100 pl of a 1:50 dilution of samples and a serial dilution of Akt standards [pS473] are loaded into wells coated in advance with anti-Akt antibody for 2 hours at room temperature (RT) or overnight 4 ° C The wells are washed and the anti-Akt antibody [pS473] is added and incubated for 1 hour. After washing, the HRP-conjugated anti-rabbit antiserum is added to the wells and incubated for 30 min. After washing, TMB chromogen is added to the wells and incubated for 30 minutes at RT before stopping the reaction with stop solution. The plate is read on a SpectraMax plate reader at 450 nm. The optical densities are plotted against the Akt patterns [pS473] and the concentration of pAkt in the C6 samples is deduced from the standard curve.

Example 6

This example illustrates the construction of a chondroitinase polypeptide fusion protein and a TAT cell transduction peptide.

The genetic sequence encoding a chondroitinase enzyme can be functionally linked to the HIV protein transduction domain called TAT peptide. The DNA construct of the ABCI TAT chondroitinase fusion of the resulting chimeric genes is shown in FIG. 1. It was noted that during bacterial expression of this construct, the TAT peptide was removed from the chondroitinase enzyme at some point during processing during bacterial growth. The removal of peptides bound to the N-terminal end was also observed during the expression of an N-terminal histidine-labeled ABCI chondroitinase enzyme.

Mutants were generated by deletion of the ABCI chondroitinase enzyme, where the mutant had lost a certain number of amino acids from the N-terminal portion of the polypeptide, but maintained proteoglycan degradation activity. It was observed that this N-terminal deletion maintained a histidine tag that was anchored to the N-terminal end. It is expected that various TAT-mutant fusion DNA constructs can be expressed by deletion of ABCI chondroitinase without the removal of the TAT polypeptide during expression. For example, the TAT peptide may be fused at the N-terminus of the deletion mutants such as ABCI-NA20 or the ABCI-NA60 deletion mutant. Without pretending to be limited by the theone, the authors of the present invention believe that the native proteoglycan degradation enzyme contains a signal sequence that is anchored to the N-terminal end. This signal sequence is eliminated during natural production in bacteria and in the production of the enzyme cloned in E. coli. It is believed that some signal within the n-terminal amino acids instructs the bacteria to eliminate any element attached to this end.

A DNA construct can be made with the TAT peptide anchored to the N-terminus of one of the mutants by chondroitinase deletion and perform a Western blot and a protein gel showing this expressed protein and its activity.

Example 7

This example illustrates the diffusion of molecules to cells and tissues using a proteoglycan degradation composition.

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An adult Sprague Dawley rat brain was removed from the skull and divided into four into right front, left front, right back and left rear sections, which roughly corresponded to the frontal (frontal) and occipito-parietal (rear) lobes. (A) The right front quarter was placed in artificial cerebrospinal fluid (Catalog No. 59-7316, Harvard Apparatus, Holliston, Massachusetts) containing the enzyme beta-galactosidase (Catalog No. G 5160; Sigma, St. Louis, MO) and Chondroitinase ABC I at 0.5 U / ml (Catalog No. C 3667; Sigma, St. Louis, MO) for 2 hours at 37 ° C. The brain room was cleared several times in phosphate buffered saline (PBS) and then processed with a Beta-Gal staining kit (Catalog No. Gal-S, Sigma, St. Louis, MO). The substrate-enzyme reaction (blue product) was allowed to develop for 1 hour, and the brain was rinsed several times in PBS and the half portions of each cut of the brain block were cut using straight parallel razors. (B) The left frontal quarter was placed in artificial cerebrospinal fluid (Catalog No. 59-7316, Harvard Apparatus, Holliston, MA) that contains the enzyme beta-galactosidase (Catalog No., G 5160; Sigma, St. Louis, MO ) for 2 hours at 37 ° C. The brain room was rinsed several times in phosphate buffered saline (PBS) and then processed with a Beta-Gal staining kit (Num. Gal-Sigma Catalog, St. Louis, MO). The substrate-enzyme reaction (blue product) was allowed to develop for 1 hour, and the brain was rinsed several times in PBS and portions of the center of each brain block were cut using straight parallel razors. (C) The right front quarter was placed in artificial cerebrospinal fluid (Catalog No. 59-7316; Harvard Apparatus, Holliston, MA) containing the enzyme beta-galactosidase (Catalog No. G 5160, Sigma, St. Louis, mO) and chondroitinase ABC I at 0.5 U / ml (Catalog No. C 3667, Sigma, St. Louis, MO) for 2 hours at 37 ° C. The brain room was cleared several times in phosphate buffered saline (PBS) and then processed with a Beta-Gal staining kit (Catalog No. Gal-S, Sigma, St. Louis, MO). The substrate-enzyme reaction (blue product) was allowed to develop for 1 hour, and the brain was rinsed several times in PBS and portions of the center of each brain block were cut using straight parallel razors. (D) The left hind quarter was placed in artificial cerebrospinal fluid (Catalog No. 59-7316, Harvard Apparatus, Holliston, MA) containing the enzyme beta-galactosidase (Catalog No., G 5160; Sigma, St. Louis, MO ) for 2 hours at 37 ° C. The brain room was rinsed several times in phosphate buffered saline (PBS) and then processed with a Beta-Gal staining kit (Catalog No. Gal-S, Sigma, St. Louis, MO). The substrate-enzyme reaction (blue product) was allowed to develop for 1 hour, and the brain was rinsed several times in PBS and portions of the center of each brain block were cut using straight parallel razors. Images of the brains were obtained on a scanner and are shown in FIG. 2 (I) (A-D).

The adult rat brain hemispheres were soaked in buffer alone or containing 33 U / ml ABCI chondroitinase (Acorda) for 2 hours at 37 degrees C. The hemispheres were washed and placed immediately in Eosin Y (Sigma) or in a saturated solution of Congo Red (Sigma) in 70% ethanol. Portions of tissue were cut and images were obtained on a scanner. See FIG. 2 (II) Eosin and FIG. 2 (III) Congo red.

FIG. 2 (III) The saturated solution of the Congo Red shows greater penetration through the cortex of a cerebral hemisphere treated with Chondroitinase compared to an untreated brain. Congo Red is a negatively charged 697 kDA dye. FIG. 2 (II) Eosin Y penetration through the cortex of a cerebral hemisphere treated with Chondroitinase is slightly more diffuse, but the penetration does not appear to be deeper compared to an untreated brain. Eosin Y is zwitterionic, has a global negative charge at the low pH at which it is used, and has 692 kDa. FIG. 2 (I) The adult rat lobes were incubated in beta-galactosidase alone (B & D), or with the addition of AbCi Chondroitinase (A, 0.5 U / ml or C, 0.005 U / m).

The results showed that chondroitinase treated tissue affected the penetration of beta-galactosidase in CNS tissue. Chondroitinase had drastic effects on the penetration rate of Congo Red dye but apparently not Eosin.

Example 8

This example illustrates an ABCI Chondroitinase Analysis Protocol that can be modified to measure the activity of mutant fusion proteins by deletion of ABCI Chondroitinase or the activity of other proteoglycan degradation polypeptide fusion proteins described herein.

The production of reaction products from the catalytic activity of a proteoglycan degradation molecule or fusion protein is determined by a measurement of the absorbance of the product at a wavelength of 232 nm. A typical reaction mixture consists of 120 p, 1 of the reaction mixture (40 mM Tris, pH 8.0, 40 mM Na acetate, 0.002% casema) combined with a substrate (5; 50 mM C chondroitin) and 1.5 pl of ABCI chondroitinase or a mutant fusion protein by deletion of ABCl-TAT chondroitinase. Reference of cABCI of Seikagaku: frozen at -20 ° C in 5 ml almuotas, use 1 ml per reaction, 50 mM C chondroitin (PM 521) in water: frozen at -20 ° C in 100 ml almuotas. Almuots of the reaction mixture of approximately 120 pl at 37 ° C can be prepared for 3 minutes or more. A wavelength of 232 nm is used with the spectrometer.

Knowing the extinction coefficient for the reaction product, measuring the change in absorbance of the reaction product at a reading of 232 nm over time after the addition of a known amount of ABCI Chondroitinase or other degradation polypeptide fusion proteins of proteoglycans to the 120 pl reaction mixture with 0.002% casema and added chondroitin substrate, the activity can be determined

specific in moles / min / mg of the proteoglycan degradation fusion protein. Seikagaku ABCI Chondroitinase has a specific activity under these test conditions of approximately 450 pmoles / min / mg.

SEQ ID NO: 1 Protema Chondroitinase ABC I ORIGIN

atsnpa fdpknlmqse iyhfaqnnpl adfssdknsi 61 Itlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg ekUstseaq 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 sdldyfhtls iqhlalllle pddqkrinlv ntfshyitga 481 Itqvppggkd glrpdgtawr hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdkd 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 atpek mgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk paivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gjkwqsadkns evkyqvsgdn teltftsyfg ipeftsyfg

1021 p

SEQ ID NO: 2 Protema NA20 ABCI (A45-N1023)

aqnnpl adfssdknsi

61 Itlsdkrsim gnqsllwkwk ggssftlhkk livptdkeas kawgrsstpv fsfwlynekp 121 idgyltidfg ekhstseaq 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 sdldyfhtls rqhlalllle pddqkrinlv ntfshyitga 481 Itqvppggkd 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 iildkls nvt gyafyqpasi edkwikkvnk paiymthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl

5 1021p

SEQ ID NO: 3 Protema NA60 ABCI (F85-N1023)

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 glrpdgtawr ltqvppggkd hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nnnegattih Iplkdldspk phtlmqrger 721 gfsgtssleg qygmmafnli ypanlerfdp nftakksvla adnhlifigs ninssdknkn 781 vettlfqhai tptlntlwin gqkiemnpyq ttlqqgdwli dsngngylit qaekvnvsrq 841 hqvsaenknr qptegnfssa widhstrpkd asyeymvfld atpekmgema qkfrenngly 901 qvlrkdkdvh iildklsnvt gyafyqpasi edkwikkvnk pa ivmthrqk dtlivsavtp 961 dlnmtrqkaa tpvtinvtin gkwqsadkns evkyqvsgdn teltftsyfg ipqeiklspl

1021 p

SEQ ID No. 4: Protema NA60 CA80 ABCI (F85-A942)

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 sdldyfhtls rqhlalllle pddqkrinlv ntfshyitga 481 glrpdgtawr ltqvppggkd hegnypgysf pafknasqli yllrdtpfsv gesgwnnlkk 541 amvsawiysn pevglplagr hpfnspslks vaqgyywlam saksspdktl asiylaisdk 601 tqnestaifg etitpaslpq gfyafnggaf gihrwqdkmv tlkayntnvw sseiynkdnr 661 ygryqshgva qivsngsqls qgyqqegwdw nrmegattih Iplkdldspk 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 Protema Chondroitinase AC Locus 1HMW_A

5 ORIGIN

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

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

qqtgtael 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 syikairgsy mdfnvegrgv srpdilnkka 301 ckkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 gsndfaggvs rpltklwgeq dgvygasaya Idydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

SEQ ID NO: 7 Protema CA220 AC (Q23-A480)

qqtgtael 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 syikairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 gsndfaggvs rpltklwgeq dgvygasaya Idydslqakk awfffdkeiv clgaginsna

SEQ ID NO: 8 Protema NA20 CA200 AC (L43-T500)

lmmdkva eknlntlqpd

61 gswkdvpykd damtnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem Iilmiygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd syikairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 gsndfaggvs rpltklwgeq dgvygasaya Idydslqakk awfffdkeiv clgaginsna 5 481 penitttlnq swlngpvist

SEQ ID NO: 9 Protema NA50 CA200 AC (T74-T500)

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

SEQ ID NO: 10 Protema NA100 CA200 AC (S123-T500)

smwwhne iatpqalgem lilmrygkkp ldealvhklt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek laifskyyrd syikairgsy mdfiivegrgv srpdilnkka 301 ekkrllvakm idlkhteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 gsndfaggvs rpltklwgeq dgvygasaya Idydslqakk awfffdkeiv clgaginsna 481 penitttlnq swlngpvist

5

10

tnwlpnn hllqletiiq ayiekdshyy gddkvfdqis kafkywydsd 121 pksmwwhne iatpqalgem lilmrygkkp ldealvhldt ermkrgepek ktganktdia 181 lhyfyrallt sdeallsfav kelfypvqfv hyeeglqydy sylqhgpqlq issygavfit 241 gvlklanyvr dtpyalstek Iaifskyyrd sylkairgsy mdfnvegrgv srpdilnkka 301 ekkrllvakm idlichteewa daiartdstv aagykiepyh hqfwngdyvq hlrpaysfnv 361 rmvskrtrrs esgnkenllg rylsdgatni qlrgpeyyni mpvwewdkip gitsrdyltd 421 rpltkl

SEQ ID NO: 12 Protema Chondroitinase B Locus Q46079

ORIGIN

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

SEQ ID NO: 13 Protema NA80 Chondroitinase B (Gi06-H5o6)

gnrai qawkshgpgl

121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyymdigrc 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 tyaltarlsa draakfkavi kmkeh

SEQ ID NO: 14 Protema NA120 Chondroitinase B (Ii46-H506)

ittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyymdigrc 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 tyaltarlsa draakfkavi kmkeh

SEQ ID NO: 15 Protema CA19 Chondroitinase B (Q26-L488)

qwas netlyqwke vkpgglvqia dgtykdvqli 61 vsnsgksglp itikalnpgk vfftgdakve lrgehlileg iwfkdgnrai qawkshgpgl 121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyymdigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq lmlkgnlffk dkpyvypffk ddyfiagkns wtgnvalgve 421 rsaykpvkik kgipvnisan diqpiegial dlnaliskgi tgkplswdev rpywlkempg 481 tyaltarl

qwas netlyqwke vkpgglvqia dgtykdvqli 61 vsnsgksglp itikalnpgk vfftgdakve lrgehlileg iwfkdgnrai qawkshgpgl 121 vaiygsynri tacvfdcfde ansayittsl tedgkvpqhc ridhcsftdk itfdqvinln 181 ntaraikdgs vggpgmyhrv dhcffsnpqk pgnagggiri gyymdigrc lvdsnlfmrq 241 dseaeiitsk sqenvyygnt ylncqgtmnf rhgdhqvain nfyigndqrf gyggmfvwgs 301 rhviacnyfe lsetiksrgn aalylnpgam asehalafdm liannafinv ngyaihfnpl 361 derrkeycaa nrlkfetphq lmlkgnlffk

SEQ ID NO: 17 Protema NA120 CA120 Chondroitinase B (I146-K39o)

ittsl tedgkvpqhc ridhcsftdk itfdqvinln

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

SEQ ID NO: 18 Coindroitinase AC nucleotide CHU27583

5 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 aagtgtttga ggcgacgata aaagctttta ccagatttcc agtattggta tgacagcgac 361 ccgaaaagcc gcaactggtg gcacaatgaa attgccactc cgcaggccct tggtgaaatg 421 ctgatcctga tgcgttacgg taaaaagccg cttgatgaag cattggtgca taaattgacc 481 agcggggcga gaaagaatga 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 aacttgccaa ggggtactga ttacgttagg gatacccctt atgctttaag taccgagaaa 781 ctggctatat tttcaaagta ttaccgcgac agttatctga aagctatccg tggaagttat

841 atggatttta acgtagaagg ccgcggagta agccggccag taaaaaggca acattctaaa 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 gggaatggga atgccggtat ggcataacca caagattcct 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 gatg tattta agctttggat caaccatggt gccaggccag aaaatgcgca gtatgcttat 1741 cgggaataaa atcgttttgc 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 Deletion NA50 C A275 (a220 - 11278) of the AC Condriotinase nucleic acid

atgccatga ccaattggtt gccaaacaac

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

SEQ ID NO: 20 CHC27584 Locus of Chondroitinase B nucleic acid ORIGIN

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

121 gtaaaacccg gtggtctggt acagattgcc gatgggactt ataaagatgt tcagctgatt 181 gtcagcaatt caggaaaatc tggtttgccc atcactatta aagccctgaa cccgggtaag 241 gtttttttta ccggagatgc taaagtagag ctgaggggcg agcacctgat actggaaggc 301 aagacgggaa atctggttta cagagctatt caggcatgga aatcacatgg acccggattg 361 gtggctatat atggtagcta taaccgcatt accgcatgtg tatttgattg ttttgatgaa 421 gccaattctg cttacattac tacttcgctt accgaagacg gaaaggtacc tcaacattgc 481 cgcatagacc attgcagttt taccgaiaag 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 taggcaatga aatttttata ccagcgattt ggatacgggg gaatgtttgt ttggggaagc 901 tagcctgtaa aggcatgtca ttattttgag ctgtccgaaa ccataaagtc gagggggaac 961 gccgcattgt attta CBTPA cggtgctatg gcttcggagc atgctcttgc tttcgatatg 1021 ttgatagcca acaacgcttt catcaatgta aatgggtatg ccatccattt taatccattg 1081 gaaaagaata gatgagcgca 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 5 1501 aaaagaaata aagagcactg a

SEQ ID NO: 21 Deletion NA120 CA120 (a436 - gn70) of the Condriotinase B nucleic acid

attac tacttcgctt accgaagacg gaaaggtacc tcaacattgc 481 cgcatagacc attgcagttt taccgataag atcacttttg accaggtaat taacctgaac 541 gagctattaa aatacagcca 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 taggcaatga aatttttata ccagcgattt ggatacgggg gaatgtttgt ttggggaagc 901 tagcctgtaa aggcatgtca ttattttgag ctgtccgaaa ccataaagtc gagggggaac 961 gccgcattgt atttaaaccc cggtgctatg gcttcggagc atgctcttgc tttcgatatg 1021 ttgatagcca acaacgcttt catcaatgta aatgggtatg ccatccattt taatccattg 1081 gaaaagaata gatgagcgca ttgtgcagcc aataggctta agttcgaaac cccgcaccag 1141 ctaatgttaa aaggcaatct tttctttaag

SEQ ID NO: 22 Locus I29953 of ABCI Chondroitinase nucleic acid ORIGIN

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

Claims (7)

1. A composition comprising: a polypeptide comprising a TAT protein transduction domain and a mutant by deletion of chondroitinase ABC I, wherein the TAT protein transduction domain is anchored to the N-terminus of the mutant 5 by deletion of chondroitinase ABC I and the mutant by chondroitinase ABC I deletion is selected Between SEQ ID NO: 2 and SEQ ID NO: 3. 2. The composition of claim 1, wherein the protein transduction domain is a TAT domain represented by SEQ ID NO: 57. 3. The composition of claim 1, wherein the TAT domain and the ABC I 10 chondroitinase deletion mutant are linked together by a linker polypeptide domain. 4. The composition of claim 3, wherein the domain of the linker polypeptide comprises a glycine-rich peptide. 5. The composition of claim 3, wherein the linker polypeptide domain comprises a sequence selected from the group consisting of connectors rich in Gly-Gly-Gly-Gly-Gly, Gly-Gly-Ala-Gly-Gly, Gly / Be, or 15 connectors rich in Gly / Ala. 6. The composition of claim 3, wherein the domain of the linker polypeptide comprises an amino acid of unnatural origin, a substituted amino acid, a beta amino acid, or a gamma amino acid. 7. The composition of claim 1, wherein the polypeptide is selected from SEQ ID NO: 42, or SEQ ID NO: 44. twenty Fusion nucleic acid of TAT with chondroitinase ABC I ggtcgtaaaaagcgtcgtcaacgtcgtcgtggtggtggtggiggtgccaccagcaatcctgcatttgatcetaaaaatctgatgcagtcagaaatttaccat tttgcacaaaataacccattagcagactt.ctcatcagataaaaactcaataclaacgttatctgataaacgtagcattalgggaaaccaatctcttttatggaaa tggaaaggiggtagtagctttactttacataaaaaactgattgtccceaccgataaagaagcatctaaagcatggggacgcteatctacccccgttttctcatt ttggctttacaatgaaaaaccgatfgaLggttatcttactatcgatticggagaaaaactcatttcaaccagfgaggctcaggcaggctttaaagtaaaattag atttcactggctggcgtgctgtgggagtctctttaaataacgatcttgaaaatcgagagatgaccttaaatgeaaccaatacetcctctgatggtactcaaga cagcattgggcgttetttaggtgctaaagtcgatagtattcgttttaaagcgccttctaatgtgagtcagggtgaaatctatatcgaccgtattatgtttfclgtcg atgatgctcgctaccaatggtctgattatcaagtaaaaactcgcttatcagaacctgaaattcaatttcacaacgtaaagccacaacuicctgtaacacctga aaatttagcggccattgatcitattcgccaacgtctaattaatgaatttgtcggaggtgaaaaagagacaaacctcgcattagaagagaatatcagcaaatta aaaagtgatttcgatgctcttaatattcacactttagcaaatggtggaacgcaaggcagacatctgatcactgataaacaaatcattatttatcaaccagagaa tcttaactcccaagataaacaactatttgataattatgttattttaggtaattac acgacatlaatgtttaatattagccgtgcttatgtgctggaaaaagatcccac acaaaaggcgcaactaaagcag atgtacttat taatgacaaa gcatttatta gatcaagget ttgttaaagggagtgcttta gtgacaaccc atcacigggg atacagttct cgttggtggt atalttccacgttattaatg tctgatgcac taaaagaagc gaacctacaa actcaagttt atgattcattactgtggtat tcacgtgagt ttaaaagtag ttttgatatg aaagtaagtg ctgatagctctgatctagat tatttcaata ccttatctcg ccaacattta gccttattat tactagagcctgatgatcaa aagcgtatca acttagttaa tactttcagc cattatatca ctggcgcattaacgcaagtg ccaccgggtg gtaaagatgg tttacgccct gatggtacag calggcgacatgaaggcaac tatccgggct actctttccc agcctttaaa aatgcctctc agcttatttattlattacgc gatacaccat tttcagtggg tgaaagtggt tggaacaacc tgaaaaaagcgatggtttca gcgtggatct acagtaatcc agaagttgga ttaccgcttg caggaagacacccttttaac teaccttcgt taaaatcagt cgctcaaggc tattactggc ttgccatgtctgcaaaatca tcgcctgata aaacacttgc atctatttat cttgcgatta tcaactgcta gtgataaaacacaaaatgaa aactattaca tttttggaga ccagcgtctt tacctcaaggtttctatgcc tttaatggcg gtgcttttgg tattcatcgt tggcaagata tataacacca at aaatggtgaeactgaaagct gittggtc atctgaaatt tataacaaag ataaccgttatggccgttac caaagtcatg gtgtcgctca aatagtgagt aatggctcgc agctttcacagggctatcag caagaaggtt gggattggaa ggggcaacca tagaatgcaa gacttagaca ctattcaccttcctcttaaa gtcctaaacc tcatacctta atgcaacgtg gagagcgtggatttagcgga acatcatccc ttgaaggtca atatggcatg atggcattcg atcttatttatcccgccaat cttgagcgtt ttgatcctaa tttcactgcg aaaaagagEg tattagccgctgataatcac ttaattttta ttggtagcaa tataaatagt agtgataaaa ataaaaatgttgaaacgacc ttattccaac atgccattac tccaacalta aatacccttt ggattaatggacaaaagata gaaaacatgc cttatcaaac aacacttcaa caaggtgatt ggttaattgatagcaatggc aatggttact taattactca agcagaaaaa gtcgccaacatcaggtttca gtaaatgtaa gcggaaaata aaaatcgcca accgacagaa ggaaacttta gctcggcatggatcgatcac agcaetcgcc ccaaagatgc cagttatgag tatatggtct ttttagatgcgacacctgaa aaaatgggag agatggcaca aaaattccgt gaaaataatg ggttatatcaggttcttcgt aaggataaag aegttcatat tattctcgat aaactcagca atgtaacgggatatgccttt tatcagccag catcaattga agacaaatgg atcaaaaagg ttaataaacctgcaattgtg atgactcatc gacaaaaaga eactcttatt gtcag tgcag ttacacctgatttaaatatg actcgccaaa aagcagcaac tcctgtcacc atcaatgtca tctgetgata cgattaatggcaaatggcaa aaaatagtga agtgaaatat caggtttctg gtgataacactgaactgacg tttacgagtt actitggtat tccacaagaa atcaaactct cgccactcccttgatttaat caaaagaacg ctcttgcgtt ccttttttat ttgcaggaaa tctgattatgctaataaaaa accctttagc ccacgeggtt acattaagcc tctgtttatc attacccgcacaagcattac ccactctgtc tcatgaagct ttcggcgata tttatctttt tgaaggtgaattacccaata ccctlaccac ttcaaataat aatcaattat cgctaagcaa acagcatgctaaagatggtg aacaatcact caaatggcaa tatcaaccac aagcaacatt aacactaaataatattgtta attaccaaga tgataaaaat acagccacac cactcacttt tatgatgtggatttataatg aaaaacetca atcttcccca ttaacgttag catttaaaca aaataataaaattgcactaa gttttaatgc tgaacttaat tttacggggt ggcgaggtat tgctgttccttttcgtgata tgcaaggctc tgcgacaggt caacttgatc aatiagtgat caccgciccaaaccaagccg gaacactctt ttttgatcaa atcatcatga gtgtaccgtt agacaatcgttgggcagtac ctgactatca aacacctlac gtaaataacg cagtaaacac gatggttagtaaaaactgga gtgcattatt gatgtacgat c.agatgtttc aagcccatta ccctactttaaacttcga ta ctgaatftcg cgatgaccaa acagaaatgg cttcgattta tcagcgctttgaatattatc aaggaattcc FIG. one image 1 image2 image3

 Effect of chondtroitinase ABC 1 on activity after spinal cord injury

 ad spinal locomotive

 at 88 s 1 5 6- il * ■ i § “■ 2 ■

 T T I T!

 ri -------------

 t t "7 I T r - (t— Penicillinase __ Chondroitinase ABC 1

 f L

 AND

 ) 23 eg m Days after the injury

image4 image5 FIG. 5