NOGO Receptor-Mediated Blockade of Axonal Growth

Abstract

Disclosed are NgR proteins and biologically active Nogo (ligand) protein fragments. Also disclosed are compositions and methods for modulating the expression or activity of the Nogo and NgR protein. Also disclosed are peptides which block Nogo-mediated inhibition of axonal extension. The compositions and methods of the invention are useful in the treatment of cranial or cerebral trauma, spinal cord injury, stroke or a demyelinating disease.

What is claimed: 
     
         1 . A nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising: (a) amino acid residues 27-309 of SEQ ID NO:2 (human NgR NTLRRCT domain), or amino acid residues 27-309 of SEQ ID NO:2 with 1-20 conservative amino acid substitutions; and (b) and less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 consecutive amino acid residues. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the encoded polypeptide comprises amino acid residues 27-309 of SEQ ID NO:2. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the encoded polypeptide comprises fewer than 50 consecutive amino acids from amino acids 310-445 of SEQ ID NO:2. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the encoded polypeptide does not comprise a functional GPI domain. 
     
     
         5 . A vector comprising the nucleic acid of  claim 1 . 
     
     
         6 . The vector of  claim 5 , wherein the nucleic acid is operably linked to an expression control sequence. 
     
     
         7 . A cell comprising the vector of  claim 5 . 
     
     
         8 . A polypeptide comprising: (a) amino acid residues 27-30 9 of SEQ ID NO:2 (human NgR NTLRRCT domain), or amino acid residues 27-309 if SEQ ID NO:2 with 1-20 conservative amino acid substitutions; and (b) fewer than 115 consecutive amino acids from amino acids 310-445 of SEQ ID NO 2. 
     
     
         9 . The polypeptide of  claim 8 , wherein the polypeptide comprises amino acid residues 27-309 of SEQ ID NO:2. 
     
     
         10 . The polypeptide of  claim 8 , wherein the polypeptide comprises fewer than 50 consecutive amino acids from amino acids 310-445 of SEQ ID NO:2. 
     
     
         11 . The polypeptide of  claim 8 , wherein the encoded polypeptide does not comprise a functional GPI domain. 
     
     
         12 . A method of producing the polypeptide of  claim 8 , comprising introducing a nucleic acid encoding the polypeptide into a host cell, culturing the host cell under conditions suitable for expression of said polypeptide, and recovering the polypeptide. 
     
     
         13 . An antibody that binds to an epitope in the CTS domain of NgR. 
     
     
         14 . A method of inhibiting binding of a Nogo polypeptide to a NgR, comprising contacting the Nogo polypeptide with an effective amount of the polypeptide of  claim 8 . 
     
     
         15 . A method of inhibiting binding of a Nogo polypeptide to a NgR, comprising contacting the NgR with an antibody that binds to the amino acid sequence con sting of SEQ ID NO:2 (NgR polypeptide). 
     
     
         16 . The method of  claim 15 , wherein the antibody binds to an epitope within amino acids 310-445 of SEQ ID NO:2 (CTS domain of NgR). 
     
     
         17 . A method of decreasing inhibition of axonal growth by a CNS neuron, comprising contacting the neuron with an effective amount of a molecule selected from the group consisting of (a) a polypeptide of  claim 8 ; and (b) an antibody that binds to the amino acid sequence consisting of SEQ ID NO:2 (NgR). 
     
     
         18 . The method of  claim 17 , wherein the antibody binds to an epitope within the amino acid sequence consisting of amino acids 310-445 of SEQ ID NO:2 (CTS domain of NgR). 
     
     
         19 . A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of an agent selected from the group consisting of: (a) an agent that inhibits binding of a Nogo polypeptide to a NgR; and (b) an agent that inhibits NgR-dependent signal transduction in a central nervous system neuron. 
     
     
         20 . The method of  claim 19 , wherein the agent that inhibits binding of a Nogo polypeptide to a NgR is selected from the group consisting of: (a) a polypeptide of  claim 8 ; (b) an antibody that binds to a polypeptide consisting of SEQ ID NO:2. 
     
     
         21 . The method of  claim 20  wherein the antibody binds to an epitope within the ammo acid sequence consisting of amino acids 310-445 of SEQ ID NO:2 (CTS domain of NgR). 
     
     
         22 . The method of  claim 19 , wherein the central nervous system disease, disorder or injury is a spinal cord injury. 
     
     
         23 . A method for identifying a molecule that decreases Nogo-dependent inhibition of axonal growth, the method comprising:
 (a) providing a NgR polypeptide;   (b) contacting the NgR polypeptide with a candidate molecule; and   (c) detecting a decrease in binding of the Nogo polypeptide to the NgR in the presence of the candidate molecule, as compared to the binding of the Nogo polypeptide to the NgR in the absence of the candidate molecule.   
     
     
         24 . A composition comprising a polypeptide of  claim 8  and a pharmaceutically acceptable carrier. 
     
     
         25 . A composition comprising an antibody of  claim 13  and a pharmaceutically acceptable carrier. 
     
     
         26 . A nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) IYKGVIQAI and (b) EELV, the polypeptide comprising 40 amino acids or fewer. 
     
     
         27 . The nucleic acid of  claim 26 , wherein the polypeptide comprises the amino acid sequences IYKGVIQAI and EELV. 
     
     
         28 . The nucleic acid of  claim 26 , wherein the polypeptide comprises fewer than 40 amino acids. 
     
     
         29 . The nucleic acid of  claim 28 , wherein the polypeptide comprises amino acid residues 2 to 34 of SEQ ID NO:21. 
     
     
         30 . The nucleic acid of  claim 26 , wherein the polypeptide encoded further comprises a heterologous amino acid sequence not present in NogoA, the heterologous amino acid sequence comprising at least five amino acid residues. 
     
     
         31 . A polypeptide comprising an amino acid sequence selected from the group consisting of: (a) IYKGVIQAI and (b) EELV, the polypeptide comprising 40 amino acids or fewer. 
     
     
         32 . The polypeptide of  claim 31 , wherein the polypeptide comprises the amino acid sequences IYKGVIQAI and EELV. 
     
     
         33 . The polypeptide of  claim 32 , wherein the polypeptide comprises fewer than 40 amino acids. 
     
     
         34 . The polypeptide of  claim 33 , wherein the polypeptide comprises amino acid residues 2 to 34 of SEQ ID NO:21. 
     
     
         35 . The polypeptide of  claim 31 , wherein the polypeptide further comprises a heterologous amino acid sequence not present in NogoA, the heterologous amino acid sequence comprising at least five amino acid residues. 
     
     
         36 . An antibody that binds to a polypeptide of  claim 31 . 
     
     
         37 . A composition comprising a polypeptide of  claim 31  or an antibody according to  claim 36  and a pharmaceutically acceptable carrier. 
     
     
         38 . A met rod of inhibiting binding of a Nogo polypeptide to a NgR, comprising contacting the NgR with an effective amount of the polypeptide of  claim 31 . 
     
     
         39 . A method of inhibiting binding of a Nogo polypeptide to a NgR, comprising contacting the Nogo polypeptide w th an effective amount of the antibody according to  claim 36 . 
     
     
         40 . A method of decreasing inhibition of axonal growth by a CNS neuron, comprising contacting the neuron with an effective amount of a polypeptide of  claim 31  or an antibody of  claim 36 . 
     
     
         41 . A method of treating, a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of a polypeptide of  claim 31  or an antibody of  claim 36 . 
     
     
         42 . The method of  claim 41 , wherein the central nervous system disease, disorder or injury is a spinal cord injury. 
     
     
         43 . A method of identifying a molecule that decreases axonal Nogo-dependent inhibition of axonal growth, the method comprising:
 (a) providing a polypeptide comprising a target sequence consisting of an amino acid sequence selected from the group consisting of IYKGVIQAI and EELV;   (b) contacting the polypeptide with a candidate molecule; and   (c) detecting binding of the candidate molecule to a target sequence in the polypeptide.