Neural regeneration peptides and methods for their use in treatment of brain damage

Abstract

The invention discloses a family of neuronal migration-inducing, proliferation-promoting and neurite outgrowth promoting factors, termed NRP compounds, and provides compositions and methods for the use of NRP compounds in the treatment of brain injury and neurodegenerative disease. NRP-1 compounds induce neurons and neuroblasts to proliferate and migrate into areas of damage caused by acute brain injury or chronic neurodegenerative disease, such as stroke, trauma, nervous system infections, demyelinating diseases, dementias, and metabolic disorders. NRP compounds may be administered directly to a subject or to a subject's cells by a variety of means including orally, intraperitoneally, intravascularly, and directly into the nervous system of a patient.

1 . A neural regeneration peptide (NRP) compound comprising at least one amino acid sequence comprising at least one [A]PG[R,S] domain, and having at least one biological effect selected from the group consisting of neuronal migration, neural proliferation, neural outgrowth promoting activities and neural survival. 
     
     
         2 . The NRP of  claim 1 , further comprising at least one additional amino acid domain selected from the group consisting of [A]PG[R,S], PE, [A,G]RR and ARG. 
     
     
         3 . The NRP of  claim 1 , wherein said peptide has a molecular weight of about 0.8 kD to about 2.7 kD. 
     
     
         4 . The NRP of  claim 1 , wherein said peptide has an isoelectric point between about 6.5 and about 10. 
     
     
         5 . The NRP of  claim 1 , having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. 
     
     
         6 . A nucleotide sequence encoding an NRP selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:19. 
     
     
         7 . A nucleic acid encoding an NRP, wherein said nucleotide sequence differs from the nucleotide sequence of  claim 6  only by virtue of redundancy in the genetic code. 
     
     
         8 . A method for promoting neural regeneration, comprising the step of delivering to a neural tissue, a pharmacologically effective amount of an NRP compound. 
     
     
         9 . The method of  claim 8 , wherein said neural regeneration is selected from the group consisting of neural survival, neural proliferation, neural migration and neurite outgrowth. 
     
     
         10 . The method of  claim 8 , wherein said NRP comprises a peptide of  claim 1 . 
     
     
         11 . The method of  claim 8 , wherein said NRP comprises a peptide of  claim 2 . 
     
     
         12 . The method of  claim 8 , wherein said NRP comprises a peptide of  claim 5 . 
     
     
         13 . The method of  claim 8 , wherein said NRP is encoded by a nucleic acid of  claim 6 . 
     
     
         14 . The method of  claim 8 , wherein said NRP is encoded by a nucleic acid of  claim 6  or a nucleic acid having a sequence that differs from that of a nucleic acid of  claim 6  only by the redundancy of the genetic code. 
     
     
         15 . A nucleic acid molecule encoding an NRP, said nucleic acid having at least 85% nucleotide sequence identity over the entire sequence to a nucleotide sequence of  claim 6 . 
     
     
         16 . An expression vector comprising a nucleotide sequence of  claim 6 , wherein said expression vector can express an NRP. 
     
     
         17 . The expression vector of  claim 16 , wherein the vector is a plasmid. 
     
     
         18 . The expression vector of  claim 16 , wherein the vector is a viral vector. 
     
     
         19 . A host cell containing the expression vector of  claim 16 . 
     
     
         20 . The host cell of  claim 19 , wherein the cell is prokaryotic. 
     
     
         21 . The host cell of  claim 19 , wherein the cell is eukaryotic. 
     
     
         22 . The nucleotide sequence of  claim 6  SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19., wherein “t” is replaced by “u.” 
     
     
         23 . A nucleotide sequence comprising a complementary strand to a strand selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:19. 
     
     
         24 . The nucleotide sequence of  claim 23 , said sequence at least 15 base pairs in length, and hybridizes under stringent hybridization conditions to a genomic DNA encoding an NRP. 
     
     
         25 . A polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity over the entire sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. 
     
     
         26 . The method of  claim 8 , wherein said neural tissue is present in an animal's basal ganglia. 
     
     
         27 . The method of  claim 26 , wherein said neural tissue is at risk for degeneration due to Parkinson's Disease, Alzheimer's disease, hypoxia, eschemia or Huntington's Disease. 
     
     
         28 . A method for treating a neurological condition characterized by neural degeneration in an organism, comprising the step of administering to affected nerves of said organism, a pharmacologically effective amount of an NRP compound. 
     
     
         29 . The method of  claim 28 , wherein said neurological condition is selected from the group consisting of Parkinson's Disease, Alzheimer's Disease, hypoxia, eschemia, and Huntington's Disease. 
     
     
         30 . A method for inducing neuronal proliferation and/or neuronal migration in a neuron, comprising administering an effective amount of an NRP compound to said neuron. 
     
     
         31 . A method for promoting neurite outgrowth in a neuron, comprising administering an effective amount of an NRP compound to said neuron. 
     
     
         32 . A method for promoting neuronal survival, comprising administering an effective amount of an NRP compound to said neuron. 
     
     
         33 . A method for inducing neuronal proliferation and/or neuronal migration in a neuron, comprising administering an effective amount of an NRP compound to said neuron. 
     
     
         34 . The method of  claim 33 , wherein said neuron is present within a living animal. 
     
     
         35 . The method of  claim 33 , wherein said neuron is not within the body of said animal. 
     
     
         36 . The method of  claim 33 , wherein said neuron is present within the central nervous system of said animal. 
     
     
         37 . The method of  claim 35 , wherein said neuron is not within the body of said animal. 
     
     
         38 . A method of treating a patient having a spinal cord injury comprising administering an effective amount of an NRP compound to said patient. 
     
     
         39 . The peptide of  claim 5 , wherein the amino acid sequence of  claim 5  differs from an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 by virtue of a conservative amino acid substitution. 
     
     
         40 . A method for determining the presence of an NRP, comprising the steps of;
 (a) providing a tissue culture system comprising:
 1. a tissue culture substrate; 
 2. a cortical tissue placed on said substrate; and 
 3. a thalamic tissue placed on said substrate; said cortical and thalamic tissues placed at least 2 mm apart; 
   (b) incubating said cortical tissue and said thalamic tissue in the presence of a test molecule;   (c) detecting one or more biological effect of neuronal survival, neuronal proliferation, neuronal migration or neurite outgrowth; and   (d) comparing said observed biological effect in the presence of said test molecule to the observed biological effect observed in the absence of said test molecule.   
     
     
         41 . The method of  claim 40 , wherein said cortical tissue and said thalamic tissues are from a rat. 
     
     
         42 . The method of  claim 40 , wherein said test molecule is an NRP compound. 
     
     
         43 . A method for determining the pharmacological efficacy of an NRP, comprising the steps of:
 (a) providing a tissue culture system comprising:
 1. a tissue culture substrate; 
 2. a cortical tissue placed on said substrate; and 
 3. a thalamic tissue placed on said substrate; said cortical and thalamic tissues placed at least 2 mm apart; 
   (b) incubating said cortical tissue and said thalamic tissue in the presence of a test molecule;   (c) detecting one or more biological effect of neuronal survival, neuronal proliferation, neuronal migration or neurite outgrowth; and   (d) comparing said observed biological effect in the presence of said test molecule to the observed biological effect observed in the absence of said test molecule.   
     
     
         44 . A method for comparing the efficacy of a test molecule, comprising the steps of:
 (a) providing a tissue culture system comprising:
 1. a tissue culture substrate; 
 2. a cortical tissue placed on said substrate; and 
 3. a thalamic tissue placed on said substrate; said cortical and thalamic tissues placed at least 2 mm apart; 
   (b) incubating said cortical tissue and said thalamic tissue in the presence of a test molecule;   (c) detecting one or more biological effect of neuronal survival, neuronal proliferation, neuronal migration or neurite outgrowth; and   (d) comparing said observed biological effect in the presence of said test molecule to the observed biological effect observed in the presence of a known amount of an NRP compound.   
     
     
         45 . A test kit for determining the presence of NRP-like activity, comprising the steps of:
 (a) providing a tissue culture system comprising:
 1. a tissue culture substrate; 
 2. a cortical tissue placed on said substrate; and 
 3. a thalamic tissue placed on said substrate; said cortical and thalamic tissues placed at least 2 mm apart; 
   (b) incubating said cortical tissue and said thalamic tissue in the presence of a test molecule;   (c) detecting one or more biological effect of neuronal survival, neuronal proliferation, neuronal migration or neurite outgrowth; and   (d) comparing said observed biological effect in the presence of said test molecule to the observed biological effect observed in the absence of said test molecule.