Hybrid tetanus toxoid proteins that migrate retrogradely and transynaptically into the CNS

Abstract

The non-toxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. A hybrid protein encoded by the lacZ-TTC gene fusion retains the biological functions of both proteins in vivo, i.e. retrograde transynaptic transport of the TTC fragment and -gal enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy is useful for the delivery of a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein can also be used to map synaptic connections between neural cells.

1 . A method for in vivo delivery of a desired composition into human or animal central nervous system (CNS) or spinal cord, wherein the method comprises administering to the human or animal a composition comprising a non-toxic, proteolytic fragment of tetanus toxin (TT) in association with at least a molecule having a biological function and said composition is capable of in vivo retrograde axonal transport and transynaptic transport into the CNS or the spinal cord of the human or animal and of being delivered at different areas of the CNS or the spinal cord. 
     
     
         2 . The method according to  claim 1 , wherein the composition is administered into a muscle. 
     
     
         3 . The method according to  claim 2 , wherein the composition is administered into a muscle in the vicinity of a neuromuscular junction. 
     
     
         4 . The method according to  claim 2 , wherein the muscle is selected in relation with the desired area of the CNS or spinal cord. 
     
     
         5 . The method according to  claim 1 , wherein the composition is administered into neuronal cells. 
     
     
         6 . The method according to  claim 1 , wherein the composition comprises a non-toxic, proteolytic fragment of tetanus toxin (TT) comprising a fragment C and a fragment B or a fraction thereof of at least 11 amino acid residues in association with at least a molecule having a biological function selected from the group consisting of a protein for compensation or modulation of functions under the control of the CNS or the spinal cord or modulation of functions in the CNS or the spinal cord or a protein to be delivered by gene therapy expression system to the CNS or the spinal cord. 
     
     
         7 . The method according to  claim 1 , wherein the composition comprises a non-toxic, proteolytic fragment of tetanus toxin (TT) comprising a fragment C and a fragment B or a fraction thereof of at least 11 amino acid residues and a fraction of a fragment A devoid of its toxic activity corresponding to the proteolytic domain having a zinc-binding motif located in the central part of the chain between amino acids 225 and 245 in association with at least a molecule having a biological function selected from the group consisting of protein for the compensation or the modulation of functions under the control of the CNS or the spinal cord or protein to be delivered by gene therapy expression system to the CNS or the spinal cord. 
     
     
         8 . The method according to  claim 6 , wherein the molecule is selected from the group consisting of protein SM, BDNF (Brain-derived neurotrophic factor), NT-3 (Neurotrophin-3), NT-4/5, GDNF (Glial cell-line-derived neurotrophic factor), IGF (Insulin-like growth factor), PNI (protease nexin .I), SPI3 (Serine Protease Inhibitor protein), ICE (Interleukin-1 converting enzyme), Bc1-2, GFP (green fluorescent protein), endonucleases like I-SceI or CRE, antibodies, or drugs specifically directed against neurodegenerative diseases such as latero spinal amyotrophy (LSA). 
     
     
         9 . The method according to  claim 8 , wherein the composition comprises a combination of at least two of said molecules. 
     
     
         10 . The method according to  claim 8 , wherein the molecule is located upstream from the fragment of tetanus toxin. 
     
     
         11 . The method according to  claim 8 , wherein the molecule is located downstream from the fragment of tetanus toxin. 
     
     
         12 - 31 . (canceled) 
     
     
         32 . The method according to  claim 7 , wherein the molecule is selected from the group consisting of protein SM, BDNF (Brain-derived neurotrophic factor), NT-3 (Neurotrophin-3), NT-4/5, GDNF (Glial cell-line-derived neurotrophic factor), IGF (Insulin-like growth factor), PNI (protease nexin .I), SPI3 (Serine Protease Inhibitor protein), ICE (Interleukin-1 converting enzyme), Bc1-2, GFP (green fluorescent protein), endonucleases like I-SceI or CRE, antibodies, or drugs specifically directed against neurodegenerative diseases such as latero spinal amyotrophy (LSA).