Receptor-mediated uptake of an extracellular bcl-xl fusion protein inhibits apoptosis

Abstract

Apoptosis-modifying fusion polypeptides, and the corresponding nucleic acid molecules, are disclosed. Pharmaceutical compositions comprising these polypeptides, and the use of these polypeptides to modify apoptosis, are also provided.

1 . A method for inhibiting apoptosis in a target cell, comprising contacting the target cell with an amount of a functional apoptosis-inhibiting fusion protein sufficient to inhibit apoptosis in the target cell, wherein the fusion protein comprises:
 a first domain capable of inhibiting apoptosis in the target cell; and   a second domain capable of specifically targeting the fusion protein to the target cell,   
       and wherein the fusion protein integrates into or otherwise crosses a cellular membrane of the target cell upon binding. 
     
     
         2 . The method of  claim 1 , wherein the protein is administered in the form of a pharmaceutical composition. 
     
     
         3 . The method of  claim 1 , wherein the target cell is a neuron. 
     
     
         4 . The protein of  claim 3 , wherein the neuron is a retinal ganglion cell. 
     
     
         5 . The method of  claim 1 , wherein the first domain of the fusion protein is a Bcl-2 family protein, or a variant or fragment thereof that inhibits apoptosis in the target cell to which the protein is exposed, and wherein the amount is sufficient to inhibit apoptosis. 
     
     
         6 . The method of  claim 5 , wherein the first domain of the fusion protein is Bcl-x L , or a variant or fragment thereof that inhibits apoptosis in the target cell to which the protein is exposed, and wherein the amount is sufficient to inhibit apoptosis. 
     
     
         7 . The method of  claim 1 , wherein the fusion protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence shown in SEQ ID NO: 2 (Bcl-x L -DTR);   (b) the amino acid sequence shown in SEQ ID NO: 8 (LF n -Bcl-x L ); and   (c) amino acid sequences that differ from those specified in (a) or (b) by one or more conservative amino acid substitutions, but which retain targeting and apoptosis-inhibiting abilities.   
     
     
         8 . The method of  claim 1 , wherein the target cell is in vivo in a subject. 
     
     
         9 . The method of  claim 1 , wherein the target cell is in vivo in a subject and the fusion protein is administered to the subject after transient ischemic neuronal injury. 
     
     
         10 . The method of  claim 9 , wherein the transient ischemic neuronal injury comprises a spinal cord injury. 
     
     
         11 . The method of  claim 9 , wherein the protein is administered in the form of a pharmaceutical composition. 
     
     
         12 . The method of  claim 9 , further comprising the step of co-administering an agent selected from the group consisting of a chemotherapeutic agent, an anti-inflammatory agent, an anti-viral agent, and an antibiotic agent. 
     
     
         13 . The method of  claim 1 , wherein the second domain comprises a receptor-binding domain derived from a bacterial toxin, a monoclonal antibody, a growth factor, or a cytokine. 
     
     
         14 . The method of  claim 13 , wherein the second binding domain comprises a receptor-binding domain derived from diphtheria toxin or anthrax toxin. 
     
     
         15 . The method of  claim 13 , wherein the second binding domain comprises a receptor-binding domain derived from epidermal growth factor 
     
     
         16 . The method of  claim 13 , wherein the receptor-binding domain comprises diphtheria toxin receptor binding domain, or a variant or fragment thereof that targets the fusion protein to the target cell to which the protein is exposed. 
     
     
         17 . The method of  claim 14 , wherein the second domain further comprises a translocation domain of diphtheria toxin.