RECEPTOR-MEDIATED UPTAKE OF AN EXTRACELLULAR BCL-XL FUSION PROTEIN INHIBITS APOPTOSIS
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.