Engineered
An engineered payload-delivery system includes a target cell binding unit, covalently bound to a pore forming unit, and a payload portion adapted with a region capable of non-covalently binding to the pore forming unit. The pore forming unit is derived from a particular sub-serotype of Clostridium toxin, while the payload region is derived from a different sub-serotype of Clostridium toxin. The disclosed chimeric protein-based composition is capable of specifically delivering payload to neural cells.
1. A composition for delivering an agent to a target cell, comprising
a target cell binding unit,
a pore-forming unit,
a rigid synthetic linker linking the target cell binding unit and the pore-forming unit, and
a payload unit comprising the agent, wherein said payload unit binds non-covalently to a pore formed by the pore-forming unit upon activation and oligomerization of the pore-forming unit;
wherein the pore-forming unit is a polypeptide derived from a first AB type of non-neurotoxic binary toxin, and wherein the target cell binding unit is a polypeptide derived from a second AB type of toxin, said second AB type being different from the first AB type, wherein the target cell binding unit binds to a neural cell, and
wherein the target cell binding unit and the pore-forming unit are covalently linked through the rigid synthetic linker to form a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4.
2. The composition of claim 1 , wherein the pore-forming unit is a polypeptide or a polypeptide oligomer derived from the pore-forming unit of Clostridium botulinum toxin C2.
3. The composition of claim 1 , wherein the composition delivers the agent to a neural cell.
4. The composition of claim 1 , wherein the target cell binding unit is a polypeptide or a polypeptide oligomer derived from the binding domain of the heavy chain of C. botulinum neurotoxin C1 (C1 Hcc).
5. The composition of claim 1 , wherein the agent comprises at least one member selected from the group consisting of a therapeutic agent, a diagnostic agent, and combinations thereof.
6. The composition of claim 1 , wherein the agent comprises at least one member selected from the group consisting of a toxin, a cell cycle blocker, an apoptosis inducing agent, an inhibitor of DNA replication, an inhibitor of RNA synthesis, an inhibitor of protein synthesis, an enzyme, a protein binding agent, an antibody, a neutralizing antibody, a labeling agent, magnetic beads, and combinations thereof.
7. The composition of claim 1 , wherein the agent comprises an ADP-ribosyltransferase.
8. The composition of claim 1 , wherein the agent comprises C2I from Clostridium botulinum toxin C2.
9. The composition of claim 1 , wherein the agent comprises a fluorescent agent.
10. The composition of claim 1 , wherein the target cell binding unit comprises the binding domain of the heavy chain of C. botulinum neurotoxin C1 (C1 Hcc), wherein the pore-forming unit comprises the pore-forming domain of Clostridium botulinum toxin C-2, and wherein the payload unit comprises C2I from Clostridium botulinum toxin C-2.
11. The composition of claim 1 , wherein the payload unit comprises a polypeptide having at least 90% sequence identity to amino acid sequence of SEQ ID NO: 6.
12. A method of delivering an agent to a target cell, comprising administering the composition of claim 1 to a target cell.
13. The method of claim 12 , wherein the composition is administered to a subject by injection, wherein the subject comprises the target cell.
14. The method of claim 12 , wherein the target cell is a member selected from the group consisting of a cell of a brain tumor, a cell of a neuroblastoma, and a cell of a retinoblastoma, peripheral neuron; motor neuron, sensory neuron, and combination thereof.
15. A composition for delivering an agent to a target cell, comprising
a target cell binding unit,
a pore-forming unit,
a rigid synthetic linker linking the target cell binding unit and the pore-forming unit, and
a payload unit comprising the agent, wherein said payload unit binds non-covalently to a pore formed by the pore-forming unit upon activation and oligomerization of the pore-forming unit;
wherein the pore-forming unit is a polypeptide derived from a first AB type of non-neurotoxic binary toxin, and wherein the target cell binding unit is a polypeptide derived from a second AB type of toxin, said second AB type being different from the first AB type, and wherein the target cell binding unit binds to a neural cell; and
wherein the rigid synthetic linker comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.
16. The composition of claim 1 , wherein the neural cell is a sensory neuron.
17. A composition for delivering an agent to a target cell, comprising
a target cell binding unit,
a pore-forming unit,
a rigid synthetic linker linking the target cell binding unit and the pore-forming unit, and
a payload unit comprising the agent, wherein said payload unit binds non-covalently to a pore formed by the pore-forming unit upon activation and oligomerization of the pore-forming unit;
wherein the pore-forming unit is a polypeptide derived from a first AB type of non-neurotoxic binary toxin, and wherein the target cell binding unit is a polypeptide derived from a second AB type of toxin, said second AB type being different from the first AB type, wherein the target cell binding unit binds to a neural cell, and
wherein the target cell binding unit comprises a polypeptide having at least 90% sequence identity to the amino acid sequence 230-426 of SEQ ID NO: 1, wherein the pore-forming unit comprises a polypeptide having at least 90% sequence identity to the amino acid sequence 1-591 of SEQ ID NO: 2, and
wherein the rigid synthetic linker comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.