Gene therapy by cell specific targeting
This invention is directed to a modified cyclosporin A and to a modified, genetically engineered version of its receptor, cyclophilin. This invention is further directed to a method for treating host versus graft disease following blood marrow transplantation by transfecting stem cells so that after introduction into a patient the stem cells will express the modified cyclophilin, and, as necessary, administer the modified cyclosporin A to the patient.
1. A method for inhibiting proliferation of genetically engineered T cells in an animal, wherein said genetically engineered T cells are introduced to the animal and said genetically engineered T cells comprise a nucleic acid encoding a mutated macrolide binding protein (MBP) selected from an FK506-binding protein (FKBP), cyclophilin, calcineurin, and FKBP:rapamycin associated protein (FRAP),
wherein said method comprises administering to the animal a macrolide which binds to the mutated MBP or forms a complex including the mutated MBP, thereby inhibiting proliferation of T cells expressing the mutated MBP,
wherein said genetically engineered T cells are autologous or allogeneic to the animal, and
wherein, relative to the wild-type MBP, the mutated MBP contains an altered amino acid sequence and has an altered specificity for binding to or forming a complex with a macrolide.
2. The method of claim 1 , wherein the macrolide binds to or forms a complex with the mutated MBP with a dissociation constant, K d , at least one order of magnitude less than its K d for binding to or forming a complex with wild-type MBP.
3. The method of claim 1 , wherein the macrolide binds to or forms a complex with the mutated MBP with a dissociation constant, K d , at least three orders of magnitude less than its K d for binding to or forming a complex with wild-type MBP.
4. The method of claim 1 , wherein the nucleic acid was introduced into said T cell ex vivo by DNA transfection.
5. The method of claim 1 , wherein the nucleic acid was introduced into said T cell ex vivo by virus-mediated transduction.
6. The method of claim 1 , wherein the nucleic acid was introduced into said T cell ex vivo by homologous recombination.
7. The method of claim 1 , wherein the macrolide is an analog of rapamycin, FK506 or cyclosporin.
8. The method of claim 1 , wherein the animal is a mammal.
9. The method of claim 8 , wherein the animal is a human.
10. The method of claim 1 , wherein the expression of the mutated nucleic acid is transcriptionally regulated by a T-cell specific transcriptional regulatory sequence.
11. A method for providing an animal comprising genetically engineered T cells, wherein the proliferation of said T cells is preferentially inhibited, said method comprising introducing into said animal said genetically engineered T cells, which comprise a nucleic acid encoding a mutated macrolide binding protein (MBP), wherein
(a) said mutated MBP is selected from an FK506-binding protein (FKBP), cyclophilin, calcineurin, and FKBP:rapamycin associated protein (FRAP); and
(b) relative to the wild-type MBP, the mutated MBP contains an altered amino acid sequence has an altered specificity for binding to or forming a complex with a macrolide.