Claims
- 1. A composition comprising a pharmaceutically acceptable carrier and at least 1.63×106 CD34+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52×106 of such CD34+ hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent.
- 2. The composition of claim 1, comprising at least 9.37×106 CD34+ hematopoietic cells per kg of body weight of a human subject, wherein at least 5×106 of such CD34+hematopoietic cells are transduced.
- 3. The composition of claim 1, wherein the anti-HIV agent is an RNA.
- 4. The composition of claim 1, wherein the anti-HIV agent is an RNAi molecule.
- 5. The composition of claim 1, wherein the anti-HIV agent is an antisense molecule.
- 6. The composition of claim 1, wherein the anti-HIV agent is a ribozyme.
- 7. The composition of claim 1, wherein the anti-HIV agent is a ribozyme comprising nucleotides having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′.
- 8. The composition of claim 1, wherein the viral construct is a retroviral construct.
- 9. The composition of claim 1, wherein the composition is substantially free of cytokines.
- 10. The composition of claim 1, wherein the composition is substantially free of virus.
- 11. The composition of claim 1, wherein the transduced CD34+ cells are capable of engraftment, and of giving rise to progeny cells for at least 12 months, in the subject.
- 12. A composition comprising a pharmaceutically acceptable carrier and at least 1.63×106 CD34+ hematopoietic cells per kg of body weight of the subject to whom the composition is to be administered, at least 0.52×106 of such CD34+ hematopoietic cells being transduced with a viral construct which expresses an anti-HIV agent,
wherein the composition is produced by a process comprising the steps of:
(a) isolating CD34+ hematopoietic cells from the subject; (b) culturing the CD34+ hematopoietic cells with at least one cytokine; (c) transducing the CD34+ hematopoietic cells with the viral construct which expresses the anti-HIV agent in the presence of an agent which enhances colocalization of the cells and the viral construct; (d) washing the CD34+ hematopoietic cells, and (e) mixing the CD34+ hematopoietic cells with a pharmaceutically acceptable carrier, to thereby obtain the composition.
- 13. The composition of claim 12, wherein the culturing of step (b) is performed in the presence of at least two cytokines.
- 14. The composition of claim 12, wherein the culturing of step (b) is performed in the presence of two cytokines.
- 15. The composition of claim 12, wherein the transduction of the cells in step (c) is performed in the presence of a recombinant fibronectin fragment.
- 16. A composition comprising a pharmaceutically acceptable carrier and at least 1.63×106 CD34+ hematopoietic cells per kg of body weight of the human subject to whom the composition is to be administered, at least 0.52×106 CD34+ of such CD34+ hematopoietic cells being transformed with a gene of interest not found in the CD34+ cells prior to transformation.
- 17. The composition of claim 16, comprising at least 9×106 CD34+ hematopoietic cells per kg of body weight of a human subject, wherein at least 5×106 CD34+hematopoietic cells are transduced.
- 18. The composition of claim 16, wherein the gene of interest expresses an RNA agent.
- 19. The composition of claim 16, wherein the subject is an adult.
- 20. A composition comprising a pharmaceutically acceptable carrier and at least 1.63×106 CD34+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52×106 CD34+ of such CD34+ hematopoietic cells being transformed with a gene of interest not found in the CD34′ cells prior to transformation,
wherein the composition is produced by a process comprising the steps of:
(a) isolating CD34+ hematopoietic cells from the subject; (b) culturing the CD34′ hematopoietic cells with at least one cytokine; (c) transforming the CD34+ hematopoietic cells with the a vector which encodes a gene of interest in the presence of an agent which enhances colocalization of the cells and the vector; (d) washing the CD34+ hematopoietic cells, and (e) mixing the CD34′ hematopoietic cells with a pharmaceutically acceptable carrier, to thereby obtain the composition.
- 21. A method of inserting into hematopoietic cells of a human subject a gene of interest comprising:
a) mobilizing CD34+ hematopoietic progenitor cells into the blood of the human subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating CD34+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) subjecting the CD34+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; e) determining the total number of CD34+ hematopoietic cells after step d), and if the total number is at least 1.63×106 cells per kg of body weight of the human subject, then proceeding to step f), and if the total number of CD34+ hematopoietic cells after step d) is less than 1.63×106 cells per kg of body weight of the human subject, then performing at least steps b)-d) and combining the CD34+ hematopoietic cells; and f) delivering to the subject the CD34+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene of interest.
- 22. The method of claim 21, wherein the agent that colocalizes the cells with a transduction vector is a fragment of fibronectin.
- 23. The method of claim 21, wherein step f) is performed without myeloablation.
- 24. The method of claim 21, wherein the step of mobilizing hematopoietic progenitor cells in the subject is performed by administering to the subject an amount of a cytokine sufficient to mobilize the hematopoietic progenitor cells.
- 25. The method of claim 21, wherein in the step of isolating the leukocytes from the subject's blood, apheresis is performed at least twice.
- 26. The method of claim 21, wherein the step of subjecting the CD34+ hematopoietic cells to a transduction process with a gene of interest is performed in the presence of a recombinant fibronectin fragment.
- 27. The method of claim 21, further comprising a step of culturing the isolated CD34+ hematopoietic cells of step c) in the presence of at least two cytokines.
- 28. The method of claim 21, wherein the human subject is an adult human subject.
- 29. The method of claim 21, wherein the gene of interest encodes an anti-HIV agent.
- 30. The method of claim 29, wherein the anti-HIV agent is an RNA.
- 31. The method of claim 29, wherein the anti-HIV agent is an antisense molecule.
- 32. The method of claim 29, wherein the anti-HIV agent is a ribozyme.
- 33. The method of claim 32, wherein the anti-HIV agent is a ribozyme comprising nucleotides having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′.
- 34. The method of claim wherein in step 21, if the total number of CD34+ hematopoietic cells after step d) is less than 1.63×106 cells per kg of body weight of the human subject, then further including a step of cryogenically storing the CD34+ hematopoietic cells from step d), repeating steps a)-d), and combining any cryogenically stored cells with the cells from step d).
- 35. A method of inserting into hematopoietic cells of a human subject a gene of interest comprising:
a) mobilizing CD34+ hematopoietic progenitor cells into the blood of the subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating CD34+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) determining the total number of CD34+ hematopoietic cells after step c), and if the total number is at least 1.63×106 cells per kg of body weight of the human subject, then proceeding to step e), and if the total number of CD34+ hematopoietic cells after step c) is less than 1.63×106 cells per kg of body weight of the human subject, then performing steps b)-c) and combining the CD34+ hematopoietic cells; e) subjecting the CD34+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; and f) delivering to the subject the CD34+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene of interest.
- 36. The method of claim 35, wherein the agent that colocalizes the cells with a transduction vector is a fragment of fibronectin.
- 37. A method of inserting into hematopoietic cells of a human subject a gene that expresses a ribozyme comprising nucleotides having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′ comprising:
a) mobilizing CD34+ hematopoietic progenitor cells into the blood of the subject by administering to the subject an amount of a cytokine sufficient to mobilize the hematopoietic progenitor cells; b) isolating leukocytes from the subject's blood by apheresis, which is performed at least twice; c) isolating CD34+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) culturing the isolated CD34+ hematopoietic cells of step c) for about one day in a culture medium in the presence of a cytokine; e) subjecting the CD34+ hematopoietic cells of step d) to a transduction process with a retrovirus comprising a vector that gives rise in the cell to a ribozyme comprising nucleotides having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′ in the presence of a recombinant fibronectin fragment; f) determining the total number of CD34+ hematopoietic cells after step e), and if the total number is at least 1.63×106 cells per kg of body weight of the human subject, then proceeding to step g), and if the total number of CD34+ hematopoietic cells after step e) is less than 1.63×106 cells per kg of body weight of the human subject, then again performing steps b)-e) and combining the CD34+ hematopoietic cells; and g) delivering to the subject, without myeloablation, the CD34+ hematopoietic cells, thereby inserting into hematopoietic cells of the human subject a gene that expresses the ribozyme.
- 38. A method of preparing the composition of claim 1 comprising:
a) mobilizing CD34+ hematopoietic cells into the blood of the subject; b) isolating leukocytes from the subject's blood by apheresis; c) isolating the CD34+ hematopoietic cells from the isolated leukocytes by an immunoselective method; d) subjecting the CD34+ hematopoietic cells of step c) to a transduction process with a gene of interest in the presence of an agent that colocalizes the cells with a transduction vector; and e) determining the total number of CD34+ hematopoietic cells after step d), and if the total number of CD34+hematopoietic cells after step d) is less than 1.63×106 cells per kg of body weight of the human subject, then again performing steps b)-d) and combining the CD34+ hematopoietic cells.
- 39. A use of a composition comprising a pharmaceutically acceptable carrier and at least 1.63×106 CD34+ hematopoietic cells per kg of body weight of a human subject to whom the composition is to be administered, at least 0.52×106 CD34+ of such cells per kg being transduced with a viral construct which expresses an anti-HIV agent, for the manufacture of a medicament for the treatment of the human subject infected with HIV.
- 40. A kit comprising elements for use in carrying out the method of claim 35.
- 41. A kit comprising
a) an amount of an agent capable of mobilizing hematopoietic progenitor cells in a human subject; b) a culture medium including at least one cytokine acceptable for culturing CD34+ hematopoietic cells; c) a retroviral vector comprising nucleotides having a sequence that in a cell gives rise to a ribozyme having the sequence 5′-UUA GGA UCC UGA UGA GUC CGU GAG GAC GAA ACU GGC UCC-3′; and d) tissue culture vessels coated on their inside with a recombinant fibronectin fragment.
- 42. A package comprising the kit of claim 41, and instructions for the use of the kit.
Parent Case Info
[0001] This application claims benefit of U.S. Provisional Application No. 60/304,127, filed Jul. 10, 2001, U.S. Provisional Application No. 60/304,283, Jul. 10, 2001, U.S. Provisional Application No. 60/343,484, filed Dec. 21, 2001, and U.S. Provisional Application No. 60/386,063, filed Jun. 4, 2002, the contents of all of which are hereby incorporated by reference.
Provisional Applications (4)
|
Number |
Date |
Country |
|
60304127 |
Jul 2001 |
US |
|
60304283 |
Jul 2001 |
US |
|
60343484 |
Dec 2001 |
US |
|
60386063 |
Jun 2002 |
US |