Methods for genetic modification of hematopoietic progenitor cells and uses of the modified cells

• US 20040072771A1
• Filed: 07/10/2002
• Published: 04/15/2004
• Est. Priority Date: 07/10/2001
• Status: Active Grant

• Current Assignee: Johnson & Johnson Research Pty Ltd. (Johnson & Johnson)
• Original Assignee: Johnson & Johnson Research Pty Ltd. (Johnson & Johnson)
• Inventors: Sun, Lun -Quan, Gerlach, Wayne, Symonds, Geoffrey P., MacPherson, Janet L., Amado, Rafael G., Fanning, Gregory C.
• Granted Patent: US 7,345,025 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/14 interfering N.A.

Abstract

Described are compositions and methods relating to gene therapy, particularly as applied to hematopoietic progenitor (HP) cells, to transduced cells and methods of obtaining them, and to methods of using them to provide prolonged engraftment of modified hematopoietic cells in human subjects. The invention particularly relates to ex vivo gene therapy of HP cells for treatment or prevention of HIV infection.

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.

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.

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.

40. A kit comprising elements for use in carrying out the method of claim 35.

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.

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.

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.