Methods for genetic modification of hematopoietic progenitor cells and uses of the modified cells
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.
1. A method for determining whether transduced CD34 + hematopoietic progenitor (HP) cells undergo T-lymphocyte development for more than 12 months in a human subject comprising:
a) mobilizing CD34 + hematopoietic progenitor cells into the blood of a human subject by administering to the subject an amount of one or more cytokines sufficient to mobilize the hematopoietic cells;
b) isolating by apheresis leukocytes from the blood of the subject of step a);
c) isolating the CD34 + hematopoietic cells from the isolated leukocytes of step b);
d) culturing the CD34 + cells in the presence of at least one cytokine;
e) transducing the CD34 + cells with a viral construct which comprises a gene of interest in the presence of an agent which enhances colocalization of the cells and the viral construct to form a composition of cells having a minimum dose of 0.52×10 6 transduced CD34 + hematopoietic progenitor cells per kg body weight of the subject in a total CD34 + cell population of at least 1.63×10 6 cells per kg of body weight of the subject, wherein the number of transduced CD34 + hematopoietic cells is based on a determination by competitive PCR of the CD34 + hematopoietic cells;
f) delivering to the subject the composition of cells;
g) at least 12 months later, isolating blood from the subject;
h) determining whether T-lymphocytes in the blood comprise the gene of interest, thereby determining whether transduced CD34 + hematopoietic progenitor cells undergo T-lymphocyte development for more than 12 months in the human subject.
2. The method of claim 1 , wherein the agent that colocalizes the cells with a transduction vector is a fragment of fibronectin.
3. The method of claim 1 , wherein step (f) is performed without myeloablation.
4. The method of claim 1 , wherein the step of subjecting the CD34 + hematopoietic cells to a transduction process with a gene of interest is performed in the presence of fibronectin fragment.
5. A method for determining whether transduced CD34 + hematopoietic progenitor (HP) cells undergo T-lymphocyte development for more than 12 months in a human subject comprising:
a) mobilizing CD34 + hematopoietic progenitor cells into the blood of a human subject by administering to the subject an amount of one or more cytokines sufficient to mobilize the hematopoietic cells;
b) isolating by apheresis leukocytes from the blood of the subject of step a);
c) isolating the CD34 + hematopoietic cells from the isolated leukocytes of step b);
d) culturing the CD34 + cells in the presence of at least one cytokine;
e) transducing the CD34 + cells with a viral construct which comprises a gene of interest in the presence of an agent which enhances colocalization of the cells and the viral construct to form a composition of cells;
f) determining whether the composition of cells has a minimum dose of 0.52×10 6 transduced CD34 + hematopoietic progenitor cells per kg body weight of the subject in a total CD34 + cell population of at least 1.63×10 6 cells per kg of body weight of the subject, wherein the number of transduced CD34 + hematopoietic cells is based on a determination by competitive PCR of the CD34 + hematopoietic cells, and if so then proceeding to step (g), and if not then again performing steps (a)-(e) and combining the compositions of cells;
g) delivering to the subject the composition of cells;
h) at least 12 months later, isolating blood from the subject;
i) determining whether T-lymphocytes in the blood comprise the gene of interest, thereby determining whether transduced CD34 + hematopoietic progenitor cells undergo T-lymphocyte development for more than 12 months in the human subject.
6. The method of claim 5 , wherein the agent that colocalizes the cells with a transduction vector is a fragment of fibronectin.
7. The method of claim 5 , wherein step (g) is performed without myeloablation.
8. The method of claim 5 , wherein the step of subjecting the CD34 + hematopoietic cells to a transduction process with a gene of interest is performed in the presence of fibronectin fragment.
9. A method for determining whether genetically protected T-lymphocytes can be produced for more than 12 months in a human subject afflicted with HIV comprising:
a) mobilizing CD34 + hematopoietic progenitor cells into the blood of a human subject by administering to the subject an amount of one or more cytokines sufficient to mobilize the hematopoietic cells;
b) isolating by apheresis leukocytes from the blood of the subject of step a);
c) isolating the CD34 + hematopoietic cells from the isolated leukocytes of step b);
d) culturing the CD34 + cells in the presence at least one cytokine;
e) transducing the CD34 + cells with a viral construct which comprises a gene of interest encoding an anti-HIV agent in the presence of an agent which enhances colocalization of the cells and the viral construct to form a composition of cells;
f) determining whether the composition of cells has a minimum dose of 0.52×10 6 transduced CD34 + hematopoietic progenitor cells per kg body weight of the subject in a total CD34 + cell population of at least 1.63×10 6 cells per kg of body weight of the subject, wherein the number of transduced CD34 + hematopoietic cells is based on a determination by competitive PCR of the CD34 + hematopoietic cells, and if so then proceeding to step (g), and if not then again performing steps (a)-(e) and combining the compositions of cells;
g) delivering to the subject the composition of cells;
h) at least 12 months later, isolating blood from the subject;
i) determining whether cells in the blood comprise the gene of interest, thereby determining whether the genetically protected T-lymphocytes can be produced for more than 12 months in the human subject.
10. The method of claim 9 , wherein the agent that colocalizes the cells with a transduction vector is a fragment of fibronectin.
11. The method of claim 9 , wherein step (g) is performed without myeloablation.
12. The method of claim 9 , wherein the step of subjecting the CD34 + hematopoietic cells to a transduction process with a gene of interest is performed in the presence of fibronectin fragment.