Methods for genetic modification of stem cells
A method of preparing a therapeutic cell population for clinical use from a starting population of cells comprising haematopoietic stem cells, said method comprising separating a population of cells that substantially do not express CD38 but which express CD34 from the starting population of cells, and transducing the separated cell population with a vector to obtain the therapeutic cell population.
1. A method of treatment comprising administering a therapeutic cell population and a support cell population to a subject in need thereof,
wherein the therapeutic cell population and the support cell population are prepared according to a method of preparing a therapeutic cell population and a support cell population for clinical use from a starting population of cells comprising haematopoietic stem cells, said method comprising separating a population of cells that substantially do not express CD38 but which express CD34 from the starting population of cells, and transducing the separated cell population with a vector to obtain the therapeutic cell population, wherein the separated CD38-expressing cells or portion thereof are retained to form the support cell population, and wherein the support cell population is not cultured ex vivo.
2. The method of claim 1 , wherein the therapeutic cell population is administered to the subject prior to administration of the support cell population.
3. The method of claim 1 , wherein the therapeutic cell population is administered to the subject contemporaneously with or simultaneously to administration of the support cell population.
4. The method of claim 1 , wherein the step of transducing a population of cells with a vector comprises culturing the cells for about 12-24 hours.
5. The method of claim 1 , wherein the step of transducing a population of cells with a vector comprises treating the population of cells with prostaglandin E2, or a prostaglandin E2 derivative.
6. The method of claim 5 , wherein the prostaglandin E2 derivative is 16, 16-dimethyl prostaglandin E2.
7. The method of claim 5 , wherein the cells are treated with prostaglandin E2 or prostaglandin E2 derivative before exposure of the cells to the vector.
8. The method of claim 1 , wherein the number of CD34 + cells administered to a subject in the support cell population is about 5-15× the number of CD34 + cells administered in the therapeutic cell population when the subject has undergone myeloablative conditioning.
9. The method of claim 1 , wherein the therapeutic and support cell populations are administered as part of an autologous stem cell transplant procedure or an allogeneic stem cell transplant procedure.
10. A method of gene therapy comprising administering a therapeutic cell population and a support cell population to a subject in need thereof,
wherein the therapeutic cell population and the support cell population are prepared according to a method of preparing a therapeutic cell population and a support cell population for clinical use from a starting population of cells comprising haematopoietic stem cells, said method comprising separating a population of cells that substantially do not express CD38 but which express CD34 from the starting population of cells, and transducing the separated cell population with a vector to obtain the therapeutic cell population, wherein the separated CD38-expressing cells or portion thereof are retained to form the support cell population, and
wherein the support cell population is not cultured ex vivo after separation from the starting population of cells.
11. The method of claim 10 , wherein the therapeutic cell population has the CD34 + CD38 − phenotype, and wherein the support cell population has the CD34 + CD38 int1 , CD34 + CD38 int2 , and/or CD34 + CD38 + phenotype.
12. The method of claim 10 , wherein the therapeutic cell population is administered to the subject prior to, contemporaneously with or simultaneously to administration of the support cell population.
13. The method of claim 10 , wherein the number of CD34 + cells administered to a subject in the support cell population is about 5-15× the number of CD34 + cells administered in the therapeutic cell population when the subject has undergone myeloablative conditioning.
14. The method of claim 10 , wherein the therapeutic and support cell populations are administered as part of an autologous stem cell transplant procedure or an allogeneic stem cell transplant procedure.
15. The method of claim 1 , wherein the method of preparing a therapeutic cell population and a support cell population comprises the steps of:
a. separating CD38-expressing cells from a starting population of cells comprising haematopoietic stem cells;
b. separating CD34-expressing cells from the population of cells obtained in step (a) that do not express CD38;
c. transducing the CD34-expressing cell population obtained in step (b) with a vector to obtain the therapeutic cell population.
16. The method of claim 1 , wherein the therapeutic cell population has the CD34 + CD38 − phenotype.
17. The method of claim 1 , wherein the starting population of cells comprising haematopoietic stem cells is obtained from mobilized peripheral blood, bone marrow or umbilical cord blood.
18. The method of claim 1 , wherein the CD38-expressing cells and/or CD34-expressing cells are separated using magnetic bead-based separation or flow cytometry.
19. The method of claim 1 , wherein the vector is a viral vector.
20. The method of claim 19 , wherein the viral vector is a lentiviral vector.
21. The method of claim 19 , wherein the viral vector comprises a nucleotide of interest.
22. The method of claim 1 , wherein the step of transducing a population of cells with a vector comprises culturing the cells for less than about 44 h.
23. The method of claim 1 , wherein the support cell population has the CD34 + CD38 int1 , CD34 + CD38 int2 , and/or CD34 + CD38 + phenotype.