Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-04-07
2001-05-01
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S030000, C435S325000, C435S376000, C435S377000, C435S383000, C435S384000
Reexamination Certificate
active
06225044
ABSTRACT:
BACKGROUND OF THE INVENTION
Generally the invention relates to a method for gene transfer into cells activated from a quiescent state, in particular hematopoietic stem cells, and the cells thus obtained.
DESCRIPTION OF THE RELATED ART
Progress in the identification and cloning of genes that are responsible for human genetic diseases has led to a major program for the improvement of gene transfer technology (Anderson, W. F. (1992) Science, 256: 808-813; Miller, A. D. (1992) Nature, 357: 455-460; Morgan, R. A., & Anderson, W. F. (1993) Annu. Rev. Biochem., 62: 191-217; Karleson, S. (1991) Blood, 78: 2481-2492). For example, different viral vectors have been used for hematopoietic stem cells, each having their own advantage (Anderson, W. F. (1992), mentioned above). The insertion of a retroviral vector into the host genome ensures its replication in the host cell. This insertion requires an active proliferative state (Anderson, W. F. (1992), mentioned above; Varmus, H. E., Padgett, T., Heasley, S., Simon, G., & Bishop, J. M. (1977), Cell., 11: 307-319; Nolta, J. A., & Kohn, D. B. (1990) Hum. Gene Ther., 1: 257-268; Miller, D. G., Adam, M. A., & Miller, A. D. (1990) Mol. Cell. Biol., 10: 4239-4242) which does not exist in the compartment of the hematopoietic stem cell which is in a quiescent state (Lajtha, L. G., & Schofield, R. (1974) Différenciation, 2: 313-320), or in other types of stem cells and somatic cells such as the hepatic cells. Using antisense oligonucleotides against inhibitor gene, in particular TGF-&bgr;-1, has shown that the early progenitors of human bone marrow can be released from their quiescent state by blockage of an autocrine TGF-&bgr; (Hatzfeld, J., Li, M. -L., Brown, E. L., Sookdeo, H., Levesque, J. -P., O'Toole, T., Gurney, C., Clark, S. C., and Hatzfeld, A. (1991), J. Exp. Med., 174: 925-929).
Moreover, where transplants of hematopoietic tissue are concerned, in the past, the capacity for transplanting samples of bone marrow has been analysed by estimating the quantity of CFU-GM (colony-forming unit —granulocyte/macrophage: granulo-monocytic progenitor) cells. However, these cells which probably play an important part in cell generation for a short period following the transplantation of a graft may not reflect the quantity of more primitive cells, and in particular of hematopoietic stem cells which are important in long-term hematopoiesis. The stem cells involved in long-term transplants represent a small sub-population of cells which may possibly have the phenotype CD34+CD38− (compartment of the hematopoietic stem cell: cells rich in membrane antigen CD34 and poor in maturation membrane antigen CD38).
Recently, human cord blood has proved to be sufficient for reconstituting hematopoiesis after transplantation in children (Gluckman, E., Broxmeyer, H. E., Auerbach, A. D., Friedman, H. S., Douglas, G. W., Devergie, A., Esperou, H., Thierry, D., Socie, G., Lehn, P., Cooper, S., English, D., Kurtzberg, J., Bard, J., & Boyse, E. A. (1989), N. Eng. J. Med., 3: 1174-1178; Broxmeyer, H. E., Douglas, G. W., Hangoe, G., Cooper, S., Bard, J., English, D., Arny, M., Thomas, L., & Boyse, R. A. (1989) Proc. Natl. Acad. Sci. USA, 86: 3828-3832; Broxmeyer, H. E., Hangoe, G., & Cooper, S. (1992) Bone Marrow Transplant., 9: 7-10). In vitro, the data from various laboratories (Broxmeyer, H. E., Hangoe, G., Cooper, S., Ribeiro, R. C., Graves, V., Yoder, M., Wagner, J., Vadhan-Raj, S., Benninger, L., Rubinstein, P., & Broun, E. R. (1992) Proc. Natl. Acad. Sci. USA, 89: 4109-4113; Lu, L., Xiao, M., Shen, R. -N., Grisby, S., & Broxmeyer, H. E. (1993) Blood, 21: 41-48; Howe, J. M., Bradley, B. A., Marsh, J. C. W., Luft, T., Coutinho, L., Testa, N. G., & Dexter, T. M. (1992) Lancet, 340: 73-76; Cardoso, A. A., Li, M. L., Ratard, P., Hatzfeld, A., Brown, E. L., Levesque, J. -P., Sookdeo, H., Panterne, B., Sansilvestri, P., Clark, S. C., & Hatzfeld, J. (1993) Proc. Natl. Acad. Sci. USA, 90: 8707-8711, suggesting that cord blood has a greater capacity for generating progenitors in culture than that of bone marrow, and consequently that cord blood could be sufficient to effect transplants of hematopoietic tissues onto adult recipients.
However, a rapid gene transfer into the whole (totality) of the compartment of the hematopoietic stem cell or of any other stem or somatic cell at rest (this state of rest also being called “quiescencene” or as corresponding to the Go phase), has not been satisfactorily achieved to date.
SUMMARY OF THE INVENTION
Gene transfer into cells activated from a quiescent state, in particular hematopoietic stem cells activated from a quiescent state, is one of the aspects of the invention.
The subject matter of the invention is also quiescent cells, in particular quiescent hematopoietic stem cells, containing a heterologous gene.
The subject matter of the invention is also hematopoietic tissue containing a heterologous gene likely to form long-term grafts.
The invention relates to cells, in particular hematopoietic stem cells, containing in their genome all or part of a heterologous gene.
An object of the invention relates to the use of means for blocking at least one inhibitor of the cell cycle for a period not exceeding 72 h (h: hours), and preferably not exceeding 36 h, and in particular less than 20 h, and preferably approximately 1 to 15 h, and preferably approximately 1 to 10 h, in a culture of stem cells, in order to release the stem cells from their quiescent state.
According to an advantageous embodiment, an object of the invention is the use of means for blocking at least one inhibitor of the cell cycle in a test comparing, on the one hand, a culture of stem cells in a medium which contains the aforementioned means for blocking at least one inhibitor of the cell cycle and, on the other hand, the same culture of stem cells as that mentioned above in a medium which does not contain the aforementioned means for blocking at least one inhibitor of the cell cycle, in order to determine the degree of maturity of the aforementioned stem cells.
According to another embodiment, an object of the invention is the use of means for blocking at least one inhibitor of the cell cycle in a culture of stem cells, in order to release the stem cells from their quiescent state, followed by a step in which genes are transferred into the aforementioned stem cells.
Within the framework of the invention, the stem cells are advantageously hematopoietic cells and the means for blocking at least one inhibitor of the cell cycle advantageously comprises anti-TGF-&bgr;.
Another object of the invention relates to cells, in particular hematopoietic stem cells, containing all or part of a heterologous gene in their genome, which are likely to be obtained according to the method comprising the following steps:
the prestimulation of quiescent cells, in particular of quiescent hematopoietic stem cells, in a medium containing:
direct or indirect means for blocking at least one inhibitor of the cell cycle of the said cells, in particular of the said hematopoietic stem cells, for a period which is sufficient to release the cells, in particular the hematopoietic stem cells, from their quiescent state, and preferably does not exceed 72 h, and preferably does not exceed 36 h, and is in particular less than approximately 20 h, and is preferably approximately 1 to 15 h, and preferably approximately 1 to 10 h, and
substances, in particular cytokines, for permitting the activation of a cell cycle and/or subsequently the passage of at least one cell cycle,
the step comprising transfer by contacting the cells, in particular the hematopoietic stem cells, obtained in the previous step, with all or part of a heterologous gene, in a medium permitting the transfer of all or part of the heterologous gene into the genome of the cells, in particular of the hematopoietic stem cells, in order to obtain cells, in particular hematopoietic stem cells, including all or part of a heterologous gene and,
the possible recovery of the cells, in particular of the hematopoietic stem cells, obtained in
Hatzfeld Jacques
Klein Antoinette
Centre National de la Recherche Scientifique
Chan Christina Y.
VanderVegt F. Pierre
Young & Thompson
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