Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-12
2001-05-01
Guzo, David (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S320100, C435S325000
Reexamination Certificate
active
06225459
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the field of genetic engineering, particularly the field of gene therapy.
BACKGROUND ART
Gene therapy attempts to treat diseases caused by congenital or acquired genetic defects, namely gene disorders, by substituting or supplementing defective genes with normal genes. Although various treatment methods for gene therapy have been investigated, only a very limited number of the methods to date have met with success, including the treatment of adenosine deaminase (ADA) deficiency. This is mainly because the methods for efficiently introducing a therapeutic gene into target cells and the methods for expressing an introduced gene in the cell have not yet been established. So far, liposomes, HVJ-liposomes, retroviruses, and the like have been employed as carriers introducing the therapeutic gene into target cells. However, none of them are satisfactory in gene introduction efficiency. Various attempts have been made to increase the expression efficiency of the introduced gene, by, for example, improving the promoter. However, in each case the expression efficiency of the desired gene was still poor, and the quantity of the gene product was insufficient to afford gene therapy. Thus, in the field of gene therapy, a vector that enables a high level expression of a therapeutic gene in a variety of target cells has been sought.
In the field of hematology, Tec tyrosine kinase, a protein thought to participate in the proliferation of hematopoietic stem cells, is highly expressed in mouse liver, and is also expressed in the kidney, heart, and ovary (Oncogene, 5, 1781-1786 (1990)). In humans, Tec tyrosine kinase is highly expressed in a wide range of blood and lymphoid cells (LEUKEMIA, 8, 1663-1672 (1994)).
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a vector having incorporated within it a promoter functioning efficiently in a wide variety of blood and lymphoid cells, and the cells of organs such as the liver, thereby providing a gene therapy technique targeting blood and lymphoid cells.
The present inventors noted that Tec tyrosine kinase, a protein thought to participate in the proliferation of hematopoietic stem cells, is highly expressed in a wide variety of blood cells, lymphoid cells, and the cells of organs such as the liver. The inventors isolated the promoter of Tec tyrosine kinase from a mouse genomic DNA, constructed a vector with the promoter incorporated within it ligated an exogenous gene adjacently downstream of it, and attempted to express the exogenous gene in the cells. As a result, they found that the exogenous gene was actually expressed in the cells at a high level, thereby completing the present invention. Thus, the present invention relates to:
(1) a DNA comprising at least a part of the nucleotide sequence of SEQ ID No:1 and having promoter activity;
(2) an expression vector comprising the DNA of (1); and
(3) a cell carrying the expression vector of (2).
In the present invention, “a DNA having promoter activity” means a DNA having activity to induce the transcription of a DNA region adjacent to the DNA. The present invention includes a DNA containing a part of the nucleotide sequence of SEQ ID No:1, and having promoter activity as well as the DNA having the nucleotide sequence of SEQ ID No:1. Preferably, the DNA of the present invention has a general length of at least about 50 bp, more preferably at least about 100 bp, even more preferably at least about 200 bp, and most preferably at least about 300 bp.
According to the present invention, any vector for use in gene introduction can basically be used as a “vector” into which the DNA having promoter activity is to be introduced. Particularly in gene therapy, viral vectors, such as retrovirus vectors, adenovirus vectors, or adeno associated virus vectors, and non-viral vectors such as liposomes should be used.
Any cell is included in the “cell carrying the expression vector” of the present invention. Cells that have been confirmed to actually express the Tec tyrosine kinase gene, including blood cells and lymphoid cells, such as hematopoietic stem cells, myeloid cells, B cells, and T cells, and the cells of internal organs such as the liver, kidney, heart, and ovary should be used.
REFERENCES:
Mano, et al. Oncogene 8: 417-424, 1993.*
Dzerzak et al., “Lineage-specific expression of a human &bgr;-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells”Nature331:35-41 (1988).
Ido et al., “Gene therapy for hempatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human &agr;-fetoprotein gene promoter”Cancer Research55:3105-3109 (1995).
Mano et al., “A novel protein-tyrosine kinase, tec, is preferentially expressed in liver”Oncogene5:1781-1786 (1990).
Mano et al., “Expression of a novel form of Tec kinase in hematopoietic cells and mapping of the gene to chromosome 5 near Kit”Oncogene8:417-424 (1993).
Hasegawa Mamoru
Mano Hiroyuki
Sakata Tsuneaki
Clark & Elbing LLP
Dnavec Research Inc.
Guzo David
Leffers Gerald G.
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