Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-01-16
2001-12-25
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091410, C435S456000, C435S320100, C435S372300, C435S377000, C514S04400A
Reexamination Certificate
active
06333171
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the use of regulatory sequences which are derived from the CD4 gene in vectors for expressing a heterologous gene or a transgene, with these sequences conferring on the vectors, when the latter are integrated into cells of the hematopoietic system, in particular stem cells, or, more generally, bone marrow cells, a specific expression within mature T lymphocytes, excluding immature T lymphocytes, in particular following repertoire selection.
BACKGROUND
During the maturation of T cells, expression of the CD4 and CD8 receptors responds to a complex mechanism of regulation; differential expression of the CD4 and CD8 glycoproteins is coupled to the choice of one of the pathways of differentiation either into helper T lymphocytes or into cytotoxic T lymphocytes; thus, thymocytes, that is the hematopoletic cells which are involved in a T differentiation pathway, first of all possess a CD4− CD8− phenotype (double negative or DN thymocytes); they then acquire joint expression of the CD4 molecule and the CD8 molecule, thereby forming double positive (DP) CD4+ CD8+ thymocytes and, finally, this population differentiates into single positive (SP) lymphocytes which express ether CD4, in the case of the helper T lymphocytes, or CD8, in the case of the cytotoxic T lymphocytes. Thymocytes which bind to class I histocompatibility molecules will become CD4+ CD8+ cytotoxic T lymphocytes while those which bind to class II molecules will become CD4+ CD8− helper T lymphocytes; after this intrathymic process of repertoire selection, the thyrnocytes leave the thymus and reach the peripheral system: i.e. blood and lymphoid organs.
This differentiation process is summarized in Nicolic-Zugic, J, (1991) Immuncol. Today 12: 65-70.
Many groups are currently studying the regulation of the expression of the CD4 gene since elucidation of the mechanism of this expression could contribute towards understanding the manner in which T cell development is controlled.
Several groups are working on the regulatory sequences of the human or murine CD4 gene. Those most recent studies which may be cited are the following: Killeen et al. (EMBO Journal Vol. 12 No. 4 p. 1547, 1993) demonstrated that a transgene carrying human CD4 of a size of approximately 35 kb possessed all the requisite genomic sequences for controlling expression during development in transgenic mice. Two distinct regulatory elements in this fragment were identified as being critical for expressing the transgene: the first is an enhancer sequence which is situated either 13 kb upstream of the cap site of the murine CD4 or 6 kb upstream of the human CD4. Blum M. D. et al. (J. Exp. Med. (1993) 177 No. 5: 1343-1358) identified and sequenced the human CD4 enhancer while Sawada et al. (Mol. Cell. Biol; 11 55: 5506-5515, 1991) identified and sequenced the murine CD4 enhancer. These two groups demonstrated that expression of this enhancer is specific for the CD4 gene in mature or immature T lymphocytes; P. Salmon et al. (Proc. Natl. Acad. Sci., USA 90: 7739-7743 (1993)) analyzed the structure and the sequence of the human CD4 promoter and compared it with that of the murine CD4 promoter. They identified a fragment of approximately 1100 base pairs which exhibits the function of a specific CD4 promoter. Aligning this sequence with that of the murine CD4 promoter indicates a very similar structure, as
FIG. 1
of this latter paper shows.
Differentiation of the T lymphocytes employs other regulatory elements of the silencer type, whose presence leads to a decrease in, or cessation of, transcription of genes when they are in their vicinity. In Cell 77: 911-929, (1994), Sawada et al. demonstrated the existence of a silencer in an initial intron of the CD4 gene, one of the functions of which silencer is to extinguish transcription of the CD4 gene in mature CD8+ T lymphocytes.
Finally, the human CD4 gene was analyzed by Z. Hanna et al. (Mol. and Cell. Biology (1994) p. 1084-1094) in a construct which comprises at least 3 introns of a total length of 12 kb.
All the abovementioned recent studies indicate that expression of the CD4 cene is controlled in a similar manner in human and murine cells; nevertheless, the relationships between the different regulatory elements and their functions have not been elucidated.
SUMMARY OF THE INVENTION
The present invention results from the discovery, which was made by the inventors, of a combination of regulatory sequences which exhibit properties which are unexpected and entirely unforeseeable: this combination, which is composed of genetic elements which are derived from sequences which are located 5′ of the CD4 gene, where appropriate combined with the cDNA of the CD4 gene, is expressed in a restricted manner in mature T cells and NK cells in a transgenic mouse model; it has not been possible to detect any expression in immature T cells, including double positive or double negative thymocytes. This group of regulatory sequences is the first to have this specificity of expression, something which is especially important, in particular for use in the context of gene therapy, for programs which would require a gene to be expressed specifically in mature T lymphocytes.
There was nothing in the state of the art to suggest that it was possible for such a combination to lead to this result.
The present invention results from the discovery that while the combination of the CD4 enhancer and promoter promotes expression of a reporter gene, in this case the cDNA of the human CD4 gene, in mature mouse thymocytes, this reporter gene is not expressed in immature CD4+ CD8+ double positive thymocytes; this result is surprising in that it implies the existence of an additional regulatory element which is as yet unknown and which would enable the CD4 gene to be expressed at the immature stage; this element, which is thought to act in “cis”, is thought to be missing from the construct since no mouse regulatory element which would be able to act in trans is effective for bringing about such expression in the immature cells.
A foreseeable result is that linking the silencer described by Sawada et al. 1994 (see above) to this combination forms a cassette for expressing a heterologous gene exclusively in CD4+ CD8− SP helper T cells.
The present invention relates to a system for expressing a protein or a heterologous gene, which system comprises a recombinant vector which can be used for transducing cells of the hematopoietic line, in particular blood or marrow stein cells, in such a way that the cell which has been thus transduced will only express the protein or the heterologous gene which is carried by the vector in mature T cells after repertoire selection, to the exclusion of expression in immature T cells, in particular CD4− CD8− and CD4+ CD8+ cells; said vector of the expression system is provided with all the sequences which are required for its expression and it is characterized in that it contains at least one enhancer of a CD4 gene which is derived from the same species or from a different species.
The vector of the expression system may also contain, combined with the enhancer, a promoter which consists of one of the following sequences:
the promoter of the human CD4 gene, as depicted in
FIG. 6
, or
the sequence contained between nucleotides −496 and +16 in the P. SALMON (1993, see above) numeration (FIG.
6
), or
the sequence contained between nucleotides −165 and +16 (P. SALMON, 1993, see above) (FIG.
6
), or
any sequence which is derived from one of the preceding sequences by means of the addition, deletion or substitution of nucleotides without substantial modification of the expression of the heterologous protein under the control of this promoter.
A specific example of an efficient enhancer, within the context of this invention, is that of murine CD4, which enhancer consists of 339, or all or part of the 339 base pairs and is described in the abovementioned Sawa
Boyer Olivier
Klatzmann David
Salmon Patrick
Gollin Michael A.
Saunders David
Tung Mary Beth
Universite Pierre Et Marie Curie
Venable
LandOfFree
CD4 gene regulatory sequences specifically expressed in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with CD4 gene regulatory sequences specifically expressed in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and CD4 gene regulatory sequences specifically expressed in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2587229