Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-11-13
2001-03-13
Fredman, Jeffrey (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06201115
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to amplifying sequences and vectors comprising these sequences, to the use of these vectors in compositions for the expression of nucleotide sequences in these transfected cells, and to the use of these products for therapeutic and vaccine applications.
2. Description of the Background
The desmine gene, one of the first muscle proteins to be detected in the developing embryo of mammals, was sequenced in 1989 by Li e. al. (Gene, 78,243-254).
Subsequently, an amplifying sequence of 280 base pairs, situated between the nucleotides −693 and −973 upstream from the transcription initiation site, was described (Li and Paulin, 1991, J.Biol.Chem., 266 6562-6570).
This amplifying sequence was identified by producing a series of plasmid constructions carrying the bacterial gene coding for chloramphenicol acetyl transferase (CAT), and by introducing them into myogenic or non-myogenic mice cells. The authors show that this amplifying region of 280 base pairs can activate both homologous and heterologous promoters regardless of their orientation, position or distance in relation to this sequence.
The study of this sequence was continued (Li et al., 1993,J.Biol.Chem., 268,10 403-10.415) and showed that this sequence contained two regions: one active in myotubes and the other active in myoblasts.
The part that is active in myotubes lies between positions −973 and −848, that is to say in a sequence of 125 nucleotides.
Within this sequence, a region lying between positions −910 and −870 is protected from the action of a DNase. This region contains binding sites of MEF2 and MyoD1 factors. No specific activity is indicated for this region of 40 nucleotides. In particular, no experiment has been carried out with constructions containing this isolated region. Mention has only been made that MyoD1 and MEF2 sites are necessary to obtain full amplifying activity in the myotubes.
The amplifying effect of the sequence upstream from the desmine gene has also been tested in a transgenic mouse (Li et al., 1993, Development, 117,947-959). A fragment of 1 kb containing the regulatory sequence upstream from the desmine gene is bound to a reporting gene coding for
Escherichia coli
&bgr;-galactosidase. The authors show that the amplifying activity of the desmine promoter is very considerable as the activity of &bgr;-galactosidase can be easily detected in tissue sections.
It arises from the prior art analysed above, that the amplifying properties of certain parts upstream from the human desmine gene are known. On the other hand, the regions responsible for this amplifying activity have not been identified.
They could therefore not be successfully modified with a view to improving their performance for the expression of genes or nucleotide sequences corresponding in whole or in part to these c-DNAs (complementary DNAs) coding for proteins of interest or for fragments of these proteins conveying epitopes able to induce protective antibodies or antibodies which recognise said epitopes. The preferable length of c-DNA or genomic DNA fragments is between 30 base pairs and 2 kb. They can be produced by chemical synthesis or by restriction enzyme cutting of DNA extracted from cells or micro-organisms selected by methods known to men of the art.
It is known that by introducing DNA into these eucaryote cells using the described methods, in particular by patent WO-90 11 092, it is not possible to obtain an extended or a high quality expression of said DNAs introduced in plasmid form.
SUMMARY OF THE INVENTION
The present invention brings an improvement in the expression level of genetic material introduced into transfected cells by means of constructions which use all or part of the activating sequences identified for the desmine gene and which can be used for the expression of any other gene or genetic material (genomic DNA fragments, c-DNA fragments etc.) in muscle tissue.
In particular, it is possible to use said sequences to express nucleotide sequences having an immunomodulatory function, such as cytokines, or immunogenic properties, such as the VPI protein of the polio virus or HBs protein of the hepatitis-B virus.
Another application lies in the area of gene therapy. In particular and in general, techniques which require the introduction of DNA into tissues necessitate the use of nucleotide sequences or vectors containing such, or liposomes strongly expressing the genes or cDNAs carried by these sequences. To date, this problem has not been solved in satisfactory manner.
The applicant therefore sought to determine which parts of the desmine gene regulatory sequences were responsible for the amplifying activity.
REFERENCES:
patent: 4983518 (1991-01-01), Schaffner et al.
patent: 5631134 (1997-05-01), Cantor
Drmanac et al, “DNA sequence determination by hybridization: A strategy for efficient large scale sequencing”, Science 260:1649-1652 (Jun. 1993).
Li et al, “Different Factors Interact with Myoblast-specific and Myotube-specific Enhancer Regions of the Human Desmin Gene”, The Journal of Biological Chemistry, vol. 268, No. 14, pp. 10403-10415, May 15, 1993.
Li et al, An E box in the desmin promoter cooperates with the E box and MEF-2 sites of a distal enhancer to direct muscle-specific transcription, The EMBO Journal, vol. 13, No. 15, pp. 3580-3589, 1994.
Li et al, “Regulation of the mouse desmin gene: transactivation by MyoD, myogenin, MRF4 and Myf5”, Nucleic Acids Research, vol. 21, No. 2, pp. 335-343, 1993.
Li et al, “Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice”, Development, No. 117, pp. 947-959, 1993.
Harris et al, “Strategies for targeted gene therapy”, Trends Genetics 12(10):400-405, (1995).
Marshall, “Gene therapy's growing pains”, Science 269:1050-1055, (1995).
Li Zhen Lin
Paulin Denise
Fredman Jeffrey
Institut Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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