Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-09-24
2003-06-10
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C536S023100
Reexamination Certificate
active
06576758
ABSTRACT:
BACKGROUND OF THE INVENTION
The present application relates to nucleic acid constructs which can be used in genetic manipulation and in particular in the prophylaxis or therapy of diseases (termed gene therapy in that which follows). In gene therapy, genes which are to be expressed in an organism are introduced into the organism. The regulation of the expression of these genes is of significance for the prophylactic or therapeutic effect of the gene therapy.
Regulators of the expression of a gene are described in Patent Applications PCT/GB95/02000, PCT/EP95/03370, PCT/EP95/03371, PCT/EP95/03368 and PCT/EP95/03339. These regulators comprise an activator sequence whose function is, for example, the cell-specific or virus-specific activation of basal transcription. The DNA sequence of this activator sequence is linked by its 3′ end to the 5′ end of a promoter module. The structural gene is in turn linked by its 5′ end to the 3′ end of the promoter module.
The promoter module is composed of nucleic acid sequences for binding the transcription factors of the CDF and CHF families or of the E2F and CHF families. In the G0 and G1 phases of the cell cycle, this binding leads to inhibition of the upstream activator sequence and consequently to inhibition or transcription of the structural gene which is located downstream (i.e. in the direction of transcription).
In the G0 and G1 phases of cell division, the DNA which is contained in the cell is in the diploid state. In the G0 phase, the cell is at rest, while in the G1 phase its cell cycle progression is inhibited. The G1 phase is followed by the S phase, in which DNA synthesis takes place and in which the genome is replicated. There then follows the G2 phase, in which the cell is in the tetraploid state. The G2 phase is followed by cell division (mitosis=M phase). The daughter cells then pass into the G0 state or G1 state.
The combination of a cell-specific or virus-specific activator sequence and a promoter module which inhibits this activator sequence in the G0 and G1 phases consequently makes it possible to regulate the expression of a structural gene in a cell-specific or virus-specific and also cell cycle-specific (i.e. restricted to the S and G2 phases) manner.
The combination of an activator sequence and a promoter module is termed a chimeric promoter. While there are many possible applications for chimeric promoters in gene therapy, there are also a number of limitations arising from shortcomings. Examples of these limitations are:
a weak activator sequence which brings about too low a transcription of the structural gene,
the use of an activator sequence which cannot be inhibited by the chosen promoter module in a sufficiently cell cycle-dependent manner,
the restriction to two (for example cell-specific or virus-specific and cell cycle-specific) regulators of the transcription of the structural gene, and
inadequate intracellular transport of the transcription product of the structural gene which has been introduced into the cell.
The present invention overcomes the shortcomings of using known chimeric promoters to express foreign genes by providing the nucleic acid constructs of the present invention, which enable the regulated expression of foreign genes in host cells.
SUMMARY OF THE INVENTION
An object of the present invention is to make available nucleic acid constructs which enable the expression of foreign genes (transgenes) to be regulated in a precise manner in the host cells. The present invention therefore relates to nucleic acid constructs in which precise regulation of the transgene is achieved by at least one nucleic acid sequence exhibiting a first mutation which inhibits the proper expression of a transgene, and in which at least one further nucleic acid sequence exhibits a second mutation which abolishes the inhibition due to the mutation in the first nucleic acid sequence(s).
More particularly, the nucleic acid construct of the present invention regulates expression of a transgene in a host cell utilizing alternative constructs. When the nucleic acid sequence containing the first mutation is a transgene (b) containing a mutation which inhibits the transcription and/or the translation of said transgene or inhibits the function of the pharmacologically active compound, then the nucleic acid construct further comprises a first promoter or enhancer sequence (a), which is located upstream from the 5′ end of the transgene, or alternatively, when the nucleic acid sequence containing the first mutation is a first promoter or enhancer sequence (a′), which contains a mutation which inhibits the function of the first promoter, then the nucleic acid construct further comprises a transgene (b′) encoding a pharmacologically active compound. In either instance, at least one nucleic acid sequence containing the second mutation abolishes the inhibition due to the first mutation.
These nucleotide sequences are under the control of identical or different promoter sequences, so that a transgene can only be expressed when all these promoter sequences are activated.
Preferably, the novel nucleic acid constructs comprise at least the following components, listed in the direction of reading, from the 5′ end to the 3′ end:
a first (I) promoter or enhancer sequence (a) which is nonspecific, cell-specific or virus-specific, or which can be activated by tetracycline or metabolically and/or cell cycle-specifically, which activates the transcription of a transgene and which can contain a mutation (a′) which inhibits the function of the promoter,
a transgene (b′) which, as structural gene, encodes an active compound and can contain a mutation (b) which stops the transcription and/or the translation of this structural gene or inhibits the function of the product of the structural gene,
a second (II) promoter or enhancer sequence (c) or (c′) which is nonspecific, cell-specific or virus-specific, or which can be activated metabolically and/or cell cycle-specifically, which activates the basal transcription of the component (d) or (d′) and which can contain a mutation which inhibits the function of the promoter,
a gene for a tRNA (suppressor tRNA) or a regulatory protein (d) or (d′) for relieving the mutation in one or more of the promoters or in the transgene.
The first (I) promoter sequence or enhancer sequence (a) and the second (II) promoter sequence or enhancer sequence (c) can be identical or different, and at least one of the components (a) and (c) can be nonspecifically, cell-specifically or virus-specifically activatable, be activatable by tetracycline or metabolically, in particular by hypoxia, or be cell cycle-specifically activatable.
The invention also relates to a nucleic acid construct wherein the component (b) exhibits a nuclear retention signal whose cDNA is linked, at the 5′ end, directly or indirectly, to the 3′ end of the structural gene, and wherein the transcription product of the nuclear retention signal exhibits a structure for binding a nuclear export factor.
The invention also relates to a nucleic acid construct which, in addition to components (a) to (d), exhibits the following components:
a further promoter or enhancer sequence (i) which activates the basal transcription of a nuclear export factor, and
a nucleic acid which encodes a nuclear export factor (k) which binds to the transcription product of the nuclear retention signal (h) and thereby mediates transport of the transcription product of the transgene out of the cell nucleus into the cytoplasm.
Within the context of the present invention, at least one of the promoter sequences or enhancer sequences (a) and (c) can be a chimeric promoter in which the promoter module CDE-CHR or E2FBS-CHR can interact with an upstream activator sequence which is cell-specifically, virus-specifically or metabolically activatable and can thereby influence, in particular inhibit, the expression of a downstream gene.
The components (a) and (c) can also be activator-responsive prom
Mueller Rolf
Sedlacek Hans-Harald
Seifart Klaus-Heinrich
Aventis Pharma Deutschland GmbH
Heller Ehrman White and McAuliffe
Loeb Bronwen M.
Yucel Remy
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