Self-enhancing, pharmacologically controllable expression...

Details Self-enhancing, pharmacologically controllable expression... Self-enhancing, pharmacologically controllable expression...

1997-12-09

2002-05-07

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S320100, C435S325000, C536S023400, C536S024100

Reexamination Certificate

active

06383785

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to a self-enhancing nucleic acid construct that comprises at least one regulatory sequence coupled to at least one structural gene and at least one transcription factor protein gene.
Despite various approaches taken in gene therapy, preclinical and clinical investigation results so far obtained indicate that two fundamental problems remain unsolved. One is insufficient transgenic expression from target cells in vitro or in vivo due to intracellular shutdown processes. The second is inadequate control of transgenic expression.
In an attempt to rectify these shortcomings of the prior art, Rivera et al. (Nature Med. 2, 1028 (1996)), Belshaw et al. (PNAS USA 93, 4604 (1996)) and Ho et al. (Nature 382, 822 (1996)) have developed the first techniques for external control of transgenic expression. These approaches are based on adding the active compound rapamycin, which couples two subunits together. The resulting coupling product acts as a transcription factor. The first subunit constitutes a fusion protein formed between a DNA-binding protein and FK506-binding protein (FKBP), which protein also binds rapamycin. The second subunit is a fusion protein which is formed between a protein FRAP, which also binds to rapamycin, and the activation sequence of transcription factor protein NF-KB.
The functional transcription factor protein that is produced by the coupling of these two subunits with rapamycin in turn activates the sequence in the transgene for activating the structural gene.
The advantage of this external approach is that the expression of a structural gene can be switched on or switched off by adding or removing, respectively, the active compound rapamycin. However, this approach does not solve the problem of inadequate expression of a structural gene. Accordingly, the need remains for an approach to increase transgenic expression.
SUMMARY OF THE INVENTION
The invention fulfills the unmet needs of the art by providing nucleic acid constructs, compositions containing the constructs and methods of their use to achieve high transgenic expression. The invention does this by incorporating a positive feedback system within the nucleic acid construct itself. The resulting system is termed herein a “self-enhancing expression system.”
In one embodiment of the invention a nucleic acid construct is provided that comprises:
at least one first structural gene that encodes an active compound;
at least one second structural gene that encodes a transcription factor protein; and
at least one activation sequence comprised of at least one sequence that binds the transcription factor protein and at least one promoter sequence;
wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein.
In another embodiment of the invention a nucleic acid construct is provided that comprises:
at least one first structural gene that encodes an active compound;
at least one second structural gene that encodes a transcription factor protein; and
at least one activation sequence comprised of at least one sequence that binds said transcription factor protein and at least one pharmacological control module that comprises, in serial order, at least one promoter, at least one fusion protein gene coding for an activation domain of a transcription factor protein and coding for a coupling substance protein, at least one promoter, at least one fusion protein gene coding for a DNA-binding protein and coding for a second coupling substance protein, and at least one activation sequence that comprises a site for the DNA-binding protein
wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein.
In still another embodiment of the invention a nucleic acid construct provided that comprises:
at least one first structural gene that encodes an active compound;
at least one second structured gene that encodes at least one first fusion protein that comprises an activation domain of a transcription factor protein, and a sequence that binds a coupling substance;
at least one third structural gene that encodes at least one second fusion protein that comprises a protein that binds a coupling substance and a DNA-binding protein;
at least one activation sequence comprised of at least one sequence that binds said second fusion protein coupled to said first fusion protein by a coupling substance and at least one promotor sequence;
wherein each activation sequence activates the expression of at least one of aid structural genes.
Further embodiments readily will be apparent to the skilled artisan, pon reading the specification and appended claims.
The Self-enhancing Expression System
In its simplest embodiment, the novel self-enhancing expression system comprises the following components:
a) at least one sequence a) and/or a′) for binding a transcription factor protein d),
b) at least one promoter sequence b) and/or b′),
c) at least one structural gene c) encoding an active compound, and
d) at least one gene encoding a transcription factor protein d) which binds to component a).
In conformity with the invention, components a) and/or a′) and b) and/or b′) constitute a sequence for activating transcription of structural gene c) and for activating expression of transcription factor protein d).
In a preferred form according to the invention, the components can be arranged as depicted in FIG.
1
.
The binding sequences a) and a′) can be identical or different and bind the transcription factor d).
The promoter sequences [components b) and b′)] can be identical or different. Low-level activation of the promoter sequences b) and b′) results in low-level expression of the structural gene [component c)] and of the gene for the transcription factor protein d) [component d)]. Transcription factor protein d) made thereby, in turn binds to binding sequences [components a) and a′)]. This binding in turn activates promoter sequences b) and b′), bringing about an enhanced expression of both the structural gene and the gene for the transcription factor protein d). This enhanced expression itself results in a higher amount of transcription factor protein d), which feeds-back and further stimulates this system.
According to the invention, the arrangement of the components as depicted in
FIG. 1
can be supplemented (i.e. “appended” at the upstream end) with genes encoding a nuclear export signal (NES) and a nuclear export factor (NEF) at the 3′ end of the structural gene. Expression of the NEF is under the control of an additional promoter (component b′″). This additional promoter sequence may be identical to or different from any part of the activation sequences [components a) and b) and/or a′) and b′)] shown in FIG.
2
.
The nuclear export signal (NES) is a nucleotide sequence that impedes the transport of a pre-messenger RNA, which is linked to it, through the nuclear membrane. The NES consequently constitutes, on its own, a nuclear retention signal (NRS). However, if the NRS binds an export protein, here termed “nuclear export factor” or “NEF”, the NRS gains the function of an NES. This is because the nuclear export factor (NEF) mediates the transport of the NES-containing premessenger or messenger RNA out of the cell nucleus and into the cytoplasm. An NES-containing premessenger or messenger RNA consequently is secreted out of the cell nucleus by its being bound to the NEF as described by Fischer et al., Cell 82, 475 (1995).
In accordance with the invention, components c) and d) also can be linked to each other (i.e. “mutually linked”) by an internal ribosome entry site (IRES) instead of through linkage with components a′) and b′). Such IRESs lead to the expression of two DNA sequences which are linked to each other by way of the IRES.
The linkage by way of an IRES can be effected, for example, as depicted in FIG.
3
.
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