Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2001-03-02
2003-08-19
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200, C424S093210, C435S320100, C435S325000, C435S455000, C435S456000, C536S024100
Reexamination Certificate
active
06608037
ABSTRACT:
BACKGROUND TO THE INVENTION
The present invention relates to a T cell factor (TCF)-responsive element, a gene and uses of the TCF-responsive element or nucleic acid construct in assays nucleic acid construct comprising a TCF-responsive element and a therapeutic and therapy.
TCFs are a family of transcription factors within the High Mobility Group (HMG) of DNA-binding proteins (Love et al., Nature, 376, 791-795,1995). The family includes TCF-1, TCF-3 and TCF-4 which are described in van der Wetering et al, (EMBO J., 10, 123-132,1991), EP-A-0 939 122 and Korinek et al. (Science, 275, 1784-1787,1997). TCF-4 has been shown to be involved in tumorigenesis related to Wnt/Wingless signalling. TCF and LEF-1 (lymphoid enhancer factor-1) are considered to mediate a nuclear response to Wnt signals by interacting with &bgr;-catenin. Wnt signalling and other cellular events that increase the stability of &bgr;-catenin are considered to result in transcriptional activation of genes by LEF-1 and TCF proteins in association with &bgr;-catenin. In the absence of Wnt signalling, LEF-1/TCF proteins repress transcription in association with Groucho and CBP (CREB binding protein).
In the absence of Wnt signalling, &bgr;-catenin is found in two distinct multiprotein complexes. One complex, located at the plasma membrane, couples cadherins (calcium dependent adhesion molecules) with the actin cytoskeleton whereas the other complex (containing the proteins adenomatous polyposis coli protein (APC), axin and glycogen synthase kinase 3&bgr; (GSK3&bgr;)) targets &bgr;-catenin for degradation. Wnt signalling antagonises the APC-axin-GSK3&bgr; complex, resulting in an increase in the pool of free cytoplasmic &bgr;-catenin. The free cytoplasmic &bgr;-catenin can translocate to the nucleus where it binds LEF-1/TCF factors and activates Wnt target genes. The regulation of LEF-1/TCF transcription factors by Wnt and other signals is discussed in Eastman et al, (Current Opin. Cell Biology, 11, 233-240, 1999).
The APC gene is a tumour supressor gene that is inactivated in most colorectal cancers. Mutations of APC are considered to cause the accumulation of free &bgr;-catenin, which then binds TCF causing increased transcriptional activation of genes including genes important for cell proliferation (e.g. cyclin D1 (Tetsu et al., Nature 398, 422-426, 1999 and Shtutman et al., PNAS USA, 96, 5522-5527, 1999) and c-myc (He et al., Science, 281, 1509-1512,1998)). The involvement of APC in tumour development is discussed in He et al, (supra).
TCFs are known to recognise and bind TCF binding elements which have the nucleotide sequence CTTTGNN, wherein N indicates A or T (van der Wetering et al, supra).
TCF reporter genes have been constructed and are described in Korinek et al, (Science, 275,1784-1787,1997), Morin et al, (Science, 275, 1787-1790, 1997), EP-A-0 939 122 and WO 98/41631. The TCF reporter gene is said to comprise three TCF binding elements upstream of either a minimal c-Fos promoter driving luciferase expression or a minimal herpes virus thymidine kinase promoter driving chloramphenicol acetyl-transferase expression. He et al (supra) discloses TCF reporter gene constructs comprising four TCF binding elements inserted into pBV-Luc.
There is a need for an effective treatment of cancers associated with a deregulation of the Wnt signalling pathway. Such cancers include most colorectal cancers, approximately 30% of melanomas and some breast, prostate and hepatocellular carcinomas.
There is also a need for a TCF response element which when linked to an expressible gene gives improved levels of expression and specificity.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid construct comprising:
a TCF response element comprising:
at least one TCF binding element having the sequence CTTTGNN, wherein N is A or T; and
a promoter,
and an expressible therapeutic gene operably linked to the TCF response element,
wherein the TCF response element enables inducible expression of the operably linked therapeutic gene.
The term “inducible expression” as used herein means the level of expression obtained using the TCF response element is induced (i.e. increased) when one or more TCF/&bgr; catenin heterodimers binds to one or more of the TCF binding elements. Preferably the level of expression is increased by at least 15 fold, more preferably at least 25 fold and most preferably at least 30 fold.
The term “operably linked” as used herein refers to a cis-linkage in which the gene is subject to expression under control of the TCF response element.
The expressible gene comprises the necessary elements enabling gene expression when operably linked to the TCF response element, such as splice acceptor sequences, internal ribosome entry site sequences (IRES) and transcription stop sites. Such elements are well known to those skilled in the art.
It has been found that by using the nucleic acid construct of the present invention that expression of the operably linked therapeutic gene is only induced when TCF/&bgr; catenin heterodimers are present and capable of activating transcription. As cells that have become cancerous due to the deregulation of the Wnt signalling pathway have TCF/&bgr; catenin heterodimers, which activate transcription, expression of the therapeutic gene will be induced. Accordingly, the nucleic acid construct of the present invention acts as a tumour selective promoter.
The nucleic acid construct of the present invention exhibits highly selective expression in that it gives no induction of expression of an operably linked gene above the background level in the absence of TCF/&bgr; catenin heterodimers or a functionally equivalent transcription activating factor.
The therapeutic gene can be any gene that on expression gives a therapeutic benefit. Preferred therapeutic genes include genes encoding toxins such as ricin and diphtheria toxin, and prodrug activating enzymes such as nitroreductases that activate CB1954, cytosine deaminase which activates 5-fluorocytosine, cytochrome P-450 which activates cyclophosphamide and paracetamol, and thymidine kinase which activates ganciclovir. Preferably the therapeutic gene encodes a nitroreductase. Suitable nitroreductases are described in EP-A-0638123 and Watanabe eta/, (NAR, 18, 1059, 1990). Other preferred therapeutic gene include genes encoding immunomodulatory agents such as IL-2, IL-12, GMCSF, B7-1 and B7-2 co-stimulatory molecules; genes encoding tumour suppressers such as RB, p53 and p16; and genes encoding apoptotic genes such as Bax, FasL and caspases.
The promoter can be any promoter that gives a desired level of expression of the operably linked gene. Suitable promoters include the SV40 promoter, the E1B promoter, and the c-Fos promoter. Preferably the promoter is the basal TATA box of the E1B promoter.
Preferably the TCF response element contains at least three and more preferably at least five TCF binding elements. It has been found that by using at least three and more preferably at least five TCF binding elements that an unexpected increase in expression can be obtained compared to a TCF response element containing fewer binding elements. This increase in expression is desirable for the production of a therapeutically effective amount of an encoded product.
Preferably the TCF response element comprises between 5 and 15 TCF binding elements, more preferably between 5 and 10 TCF binding elements and most preferably 5 TCF binding elements.
The TCF binding elements are preferably separated from each other by between 3 and 20 nucleotides, more preferably by between 3 and 14 and most preferably by between 10 and 12 nucleotides.
It is further preferred that the TCF binding elements are so spaced from each other as to be equally distributed radially around the DNA helix, especially when the promoter is the E1B promoter.
It is preferred that the TCF binding element closest to the promoter is between 140 and 10 nucleotides from the TATA box of the promoter. It is further preferred that the TCF binding element closest to the promoter is between 100 and 1
Lipinski Kai S.
Wrighton Christopher J.
Young Lawrence S.
Cozen O'Connor P.C.
M.L. Laboratories PLC
Priebe Scott D.
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