Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1996-05-16
2001-02-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S320100, C435S325000, C536S023100, C536S023200, C536S023400, C536S023500, C536S023700, C424S093100, C424S093200, C424S093600
Reexamination Certificate
active
06194211
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a transcriptional regulatory sequence useful in gene therapy.
BACKGROUND
Colorectal carcinoma (CRC) is the second most frequent cancer and the second leading cause of cancer-associated deaths in the U.S. and Western Europe. The overall five-year survival rate for patients has not meaningfully improved in the last three decades. Prognosis for the CRC cancer patient is associated with the depth of tumor penetration into the bowel wall, the presence of regional lymph node involvement and, most importantly, the presence of distant metastases. The liver is the most common site for distant metastasis and, in approximately 30% of patients, the sole initial site of tumor recurrence after successful resection of the primary colon cancer. Hepatic metastases are the most common cause of death in the CRC cancer patient.
The treatment of choice for the majority of patients with hepatic CRC metastasis is systemic or regional chemotherapy using 5-fluorouracil (5-FU) alone or in combination with other agents such as leviamasole. However, despite extensive effort, there is still no satisfactory treatment for hepatic CRC metastasis. Systemic single- and combination-agent chemotherapy and radiation are relatively ineffective emphasizing the need for new approaches and therapies for the treatment of the diseases.
A gene therapy approach is being developed for primary and metastatic liver tumors that exploits the transcriptional differences between normal and metastatic cells. This approach involves linking the transcriptional regulatory sequences (TRS) of a tumor associated marker gene to the encoding sequence of an enzyme, typically a non-mammalian enzyme, to create an artificial chimaeric gene that is selectively expressed in cancer cells. The enzyme should be capable of converting a non-toxic prodrug into a cytotoxic or cytostatic drug thereby allowing for selective elimination of metastatic cells.
The principle of this approach has been demonstrated using an alpha-fetoprotein/Varicella Zoster virus thymidine kinase chimaera to target hepatocellular carcinoma with the enzyme metabolically activating the non-toxic prodrug 6-methoxypurine arabinonucleoside ultimately leading to formation of the cytoxic anabolite adenine arabinonucleoside triphosphate (see Huber et al, Proc. Natl. Acad. Sci U.S.A., 88, 8039-8043 (1991) and EP-A-0 415 731).
For the treatment of hepatic metastases of CRC, it is desirable to control the expression of an enzyme with the transcriptional regulatory sequences of a tumor marker associated with such metastases.
CEA is a tumor associated marker that is regulated at the transcriptional level and is expressed by most CRC tumors but is not expressed in normal liver. CEA is widely used as an important diagnostic tool for postoperative surveillance, chemotherapy efficacy determinations, immunolocalisation and immunotherapy. The TRS of CEA are potentially of value in the selective expression of an enzyme in CEA
+
tumor cells since there appears to be a very low heterogeneity of CEA within metastatic tumors, perhaps because CEA may have an important functional role in metastasis.
The cloning of the CEA gene has been reported and the promoter localised to a region of 424 nucleotides upstream from the translational start (Schrewe et al, Mol. Cell. Biol., 10, 2738-2748 (1990) but the full TRS was not identified.
SUMMARY
In the work on which the present invention is based, CEA genomic clones have been identified and isolated from the human chromosome 19 genomic library LL19NL01, ATCC number 57766, by standard techniques described hereinafter. The CEA enhancers are especially advantageous for high level expression in CEA-positive cells and no expression in CEA-negative cells.
According to one aspect, the present invention provides a DNA molecule comprising the CEA TRS but without associated CEA coding sequence.
According to another aspect, the present invention provides use of a CEA TRS for and targeting expression of a heterologous enzyme to CEA
+
cells. Preferably the enzyme is capable of catalysing the production of an agent cytotoxic or cytostatic to the CEA
+
target cells.
As described in more detail hereinafter, the present invention have sequenced a large part of the CEA gene upstream of the coding sequence. As used herein, the term “CEA TRS” means any part of the CEA gene upstream of the coding sequence which has a transcriptional regulatory effect on a heterologous coding sequence operably linked thereto.
Certain parts of the sequence of the CEA gene upstream of the coding sequence have been identified as making significant contributions to the transcriptional regulatory effect, more particularly increasing the level and/or selectivity of transcription. Preferably the CEA TRS includes all or part of the region from about −299b to about +69b, more preferably about −90b to about +69b. Increases in the level of transcription and/or selectivity can also be obtained by including one or more of the following regions: −14.5 kb to −10.6 kb, preferably −13.6 kb to −10.6 kb, and/or −6.1 kb to −3.8 kb. All of the regions referred to above can be included in either orientation and in different combinations. In addition, repeats of these regions may be included, particularly repeats of the −90b to +69b region, containing for example 2,3, 4 or more copies of the region. The base numbering refers to the sequence of FIG.
6
. The regions referred to are included in the plasmids described in FIG.
5
B.
DETAILED DESCRIPTION
Gene therapy involves the stable integration of new genes into target cells and the expression of those genes, once they are in place, to alter the phenotype of that particular target cell (for review see Anderson, W. F. Science 226, 401-409 (1984) and McCormick, D. Biotechnology 3, 689-693, (1985)). Gene therapy may be beneficial for the treatment of genetic diseases that involves the replacement of one defective or missing enzyme, such as; hypoxanthine-guanine phosphoribosyl transferase in Lesch-Nyhan diseases, purine nucleoside phosphorylase in severe immunodeficiency disease, and adenosine deaminase in severed combined immunodeficiency diseases.
It has now been found that it is possible to selectively arrest the growth of, or kill, mammalian carcinoma cells with prodrugs, i.e. chemical agents capable of selective conversion to cytotoxic (causing cell death) or cytostatic (suppressing cell multiplication and growth) metabolites. This is achieved by the construction of a molecular chimaera comprising a “target tissue-specific” TRS that is selectively activated in target cells, such as cancerous cells, and that controls the expression of a heterologous enzyme. This molecular chimaera may be manipulated via suitable vectors and incorporated into an infective virion. Upon administration of an infective virion containing the molecular chimaera to a host (e.g., mammal or human), the enzyme is selectively expressed in the target cells. Administration of prodrugs (compounds that are selectively metabolised by the enzyme into metabolites that are either further metabolised to or are, in fact, cytotoxic or cytostatic agents) can then result in the production of the cytotoxic or cytostatic agent in situ in the cancer cell. According to the present invention CEA TRS provides the target tissue specificity.
Molecular chimaeras (recombinant molecules comprised of unnatural combinations of genes or sections of genes), and infective virions (complete viral particles capable of infecting appropriate host cells) are well known in the art of molecular biology.
A number of enzyme prodrug combinations may be used for the above purpose, providing the enzyme is capable of selectively activating the administered compound either directly or through an intermediate to a cytostatic or cytotoxic metabolite. The choice of compound will also depend on the enzyme system used, but must be selectively metabolised by the enzyme either directly or indirectly to a cytotoxi
Huber Brian
Richards Cynthia Ann
Bennett Virginia C.
Glaxo Wellcome Inc.
Guzo David
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