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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

Definitions

• Cancer is a complex biological phenomenon that is thought to arise out of a multi- step process of genetic and epi-genetic alterations in the cellular DNA, ultimately resulting in the transformation of the cell and its uncontrolled growth, division and migration. Identifying the aberrant molecular pathways that mediate cellular transformation has been a major challenge in understanding how malignancy develops. The advent of functional genomics has given scientists the prospect of examining global changes in gene expression, providing molecular phenotypes that could potentially help in establishing more effective techniques of diagnosis and prognosis in a variety of cancers 1"3 .
• microarrays to decipher the molecular events that result in tumour progression has proven a more difficult task, particularly since microarray data only provides a snapshot into a cell's transcriptome at a specific point in time.
• microarray data can have wider applications in the study of cancer, particularly with the advent of comparative genomic microarray analysis 4 .
• gene expression data can be mapped to chromosomes, revealing potential sites of chromosomal aberrations, e.g. amplifications or deletions, which may predominate in particular types of cancer.
• the present invention provides a method of producing a promoter element capable of regulating gene expression in a cell or tissue type of interest, said method comprising:
• each vector comprises two or more of said transcription factor regulatory elements of (a) and a minimal promoter operably linked to an antibiotic resistance gene;
• the invention also provides vectors capable of gene expression and promoter elements capable of regulating gene expression in a cell or tissue of interest, wherein said vector or promoter element is identified by a method of the invention. Also provided is the use of such a vector or promoter element for targeting expression of a gene to a cell or tissue of interest.
• the invention provides a method of expressing a gene in a cell or tissue of interest, comprising (a) identifying a promoter element capable of directing gene expression in said cell or tissue using a method of the invention; (b) generating an expression vector comprising said promoter element operably linked to said gene; and (c) transfecting said cell or tissue of interest with said vector and allowing gene expression to occur.
• Figure 1 shows a schematic diagram of one way in which the method of the invention may be carried out.
• Microarray analysis of cancer tissue/cells compared to normal tissue/cells is conducted in order to identify differentially expressed genes, from the whole genome, which are specifically upregulated in cancer cells.
• the cis-regulatory elements driving the expression of upregulated genes are identified and synthesised as double stranded DNA oligonucleotides .
• the DNA oligonucleotides are randomly ligated together and cloned into a minimal promoter expression cassette driving the transcription of an antibiotic selection marker.
• a library of clones with randomly ligated cis regulatory elements is thus created.
• the library of clones is either transfected in the context of a retroviral vector into recipient cancer cells, or the plasmids comprising the expression cassette are first isolated from individual clones and transfected into recipient cancer cells.
• Retro virally transduced or plasmid-transfected recipient cancer cells are subjected to high concentrations of the relevant antibiotic and synthetic promoter sequences are rescued from highly-expressing recipient cancer cells by PCR.
• microarray technology is to truly result in the design of tailored therapies to individual cancers or even patients, as has been heralded, it is important that the functional genomics methodology that was designed for the identification of signalling and transcription networks be applied to the design of cancer-specific promoters so that effective gene therapeutic strategies can be formulated.
• Described herein are methods whereby data obtained from functional genomics experiments, such as microarray analysis, are analysed using widely available bioinformatics software tools, which function to find over-represented cis promoter elements, in order to design synthetic promoters that are only active in cancer cells. This represents a major leap forward in the design of cancer-specific promoters that can subsequently be used in the study of cancer, or in the design of safe and effective genetic therapy of human malignancies.
• the regulation of gene expression in eukaryotes is highly complex and often occurs through the coordinated action of multiple transcription factors.
• the use of trans- factor combinations in the control of gene expression allows a cell to employ a relatively small number of transcription factors in the regulation of disparate biological processes.
• the present invention is based on the premise that the elucidation of disease- specific transcriptional programs will afford us the opportunity to construct synthetic conditional promoter elements that can be used in gene therapy to drive restricted gene expression in pathologic sites of interest. Integrative computational approaches may be used to identify transcriptional programs active in specific cancer indications, and will consequently allow for the rational design of synthetic promoter elements designed to drive highly cytotoxic genes for an effective anti-cancer therapeutic approach.
• microarray data obtained by experimentation may be used in order to identify the regulatory sequences overrepresented in clusters of genes found to be upregulated in cancer stem cells.
• bioinformatics tools examples of which are given in table 1, may be used to screen for cw-regulatory elements.
• such tools function by comparing gene expression profiles between differentially regulated genes and examining upstream sequences, available through genome sequence resources.
• the untranslated regions of specific genes are compared between species and the most highly conserved sequences are returned and proposed to be potential czs-elements.
• a combination of all available approaches may employed in order to identify regulatory sequences that predominate in the profile of specific cell or tissue types, for example in cancer stem cells. The most common sequences identified are then used as the building blocks employed in the design of synthetic promoters.
• the invention relates to assays carried out on a cell or tissue type of interest.
• a cell or tissue type of interest may be any type of cell, or plurality of cells such as a tissue.
• This may be a prokaryotic cell or cells or a eukaryotic cell, cells or tissue.
• a suitable eukaryotic cell may be derived from an organism, such as an animal, such as a mammal and preferably a human. Such a cell or tissue may have been taken directly from such an organism or may be derived therefrom.
• the cell or tissue may be from a primary, secondary or immortalised cell line or culture that is derived from such an organism.
• the cell or tissue may be a naturally occurring cell or tissue or may have been artificially manipulated.
• a cell or tissue may be manipulated by exposure to altered environmental or disease-specific conditions.
• a cell or tissue may be manipulated by exposing it to an agent, such as a biological ligand or chemical agent.
• the biological ligand may be any biological molecule that is capable of having an effect on the cell, particularly an effect on gene transcription.
• a biological ligand may be a molecule that is capable of binding to the cell or acting within the cell.
• a biological ligand may, for example, be a polypeptide, protein, nucleic acid or carbohydrate molecule. Suitable biological ligands include hormones, growth factors and neurotransmitters.
• the chemical agent may be any agent capable of acting on the cell, preferably leading to a change in gene transcription within the cell.
• the chemical agent may, for example, be a chemotherapeutic drug or a therapeutic small molecular drug.
• the cell or tissue may from an abnormal or disease source.
• the cell or tissue may be taken from, or derive from, an organism suffering from a disease.
• the cell or tissue is from a tissue or organ that is affected by the disease.
• the cell or tissue may be taken from a tumour.
• the cell may be from, or derived from, a tumour cell line in vitro.
• TFREs transcription factor regulatory elements
• a suitable TFRE is a nucleic acid molecule that is recognised by a transcription factor.
• a TFRE may comprise a sequence to which a transcription factor can bind.
• a TFRE may comprise a cis-acting region.
• transcription factor is meant any factor, such as a protein, that can bind to such a cis-acting region and regulate either positively or negatively the expression of a gene.
• a transcription factor may bind upstream of the coding sequence of a gene to either enhance or repress transcription of the gene by assisting or blocking RNA polymerase binding.
• Many transcription factors are well known in the art and include STAT, E2F, Oct-4, Nanog, Brachury, Pax genes, Sox2 and MCEF.
• a TFRE comprises a nucleic acid sequence preferably, a double stranded DNA sequence.
• a TFRE may comprise a cis-acting region and may also comprise additional nucleic acids.
• the core six to eight nucleotides of promoter and enhancer elements may be sufficient for the binding of their corresponding frvms-activating factors. Indeed, in some cases this short oligonucleotide element is sufficient to drive gene expression alone 9 .
• a transcription factor binding site may consist of 6 to 8 nucleic acids.
• a TFRE comprising that site will be at least 6 to 8 nucleic acids in length.
• a TFRE of the invention is preferably 6 or more, 8 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more nucleic acids in length.
• a TFRE of the invention may be 100 or less,
• a suitable TFRE is one that is active in the cell or tissue of interest.
• a TFRE may be identified as being associated with a gene that is expressed in the cell or tissue of interest.
• a TFRE may be associated with a gene that is differentially expressed in that cell or tissue, when compared with another cell or tissue.
• differential expression of a gene may be seen by comparing the expression of the gene in two different cells or tissues, or in the same cells or tissues under different conditions. Expression in one cell or tissue type may be compared with that in a different, but related, tissue type.
• the expression of genes in that cell or tissue may be compared with the expression of the same genes in an equivalent normal or untreated cell or tissue. This may allow the identification of genes that are differentially regulated between the two cell or tissue types.
• a TFRE that is associated with such a gene is generally located close to the coding sequence of the gene within the genome of the cell. For example, such a TFRE may be located in the region immediately upstream or downstream of that coding sequence. Such a TFRE may be located close to a promoter or other regulatory sequence that regulates expression of the gene. The location of a TFRE may be determined by the skilled person using his knowledge of this field and the methods described herein.
• Suitable TFREs may thus be identified by analysis of the cell or tissue of interest.
• Genes that are differentially expressed in the cell or tissue of interest may be identified by routine methods. For example, routine methods may be used to compare the expression profile of genes in the cell or tissue of interest with that in other cell or tissue types which may act as a control. Genes that are up-regulated or down-regulated in the cell or tissue of interest may thus be identified.
• Such an analysis may make use of, for example, microarray analysis or serial analysis of gene expression (SAGE).
• Such an analysis may be carried out using a sample of expressed molecules from the cell or tissue of interest or using all the expressed molecules from the cell or tissue of interest. For example, in one embodiment, such an analysis may be carried out using the total RNA content of the cell or tissue of interest.
• the methods of the invention may thus be used to analyse expression from the entire genome of the cell or tissue of interest.
• Such an analysis may be used to assess the expression of a wide variety of genes, or a subgroup of genes.
• a selection of genes may be used that is known to be regulated by a wide variety of different transcription factors, or each gene by only one or two transcription factors.
• the sequences upstream of the differentially expressed genes may be screened for cis-regulatory elements.
• Those cis-regulatory elements which control expression of differentially expressed genes are considered to be active in the cell or tissue of interest.
• the transcription factor(s) which control their activity must be present in that cell type. This therefore allows the identification of TFREs that are active in the cell or tissue of interest.
• TFREs and cis-elements may be identified using known methods, for example by screening using known bioinformatics techniques.
• Gene sets for comparative analysis can be chosen based on clustering, e.g. hierarchical and k-means 25 , from simple expression ratio 26 or functional analysis of gene products 27 . This provides scientists with the opportunity to identify promoter elements that are responsive to certain environmental conditions, or those that play a key role in mediating the differentiation of certain tissues or those that may be particularly active in mediating pathologic phenotypes.
• Phylogenetic footprinting, or comparative genomics is now being applied to identify novel promoter elements by comparing the evolutionary conserved untranslated elements proximal to known genes from a variety of organisms 28 .
• the availability of genome sequences between species has notably advanced comparative genomics and the understanding of evolutionary biology in general.
• the neutral theory of molecular evolution provides a framework for the identification of DNA sequences in genomes of different species.
• bioinformatics tools operate by comparing non-coding regulatory sequences between the genomes of various organisms to enable the identification of conserved transcription factor binding sites that are significantly enriched in promoters of candidate genes or from clusters identified by microarray analysis.
• Examples of these software suites include TRAFAC 32 , CORG 33 , CONSITE 34 , CONFAC 35 , VAMP 36 and CisMoIs Analyser 37 .
• these tools work by aligning the upstream sequences of target genes between species thus identifying conserved regions that could potentially function as cw-regulatory elements and have consequently been applied in the elucidation of transcription regulatory networks in a variety of models.
• Table 1 Databases employed in the identification of m-elements
• TFREs such as cis-regulatory elements
• genes that are expressed in the cell or tissue of interest preferably genes that are differentially expressed in the cell or tissue of interest.
• duplex oligonucleotides from the binding sites of muscle-specific and nonspecific transcription factors were randomly ligated and cloned upstream of a minimal muscle promoter driving luciferase 46 .
• Approximately 1,000 plasmid clones were individually tested by transient transfection into muscle cells and luciferase activity was determined in 96-well format by luminometry.
• luciferase activity was determined in 96-well format by luminometry.
• a promoter element consists of a DNA sequence that includes components that allow for the transcription of a gene.
• a promoter element may include one or more transcription regulatory elements, a minimum promoter region, sequences from the 5' untranslated region of the gene or introns. In one embodiment, a promoter element may also comprise one or more cis- elements that allow the binding of one or more ubiquitously expressed transcription factors. A promoter element may comprise one or more regulatory elements that allow for transient gene expression. A promoter element may comprise one or more regulatory elements that allow for inducible gene expression.
• a minimal promoter refers to a DNA sequence which is inactive alone, but can mediate gene transcription when combined with other transcription regulatory elements.
• Minimal promoter sequences can be derived from various sources, such as prokaryotic and eukaryotic genes. Examples of minimal promoters include the dopamine beta-hydroxylase promoter and the cytomegalovirus immediate early gene minimal promoter.
• two or more TFREs are combined with a minimal promoter in a single promoter element. This may be achieved by mixing a number of TFREs as described herein under ligation reaction conditions.
• the TFREs may be directly linked to each other.
• the TFREs may be separated by spacer nucleotides.
• the TFREs may be separated by 1 or more, 2 or more, 5 or more, 10 or more or 20 or more nucleotides.
• the TFREs combined in this way may be identified by a method described herein or may already have been identified as being active in the cell or tissue of interest.
• a promoter element preferably contains two or more TFREs.
• the number of TFREs in each promoter element may be variable, or each promoter element may comprise the same number of TFREs.
• a promoter element may comprise 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more TFREs.
• the promoter element may be arranged so that the TFREs are located upstream to the minimal promoter. Alternatively, the TFREs may be located downstream to the minimal promoter.
• a plurality of promoter elements as described herein is used to create a library of expression vectors.
• Each expression vector comprises an antibiotic resistance gene.
• expression of the gene may confer resistance to neomycin, zeocin, hygromycin or puromycin.
• a promoter element as described herein is included in a vector such that it is operably linked to the gene. That is, the promoter element is located such that it is capable of expressing the coding sequence of the gene in a cell of interest.
• the vector preferably includes no promoter or regulatory sequences other than those present in the promoter element. This ensures that any gene transcription from the promoter must have been regulated by the promoter element introduced into the vector.
• the vector may be any vector capable of expression of an antibiotic resistance gene in the cell or tissue of interest.
• the vector may be a plasmid or a viral vector.
• the vector may be a vector that integrates into the host genome, or a vector that allows gene expression while not integrated.
• a plurality of different vectors as described herein may be provided. These may form a library. For example, where analysis of differential expression as described above has led to the identification of multiple TFREs for a cell or tissue type of interest, a plurality of promoter elements may be produced which comprise those TFREs. A mixture of multiple copies of the TFREs may be combined to produce a variety of different promoter elements. These may each be included in a vector to produce a library of vectors for the cell or tissue type of interest.
• a library of vectors as described herein may be assayed for vectors that are capable of expressing the antibiotic resistance gene in the cell or tissue of interest. Briefly, such an assay will comprise the steps of: transfecting cells of the cell or tissue of interest with vectors from the library; culturing said cells under conditions suitable for gene expression; and screening the cells for antibiotic resistance.
• Transfection may be achieved using any suitable method.
• a variety of transfection methods are known in the art and the skilled person will be able to select a suitable method depending on the type of vector and type of cell or tissue that it is desired to use.
• the culturing step may involve maintaining the transfected cells under suitable conditions to allow gene expression to occur. Where an inducible regulatory sequence has been included in the promoter elements, it may also be necessary to expose the cells or tissues to the relevant inducing agent. The relevant antibiotic should then be added to the medium, hi those cells where the promoter element does contain a suitable combination of TFREs to allow gene expression, the antibiotic resistance gene will be expressed and the cells will be resistant to the application of the antibiotic. For example, where the cell or tissue of interest includes the particular combination of transcription factors needed to activate the cis- acting factors within the promoter element, that promoter element may be capable of regulating expression of the antibiotic resistance gene.
• the cell will not have antibiotic resistance and will be killed by the presence of antibiotic.
• the antibiotic resistance gene may not be expressed.
• the method may comprise a further step.
• a further assay step may be carried out to determine whether the antibiotic resistance gene will also be expressed when the vector is transfected into a different cell type. For example, where the cell or tissue of interest has been treated with a particular biological ligand or chemical agent, the activity of the promoter element may also be assessed in untreated cells to determine whether the promoter element will be generally active in that cell type or only on those cells following such a treatment.
• the activity of the promoter element in a "normal" equivalent tissue type may be assessed to determine whether the promoter element is generally active in that tissue type, or only in the disease state.
• Regulatory elements corresponding to the transcription programs found to be upregulated in cancer cells using comparative genomics and integrative bioinformatics approaches detailed above are randomly ligated together with a minimal promoter upstream of the antibiotic selection gene in a promoter-less mammalian expression vector.
• Duplex oligonucleotides are designed so that when linked together the regulatory elements are present on the same face of the double helix and contain SpI -elements to prevent promoter silencing by methylation.
• the oligonucleotides that represent promoter elements are ligated together using different ratios and each ligation mix typically comprises five or six different c/s-elements.
• Resultant plasmid constructs are then used to transfect corresponding cancer cell lines in 96-well format in order to find the optimal promoters by antibiotic selection, and promising candidate promoters are isolated and sequenced before being further transfected into control cell lines in order to ascertain tumour cell specificity. Clones containing synthetic promoters that display restricted expression in cancer cell lines are then selected.
• Duplex oligonucleotides are designed as described above and are ligated into a self-inactivating (SIN) mouse moloney retroviral vector containing a minimal promoter driving the expression of the antibiotic selection gene.
• Bacterial clones are pooled and a mixed library of retroviral vectors is constructed and used to stably transduce selected cancer cell lines. Cancer cells are infected so that only 50% of the cells express the antibiotic selection gene and very high concentrations of antibiotic are used to sort the strongest expressing cells from the remaining population.
• Single clones of cancer cell lines transduced with the optimal synthetic promoter elements are then isolated by dilution cloning approaches. Genomic DNA is isolated, the synthetic promoter rescued by PCR and cloned into a promoter-less mammalian expression vector containing eGFP to evaluate expression in control cell lines thus confirming tumour specificity.
• the invention also extends to promoter elements and vectors of the invention, such as promoter elements and vectors that have been identified by the methods of the invention and to their uses.
• Promoter elements or vectors identified by the methods of the invention as being active in a cell or tissue type of interest may be used to target genes to that cell or tissue type. For example, where the methods of the invention show that a promoter element is active specifically in a particular cell type, but not in a control cell type, then that promoter element may be used to specifically direct expression in the cell type of interest.
• a promoter element of the invention may be combined with a gene that it is desired to express in a particular cell type.
• a vector may be produced in which a promoter element of the invention is operably linked to the coding sequence of a gene. That vector may then be used to transfect a cell of interest.
• the vector may be any vector type as described herein, for example a plasmid or a viral vector. Alternatively, such a vector may be produced by replacing the antibiotic resistance gene in a vector identified by a method of the invention with the gene of interest.
• the invention thus provides a method of expressing a gene in a cell or tissue of interest, comprising the steps of: identifying a promoter element capable of regulating gene expression in said cell or tissue by a method of the invention; generating an expression vector comprising said promoter element operably linked to a gene; and rransfecting the cell or tissue with the vector and allowing gene expression to occur.
• a promoter element or vector of the invention such as a promoter element or vector that has been identified as described herein as being capable of regulating gene expression in a cell or tissue of interest, may be provided for use in a method of therapy or diagnosis to be carried out on the human or animal body.
• a promoter element or vector may be used in the manufacture of a medicament for the therapeutic treatment of the cell or tissue of interest.
• the promoter element or vector may be used for the treatment of that disease, such as cancer.
• the promoter element or vector may be used to direct expression in the particular disease tissue of a polypeptide having a therapeutic effect.
• the invention may be used to provide a method of treating a disease such as cancer, the method comprising delivering a promoter element or vector of the invention, such as a promoter element or a vector that has been identified by a method of the invention, to a patient suffering from said disease, wherein the promoter element or vector directs expression in the disease cells or tissue of a therapeutic agent.
• CisMols Analyzer identification of compositionally similar cis-element clusters in ortholog conserved regions of coordinately expressed genes. Nucleic Acids Res 33, W408-W411 (2005).

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