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WO2003038101A1 - Expression genique - Google Patents

Expression genique

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Publication number
WO2003038101A1
WO2003038101A1 PCT/GB2002/004849 GB0204849W WO03038101A1 WO 2003038101 A1 WO2003038101 A1 WO 2003038101A1 GB 0204849 W GB0204849 W GB 0204849W WO 03038101 A1 WO03038101 A1 WO 03038101A1
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WIPO (PCT)
Prior art keywords
cell
promoter
emrl
sequence
cassette
Prior art date
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Ceased
Application number
PCT/GB2002/004849
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English (en)
Inventor
David Robert Greaves
Andrew John Mcknight
Siamon Gordon
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication date
Priority claimed from GB0126030A external-priority patent/GB0126030D0/en
Priority claimed from GB0209138A external-priority patent/GB0209138D0/en
Application filed by Oxford University Innovation Ltd filed Critical Oxford University Innovation Ltd
Publication of WO2003038101A1 publication Critical patent/WO2003038101A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to an expression cassette for directing gene expression in antigen presenting cells and in other cell types. It also relates to methods of gene expression using the expression cassette, for example in methods of target ' validation, gene therapy and genetic vaccination.
  • F4/80 monoclonal antibody was first described in 1981 and it has proven to be a useful marker for cells of the mononuclear phagocyte lineage.
  • F4/80 + cells in murine tissues include tissue resident macrophages, for instance the Kupffer cells of the liver, micro glia in the brain and important antigen presenting cells such as Langerhans' cells of the epidermis.
  • tissue resident macrophages for instance the Kupffer cells of the liver, micro glia in the brain and important antigen presenting cells such as Langerhans' cells of the epidermis.
  • Molecular cloning of cDNAs encoding the antigen recognised by the F4/80 mAb revealed this antigen to be an unusual cell surface protein with multiple extracellular EGF repeats linked by a mucin stalk to seven transmembrane spanning domains.
  • Emrl This gene, termed Emrl, helped to define a small family of cell surface proteins some of which are expressed on leukocytes e.g. CD97, Emr2 and Emr3, some on dendritic cells e.g. FIRE and others that, are expressed on non haematopoietic cell types.
  • the transcriptional regulation of macrophage-expressed genes has been the subject of intense investigation, both with a view to understanding macrophage differentiation and identifying sequences that can be used to specifically direct transgene expression to cells of the mononuclear phagocyte lineage.
  • Analysis of many macrophage gene promoters has revealed an important role for the transcription factor PU.1, which is a member of the ETS family of transcription factors.
  • Other transcription factors shown to be important for macrophage gene expression include AP-1, Spl, Elf-1 and C/EBP transcription factors.
  • This purine-rich sequence is a potent macrophage-specific enhancer element that can act on a heterologous promoter containing a TATA box, eukaryotic initiation factor 4A1, and the non TATA box- containing promoter of the human CD68 gene.
  • the purine-rich sequence of the Emrl promoter binds the transcription factor PU.l and the Emrl proximal promoter can be significantly transactivated by the transcription factor C/EBP ⁇ .
  • the present invention provides: an expression cassette comprising: (a) a control sequence comprising a purine-rich fragment of an Emrl promoter which fragment has enhancer activity and a promoter; and (b) a heterologous protein coding sequence operably linked to the control sequence; an expression vector comprising -an- expression cassette- according to the invention; a cell comprising an expression cassette or vector according to the invention; a method of expressing a protein in a cell, which method comprises delivering an expression cassette according to the invention or a vector according to the invention to a cell and maintaining the cell under conditions suitable for expression of the protein; a method of studying the function of a protein, the method comprising:
  • the skin disease is selected from psoriasis, allergic dermatitis, eczema, cutaneous leishmaniasis and skin cancers such as melanoma.
  • Fig. IA Promoter sequence of the murine Emrl gene (1791-2070 of SEQ ID NO: 3).
  • the DNA sequence of the Emrl promoter is numbered taking the A of the ATG translation initiation codon as position +1.
  • the major transcription initiation start sites of the Emrl promoter were mapped by 5' RACE and shown to be clustered between positions -10 and -20 of the Emrl promoter.
  • the DNA sequence in bold text contains the Emrl purine-rich sequence (PuRS) (SEQ ID NO: 1) which was PCR amplified and used in the experiments of Figures 3 and 4.
  • PuRS Emrl purine-rich sequence
  • Fig. IB Duplication of the Emrl promoter purine rich sequence.
  • the purine rich sequence (PuRS) of the Emrl promoter between positions -278 and -159 was PCR amplified and cloned into the unique Kpn I cloning site 5' of the murine Emrl -278 promoter in the luciferase reporter vector pGL3 Basic in the forward orientation to give the plasmid PuRsx2 Emrl (SEQ ID NO: 11).
  • Emrl promoter sequences present in the plasmid PuRsx2 Emrl are shown, the PuRS sequence is denoted in bold type, GGAA core PU.l binding consensus sequences are double underlined and a Kpn I restriction enzyme cleavage site introduced during cloning is shown in lower case type.
  • Fig. 2 Deletion analysis of the murine Emrl promoter. A 5' deletion series of the Emrl promoter was constructed in the luciferase reporter plasmid pGL3Basic (Promega). Panel A shows a diagrammatic representation of the promoter plasmids used in this study.
  • Panel B shows luciferase enzyme activity obtained with each of the indicated plasmids normalised for ⁇ galactosidase enzyme activity. Luciferase activities are expressed as a multiple of the activity obtained with the promoterless reporter plasmid pGL3Basic analysed in the same transfection. Error bars represent the standard error of the mean for two independent experiments.
  • Fig. 3 The purine-rich sequence of the murine Emrl gene enhances expression from a human CD68 minimal promoter.
  • Panel A shows a diagrammatic representation of the promoter plasmids used in this study. The arrow above the CD68 promoter indicates the position of the clustered CD68 transcription start sites and the arrow below the Emrl purine-rich sequence indicates the orientation of this sequence in each plasmid. •Panel-B--shows.t]hf?4uciferase-en_ayme activity (in. relative light units) obtained with the- indicated plasmids normalised for ⁇ galactosidase enzyme activity. The -277Emrl plasmid was included in the same RAW cell transfection for comparison of relative promoter activity. Error bars represent the standard error of the mean for two independent experiments.
  • Fig. 4 The purine-rich sequence of the murine Emrl gene enhances expression from a human EIF4A1 minimal promoter.
  • Panel A shows a diagrammatic representation of the promoter plasmids used in this study. The arrow above the EIF4A1 promoter indicates the position of the major EIF4A1 transcription start site and the arrow below the Emrl purine-rich sequence indicates the orientation of this sequence in each plasmid.
  • Panel B shows the luciferase enzyme activity obtained with each of the indicated plasmids normalised for ⁇ galactosidase enzyme activity. Luciferase activity is expressed as a multiple of the activity obtained with the promoterless reporter plasmid pGL3Basic analysed in the same transfection. Error bars represent the standard error of the mean for two independent experiments.
  • Fig. 5 The Emrl promoter is transactivated by C/EBP ⁇ in transfected CHO cells.
  • the -71 Emrl promoter reporter plasmid (l ⁇ g ) was transfected into Chinese Hamster Ovary (CHO) cells with the indicated amounts (in ⁇ g) of a mammalian expression vector encoding the transcription factor C/EBP ⁇ . Luciferase enzyme activities of transfected cell lysates were determined and are expressed as a multiple of the luciferase activity obtained using the empty expression vector pcDNA3. Error bars represent the standard error of the mean for two independent experiments.
  • Fig. 6 ETS factors and C/EBP ⁇ synergistically activate transcription of the Emrl promoter.
  • the -71 Emrl promoter reporter plasmid (l ⁇ g ) was transfected into Chinese Hamster Ovary (CHO) cells with mammalian expression vectors encoding the transcription factors C/EBP ⁇ , PU.l and Elf-1 (all 1 ⁇ g).
  • the total amount of plasmid DNA was adjusted to 5 ⁇ g with the plasmid pcDNA3.
  • Luciferase enzyme activities of transfected cell lysates were determined and are expressed as a multiple of the luciferase activity obtained using the empty expression vector pcDNA3. Error bars represent the standard error of the mean for two independent experiments.
  • Fig. 7 Purine rich sequence of the murine Mcll promoter.
  • This sequence was PCR amplified and cloned into the unique Mlu I cloning site 5' of the human -110 CD68 minimal promoter in the luciferase reporter vector pGL3 Basic in both orientations to give the plasmids, MclPuRS-F-110CD68 and MclPuRS-R-110CD68.
  • Plasmids were used to transiently transfect murine RAW and simian Cos-7 cells in the presence of a beta-galactosidase expression vector. Cell lysates prepared 24 hours later were assayed for luciferase and beta-galactosidase activity and results are expressed as relative light units (RLU) normalised for beta galactosidase expression.
  • RLU relative light units
  • Fig. 8 shows the constructs used in Example 7 and summarises the relative activity obtained.
  • Fig. 9 shows the constructs used in Example 8 and summarises the relative activity obtained.
  • SEQ ID NO: 1 is a purine-rich fragment of the murine Emrl promoter which enhances macrophage specific gene expression.
  • SEQ ID NO: 2 shows the human Emrl promoter.
  • the start of the human Emrl cDNA is at position 1030 and the ATG translation initiation codon is at positions 1068 to 1070.
  • SEQ ID NO: 3 is the murine Emrl promoter.
  • the purine-rich fragment shown in SEQ ID NO: 1 is present at positions 1791 to 1912.
  • the ATG translation initiation codon is at positions 2068 to 2070.
  • SEQ ID NO ' : 4 is the fragment of the murine Emrl promoter 3' to the purine-rich fragment shown in SEQ ID NO: 1.
  • SEQ ID NO: 5 and 6 are primers used to generate an Emrl probe.
  • SEQ ID NO: 7 and 8 are primers used to amplify the Emrl promoter.
  • SEQ ID NO: 9 and 10 are primers used to amplify the b-myb promoter.
  • SEQ ID NO: 11 is a duplication Emrl promoter purine rich sequence.
  • SEQ ID NO: 12 - SEQ ID NO: 15 are oligonucleotide fragments of the Emrl purine rich fragment.
  • SEQ ID NO: 16 is the murine Mel purine rich promoter sequence. Detailed Description of the Invention
  • the present invention provides an expression cassette consisting essentially of:
  • control sequence comprising (i) a purine-rich fragment of an Emrl promoter which fragment has enhancer activity and (ii) a promoter sequence;
  • control sequence is operably linked to the protein coding sequence.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner, thus the control sequence operably linked to the coding sequence is positioned in such a way that control of expression of the coding sequence is achieved as a result of transcription factor binding to, or otherwise activating, the control sequence.
  • An expression cassette of the invention may be used to direct expression of the heterologous protein in cells in vitro or in vivo.
  • the purine-rich fragment acts as an enhancer to direct high-level expression of the protein from the promoter sequence.
  • an expression cassette of the invention is primarily useful for directing high-levels of protein expression in antigen presenting cells, dendritic cells, especially immature dendritic cells, Langerhans' cells, microgial, Kupffer cells and macrophages.
  • An expression cassette may also be used to direct expression of proteins in other cell types but delivery of transcription factors to the cells may also be necessary to facilitate such expression.
  • the purine-rich fragment of the Emrl promoter is capable of interacting with one or more transcription factors, preferably a transcription factor of the ETS family, such as PU.l, Elf, AP-1 or Spl.
  • a transcription factor of the ETS family such as PU.l, Elf, AP-1 or Spl.
  • the promoter interacts with high affinity to PU.l and only weakly interacts or interacts less strongly with other transcription factors such as Elf, compared to PU.l.
  • the control sequence may typically be transactivated by a transcription factor, preferably by C/EBP ⁇ .
  • the transcription factor(s) is one found in a cell of the mononuclear phagocyte lineage.
  • Transcription factors of the ETS family interact with nucleotides comprising a GGAA motif.
  • the purine-rich sequence comprises at least one GGAA motif.
  • the fragment comprises 2, 3, 4, 5 or 6 GGAA motifs, more preferably 7, 8 or 9 GGAA motifs.
  • the fragment comprises SEQ ID NO: 13 and or SEQ ID NO: 14.
  • the purine-rich fragment of the Emrl gene may be of any suitable length, provided that it retains enhancer activity.
  • the purine-rich fragment will be from 40 to 200 nucleotides in length, preferably from 60 to 150, 70 to 120, more preferably from 80 to 100 nucleotides in length.
  • the purine-rich enhancer fragment may be present in the control sequence as a single copy or multiple copies may be present, for example 2 to 6 or 3 to 4 copies such as that of SEQ ID NO: 11. Where multiple copies of the enhancer fragment are present in the control sequence each enhancer fragment may be identical or different. For example the different fragment sequences may be of different lengths and/or may be variants of the same-sequence, for example allelicvariants-.- A preferred purine-rich fragment of the murine Emrl promoter is shown in SEQ
  • the fragment shown in SEQ ID NO: 1 is the purine-rich sequence between positions -278 and -187 of the murine Emrl promoter.
  • the purine-rich fragment may be a variant of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or said variant. Suitable variants include allelic variants and species variants.
  • a preferred variant is a fragment of the human Emrl sequence shown in SEQ ID NO: 2, which fragment has enhancer activity.
  • a preferred fragment of the human Emrl sequence is from position 797 to position 841 of SEQ ID NO: 2.
  • the present invention encompasses the sequence from 797 to 841 of SEQ ID No 2, and may comprise multimers, thereof, larger fragments of SEQ ID No. 2 incorporating the fragment together with other promoter sequences as described below.
  • the purine-rich sequence may comprise variants of the sequence shown in SEQ ID NO: 1 that include complete and partial duplications of sequences, such as the GGAA sequence, from within this region.
  • the purine-rich sequence may comprise one or more repeats of the sequence of SEQ ID NO: 1, a number of different fragments of the sequence shown in SEQ ID NO: 1 or a combination of complete copies of the sequence of SEQ ID NO: 1 and fragments thereof.
  • the purine-rich sequence may be present in the expression cassette in either orientation. Where multiple copies of purine-rich sequences are present, each copy may be directed in either orientation.
  • the expression cassette is used to express a protein in a cell which is not of the mononuclear phagocyte cell lineage, the sequence shown in SEQ ID NO: 1 is present on the sense strand.
  • the control sequence may further comprise a promoter.
  • the promoter may or may not contain a TATA box.
  • the promoter may be a macrophage lineage promoter such as the remaining sequence of the Emrl promoter, or a fragment thereof which has promoter activity, or another macrophage promoter such as CD68, or a fragment thereof which has promoter activity.
  • a preferred fragment of the CD68 promoter is a minimal promoter- such. s thefragment-from l-10>to-+l ⁇ .of the CD68 promoter.
  • the promoter may be a promoter that is not specific to cells of the mononuclear phagocyte cell lineage, for example, a non-macrophage promoter.
  • a suitable non-macrophage promoter is the EIF4A1 promoter.
  • Suitable fragments of this promoter typically comprise a CCAAT box, an SP1 site and a TATA box.
  • a suitable fragment is from -69 to +1 of the EIF4A1 gene.
  • the expression cassette may comprise the entire sequence of the Emrl promoter shown in SEQ ID NO: 3.
  • the promoter may comprise, in addition to the enhancer fragment shown in SEQ ID NO: 1, a fragment or variant of the sequence shown in SEQ ID NO: 3 which fragment or variant has promoter activity.
  • Suitable Emrl promoter fragments include fragments from -71 to +1, -117 to +1-187 to +1, -277 to +1 and -668 to +1 of the murine Emrl gene.
  • a preferred expression cassette comprises the fragment of SEQ ID NO: 3 shown in SEQ ID NO: 4, or a variant or fragment thereof which has promoter activity.
  • Promoter fragments -277 to +1 and -668 to +1 include the purine- rich enhancer sequence of SEQ ID NO: 1. Additional purine-rich sequence may or may not be present where the promoter comprises either of these fragments.
  • Suitable promoters include the human lysozme promoter, for example from -510 to +26 of the human lysozyme gene, and the murine macosialin (cd68) promoter, for example the sequence from -876 to +1, -333 to +1, -144 to +1 or -80 to +1 of the murine macro sialin gene .
  • Variants of the sequences shown in SEQ ID NO:l, 3 or 4 are typically allelic variants or homologous sequences from other species.
  • the sequence shown in SEQ ID NO: 1 is from Svl29 mouse DNA.
  • a variant of the invention may be derived from other strains of mice.
  • Other preferred variants of the invention are polymorphic variants of the Emrl sequence in genomic DNAs of different human populations. Such variants and fragments thereof may be identified by any suitable method.
  • a variant or fragment can hydridize to the sequence of SEQ ID NO: 1, 2, 3 or 4 or to- the -complement of the .sequence shewn in- SEQ-ID- NO: 1, 2, 3 or 4 at a- level. significantly above background. Background hybridization may occur, for example, because of other genomic DNA fragments present in a genomic DNA library.
  • the signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1, 2, 3 or 4 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ TD NO: 1, 2, 3 or 4.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60 °C up to 65 °C. If lower stringency is required suitable conditions include 2 x SSC at 60 °C.
  • control sequence of SEQ ID NO: 1, 2, 3 or 4 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10 substitutions.
  • the polynucleotide of SEQ TD NO: 1, 2, 3 or 4 may alternatively or additionally be modified by one or more insertions and/or deletions and or by an extension at either or both ends.
  • the modified sequence retains regulatory activity.
  • a nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1, 2, 3 or ' 4 will generally have at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1, 2, 3 or 4 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1, 2, 3 or 4.
  • Methods of measuring nucleic acid homology are well known in the art.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to. calculate. •ho.m.oJogy oj-line up. sequences (typically on their default settings); for example as described in Altschul (1993) I. Mol. Evol. 36:290-300; Altschul et al (1990) J. Mol. Biol. 215:403-10.
  • HSPs high scoring sequence pair
  • Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance.
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about ,0,00.1, Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define control sequences of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred.
  • a control sequence which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.
  • a variant or fragment of the sequences set out in SEQ ED NO: 1, 2 or 3 maintains the ability to enhance expression of a gene under the control of a minimal promoter.
  • the fragment or variant is able to enhance expression in an orientation independent manner when the control sequence comprises a macrophage promoter.
  • Methods for determining enhancer activity are known in the art. For example, enhancer activity may be determined by transforming an appropriate cell line, such as RAW or CHO cells, with a reporter gene construct comprising the putative enhancer sequence and a promoter operably linked to a reporter gene and monitoring expression of the reporter gene. Comparison may be made between reporter constructs containing the putative enhancer sequence and promoters lacking this sequence.
  • the purine- rich sequence is capable of enhancing expression in cells of the mononuclear phagocyte cell lineage such as antigen presenting cells, dendritic cells, immature dendritic cells, Langerhans' cells, microglia and macrophages.
  • a variant or fragment of the sequences set out in SEQ ID NO: 2, 3 or 4 maintains the ability to act as at least a basal promoter, i.e. in the absence of any other control sequences the variant or fragment is able to direct at least low levels of gene expression.
  • Methods for determining promoter activity are known in the art. For example, promoter activity may be determined by transforming an appropriate cell line, such as RAW or CHO cells, with a reporter gene construct comprising the putative promoter linked to a reporter gene and monitoring expression of the reporter gene. Comparison may be made with a promoterless reporter plasmid such as pGL3Basic.
  • the promoter is .capable of directing gene expression i cells of the mononuGlear phagocyte cell lineage, ⁇ such as antigen presenting cells, dendritic cells, immature dendritic cells, Langerhans' cells, microglia and macrophages
  • the promoter may be a basal promoter which shows minimal promoter activity,.
  • a basal promoter may be a fragment of a full-length promoter. Suitable fragments may be from 40 to 500 nucleotides in length, preferably from 50 to 300, 60 to 200 or 70 to 100 nucleotides in length.
  • the present invention also encompasses expression cassettes comprising additional control sequences such as additional enhancer and/or additional promoter sequences.
  • An additional enhancer sequence may be any type of enhancer sequence and need not be derived from the Emr-1 gene. It is however preferred that the additional enhancer sequence is capable of enhancing gene expression in cells of the mononuclear phagocyte cell lineage such as antigen presenting cells, dendritic cells, immature cells, Langerhans' cells and macrophages.
  • the additional control sequence may comprise a repressor sequence that inhibits expression in a cell type not of this lineage.
  • the expression cassette will be DNA. Protein Coding Sequence
  • the heterologous protein coding sequence in the expression cassette may be any sequence that encodes a string of amino acids provided that the sequence does not encode a full-length Emrl protein.
  • the sequence may encode a fragment of the Emrl protein or a chimeric protein comprising a fragment of the Emrl protein fused to a heterologous protein. However, it is preferred that the coding sequence does not encode a fragment of the Emrl protein.
  • the term "protein” is intended to include peptides, polypeptides and proteins.
  • the protein encoding sequence may be a cDNA sequence or genomic DNA.
  • a polypeptide coding sequence may therefore comprise both introns and exons.
  • the protein coding sequence begins with a start codon (ATG) and terminates in a stop codon (TAA, TAG or TGA).
  • the .protein will typically encounterbe of .therapeutic, use or of iirimunogenic use.
  • the protein may encode an antigen or a protein with therapeutic activity.
  • antigen is a protein molecule or portion thereof which comprises one or more epitopes which can elicit an immune response in an individual.
  • An epitope may be a T-cell epitope or a B-cell epitope.
  • the immune response generated in response to the antigen may therefore be a humoral and/or cellular immune response.
  • the protein may comprise one, two or more antigens.
  • the antigens may be from the same pathogenic protein, from different proteins of a single pathogen or from proteins from different pathogens.
  • the protein may be a tumour antigen or may comprise two or more tumour antigens.
  • a tumour antigen may be specific to a tumour cell, i.e.
  • tumour antigen is expressed on the surface of a tumour cell, for example is a cell surface receptor or cell adhesion protein.
  • tumour antigens include the MUC-1 gene product which is over expressed in a number of tumours including ovarian cancers, human papillomavirus proteins, E6 and E7, which are associated with cervical cancer, MART-I, MAGE-I, gplOO and tyrosinase which are expressed in melanoma, PSA which is expressed in prostate cancer, CEA which is expressed in a number of different types of tumour and Her2neu which is expressed in various cancers including breast cancer.
  • MUC-1 gene product which is over expressed in a number of tumours including ovarian cancers, human papillomavirus proteins, E6 and E7, which are associated with cervical cancer, MART-I, MAGE-I, gplOO and tyrosinase which are expressed in melanoma
  • PSA which is expressed in prostate cancer
  • CEA which is expressed in a number of different types of tumour
  • Her2neu which is expressed in various cancers including breast cancer.
  • the protein may be capable of modifying an immune response and/or modulating inflammation.
  • the protein may, for example, be a cytokine such as -, ⁇ - or ⁇ -interferon, an interleukin (IL), such as IL-1, IL-2, IL-4, IL-10, IL-13, IL-15, transforming growth factor (TGF- ⁇ ) or tumour necrosis factor (TNF), or an insulin-like growth factor (I or II).
  • IL interleukin
  • TGF- ⁇ transforming growth factor
  • TNF tumour necrosis factor
  • I or II insulin-like growth factor
  • Other suitable immunomodulatory proteins include chemokines such as RANTES and co-stimulatory molecules such as CD80, CD86, CD40 and CD40 ligand.
  • “Viral- 'chemokines 1 such as”vMIP-II of herpes -virus and "viral cheinokine binding proteins such as the 35K protein of vaccine virus.
  • a protein that has inflammatory modulatory activity acts to reduce inflammation.
  • the protein may be an antigen of pathogenic origin.
  • proteins of pathogenic origin are derived from pathogenic organisms, for example parasites, bacteria or viruses.
  • antigenic polypeptides include hepatitis C virus antigens, hepatitis B surface or core antigens, papillomavirus antigens, HTV antigens and malaria antigens.
  • the protein may be a cytotoxic protein that may be effective in killing cancer cells.
  • An expression cassette of the invention may be inserted into a vector.
  • the present invention thus provides both cloning and expression vectors comprising an expression cassette of the invention.
  • an expression cassette is inserted in an expression vector.
  • the expression vector may be a plasmid vector or a recombinant viral vector such as a vaccinia, adenovirus, recombinant adeno associated virus (AAN), herpes virus or retrovirus vector or an amplicon vector.
  • Suitable expression vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid D ⁇ A and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression.
  • plasmid D ⁇ A and other elements such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression.
  • an expression cassette or vector of the invention may be used to express the heterologous protein in a cell.
  • the present invention provides a cell comprising inter alia an expression cassette of the invention or a vector of the-in-vention.
  • The. cell type- is preferably -mammalian cell, more preferably.. a human cell.
  • the cell may be a haematopoietic cell, in particular a cell of the mononuclear phagocyte cell lineage.
  • the cell is an antigen-presenting cell and in particular a dendritic cell or a macrophage.
  • the cell may be a Kupffer cell, a microglial cell or a Langerhans' cell.
  • Especially preferred cells are immature dendritic cells, for example, Langerhans' cells.
  • the cell may be in vivo or in vitro.
  • a cell in vivo may be from a cell line or may be an ex vivo cell in culture. It is preferred that when the cell is in vivo, that it is an antigen presenting cell, dendritic cell, immature dendritic cell, Langerhans' cell, microglial cell or macrophage.
  • a method of expressing a protein in a cell which method comprises introducing an expression cassette according to the invention or a vector according to the invention into the cell and maintaining the cell under conditions suitable for the expression of the protein is also provided by the invention.
  • the cell may be in vivo or in vitro.
  • the invention therefore provides a method of expressing a protein in cultured cells or in cells of a human or animal body. More specifically, the present invention provides a method of expressing a heterologous protein in an antigen presenting cell, a dendritic cell, an immature dendritic cell, a Langerhans' cell, a microglial cell, a Kupffer cell or a macrophage.
  • the expression vector may be delivered to such cells ex vivo and the cells administered to a human or animal body. Alternatively, the expression vector may be administered directly to a human or animal body.
  • an enhancer sequence which depends on the presence of transcription factors specific to cells of this lineage, for example macrophage specific transcription factors, for its activity will result in high-level expression of the heterologous protein only in those cell types.
  • the method may further comprise introducing one or more transcription factor to the cell.
  • the transcription factor is preferably a mononuclear phagocyte cell lineage-specific transcription factor, for example macrophage-specific transcription factor.
  • the transcription factor may typically bind to the GGAA sequence motif in the purine-rich control sequence.
  • the transcription factor may be a member of the ETS family of transcription factors.
  • the transcription factor may be capable of transactivating the control sequence.
  • Preferred transcription factors are PU.l, Elf-1 and C/EBP ⁇ .
  • One or more transcription factors for example two or three transcription factors may be delivered to the cell.
  • the transcription factors are capable of interacting synergistically at the purine-rich enhancer sequence to enhance expression of the protein.
  • a synergistic interaction is one which results in expression of the heterologous protein at a level greater than one would expect by simply adding the expression levels achieved when using each transcription factor individually.
  • a preferred combination of transcription factors comprises PU.l and C/EBP ⁇ .
  • a suitable transcription factor may be introduced to a cell by delivering a polynucleotide encoding the transcription factor to the cell.
  • the present invention further provides a method of studying the function of a protein, the method consisting essentially of delivering an expression cassette of the invention or a vector of the invention to a cell and determining the effect, if any, of expression of the protein in the cell.
  • An expression cassette of the invention may also express antisense mRNA to specifically knock-out a protein in a cell. This is another means by which the expression cassette may be used to study protein function.
  • the cell is an antigen presenting cell, a dendritic cell, an immature dendritic cell, a Langerhans' cell, a microglia or a macrophage.
  • the effect of the. protein- may-be detem ⁇ ned-by monitoring -any-suitable- activity-.. -For example, - phagocytic activity, antigen presentation, activation of CD4T cells, responsiveness to cytokines, production of nitric oxide (NO), production of reactive oxygen intermediates (ROI), production of antibacterial enzymes or production of anti-bacterial peptides.
  • a method of studying the function of a protein may comprise studying the role of a protein in a disease. Accordingly, the function of the protein may be determined by monitoring an effect on a disease process or on a symptom of a disease.
  • Disease is used herein to describe any malfunction of the human or animal body and encompasses genetic disorders, diseases caused by a pathogen or other environmental factors, including injury and cancers.
  • a method of the invention may be used to investigate the effect of a protein on an autoimmune disease or a skin disease.
  • the disease may be a disease of the skin, such as allergic dermatitis, eczema, cutaneous leishmamasis, melanoma or other skin cancer and, more especially, psoriasis.
  • a preferred means of delivery is by topical application to the skin.
  • a method of studying the effect of a protein on a disease is preferably carried out on a non-human animal.
  • the non-human animal is an animal model of a human disorder.
  • Suitable animal models include xenograft models and chemically induced models.
  • an animal model of psoriasis may be generated by engrafting human psoriatic skin onto immune deficient mice.
  • the use of contact sensitizers, such as DNCB, TNCB, fluorescein and isothiocyanate on mouse skin may be used to induce dermatitis.
  • Expression cassettes of the invention may also be used in methods of screening potential theraputic agents. Following identification of a protein target for a particular disease state, potential therapeutic agents may be tested by expressing the target protein in a macrophage using an expression cassette of the invention and contacting the macrophages, in vitro or in vivo, with the test agent. An effect of the test agent on the function of the protein may be monitored to determine the activity of a test agent.
  • Expression cassettes of the invention are especially useful for facilitating high- level expression of antigenic proteins in antigen presenting cells of the immune system such as macrophages, B-cells and dendritic cells. Therefore an expression cassette of the invention is useful in a method of genetic vaccination since expression of the antigenic protein in such antigen-presenting cells will result in the efficient presentation of antigen to the immune system and subsequent generation of an immune response (cellular and/or humoral) to the antigen. Accordingly, the present invention also provides an expression cassette as described herein or a vector comprising such an expression cassette for use in a method of genetic vaccination.
  • a method of genetic vaccination typically comprises administering an effective amount of an expression cassette of the invention to a subject in need thereof.
  • An effective amount of an expression cassette is an amount that facilitates the expression of the protein in an antigen-presenting cell such that a protective immune response directed to the protein is stimulated.
  • a subject in need thereof is typically a subject at risk of infection by a pathogen or a subject at risk of cancer or suffering from cancer.
  • a protein for use in a method of genetic vaccination is typically an antigen.
  • the protein may comprise one or more antigen of a pathogenic organism as disclosed herein and may be used in a genetic vaccine against infection by the pathogenic organism.
  • Expression of the antigen in a macrophage or other antigen presenting cell using a cassette of the invention may stimulate the host immune system to produce an immune response to a pathogen, either prior to infection or after infection of the host by the pathogen.
  • the protein may comprise one or more tumour antigen.
  • An expression cassette of the invention may be used in the prevention and/or treatment of cancer and especially cancers for which-cancer-.tissue specifiG-or-tumour specific antigens canbe identified. .. Vaccines may also be useful in the prevention of other diseases such as autoimmune diseases including multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythromatosus etc.
  • An expression cassette of the present invention for use as a vaccine may effectively be used with any suitable adjuvant or combination of adjuvants.
  • suitable adjuvants include adjuvants formed from aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; oil-in-water and water-in-oil emulsion formulations, such as Complete Freunds Adjuvants (CFA) and Incomplete Freunds Adjuvant (IF A); adjuvants formed from bacterial cell wall components such as adjuvants including lipopolysaccharides, trehalose dimycolate (TDM), and cell wall skeleton (CWS); heat shock protein or derivatives thereof; adjuvants derived from ADP-ribosylating bacterial toxins, including diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the E.
  • alum aluminum salts
  • alum such as aluminum hydroxide, aluminum phosphate, aluminum
  • coli heat-labile toxins LT1 and LT2
  • Pseudomonas endotoxin A Pseudomonas exotoxin S
  • B. cereus exoenzyme B. sphaericus toxin
  • C. botulinum C2 and C3 toxins C. limosum exoenzyme
  • toxins from C. perfringens C. spiriforma and C. difficile, Staphylpcoccus aureus ⁇ DIN
  • ADP-ribosylating bacterial toxin mutants such as CRM 197 , a non-toxic diphtheria toxin mutant
  • saponin adjuvants such as Quil A (U.S.
  • chemokines and cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, etc.), interferons (e.g., gama interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), defensins 1 or 2, chemokines such as RANT ⁇ S, MlPl- ⁇ and MIP-2, etc; muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl- L -alanyl- D -isoglutamine (nor-MDP), N- acetylmuramyl-
  • adjuvants are commercially available from a number of distributors such as Accurate Chemicals; Ribi Immunechemicals, Hamilton, MT; GIBCO; Sigma, St. Louis, MO.
  • Preferred adjuvants are those derived from ADP-ribosylating bacterial toxins, with cholera toxin and heat labile toxins being most preferred.
  • Oligonucleotides containing a CpG motif are also preferred.
  • the adjuvant may be delivered individually or delivered in a combination of two or more adjuvants.
  • combined adjuvants may have an additive or a synergistic effect in promoting a desired immune response.
  • a synergistic effect is one where the result achieved by combining two or more adjuvants is greater than one would expect than by merely adding the result achieved with each adjuvant when administered individually.
  • a preferred adjuvant combination is an adjuvant derived from an ADP- ribosylating bacterial toxin and a synthetic oligonucleotide comprising a CpG motif.
  • Unfortunately a majority of the above-referenced adjuvants are known to be highly toxic, and are thus generally considered too toxic for human use.
  • chemokines and cytokines are immunostimulatory proteins provided in nucleic acid form, for example nucleic acid sequences that encode chemokines and cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), defensins 1 or 2, chemokines such as RANTES, MlPl- ⁇ and MIP-2 molecules and adjuvants derived from immune cells such as surface markers which are capable of boosting an immune response.
  • chemokines such as RANTES, MlPl- ⁇ and MIP-2 molecules and adjuvants derived from immune cells such as surface markers which are capable of boosting an immune response.
  • Such immunostimulatory proteins may be administered using an expression cassette of the invention.
  • the i ⁇ nmunostirnulatory,proteinma he administered prior to .administration- of an antigen or after antigen administration.
  • the immunostimulatory protein is co- admimstered with the antigen.
  • the immunostimulatory protein and antigen may be present in the same nucleic acid construct or vector or may be present in different nucleic acid constructs or vectors.
  • the use of such immunomodulatory proteins may serve to alter the, nature of the immune response to an antigen.
  • an immunomodulatory protein may direct a predominantly Thl response or a predominantly Th2 response.
  • a vaccine composition comprising an effective amount of an expression cassette of the invention, or of a vector of the invention, and a pharmaceutically acceptable carrier or diluent is also provided by the invention.
  • Macrophages, and other antigen presenting cells may also be used to direct an immune response to a pathogen following infection, or to direct an immune response to a tumour as part of cancer therapy.
  • Expression of immunomodulatory proteins in such cells may also be used to alter the nature of an immune response, for example, from a predominantly Th2 to a predominantly Thl immune response or vice versa.
  • an expression cassette of the invention may be used to deliver therapeutic genes to a human or animal subject in need of treatment.
  • a human or animal subject in need of treatment may be suffering from a pathogenic infection.
  • the therapeutic protein encoded by the expression cassette or vector used in such treatment may be an antigen derived from the relevant pathogen or may be a suitable anti-pathogenic agent, such as an an ⁇ -microbial peptide or-an anti- microbial enzyme.
  • An effective amount of a cassette or vector encoding an antigen is an amount sufficient to generate an effective immune (humoral and/or cellular) response against the pathogen.
  • An effective immune response is an immune response which results in the inactivation of the pathogenic organism and preferably a reduction in symptoms of the pathogenic infection.
  • An effective amount of a therapeutic agent is an amount sufficient to improve the condition of a patient, for example by reducing the severity of a symptom of the infection.
  • a human or animal subject in need of treatment may be suffering from a cancer.
  • the therapeutic protein encoded by the expression cassette or vector used in such treatment may be an antigen expressed on tumour cells which enables the tumour cells to be distinguished from normal cells or may be a protein with anti-tumour activity.
  • An effective amount of a cassette or vector encoding a tumour antigen is an amount sufficient to generate an effective immune (humoral and/or cellular) response against the tumour.
  • An effective immune response is an immune response which results in the inactivation of the destruction of tumour cells and preferably a reduction in symptoms of the cancer.
  • An effective amount of a therapeutic agent is an amount sufficient to improve the condition of a patient, for example by reducing the severity of a symptom of the cancer.
  • a human or animal subject in need of treatment may be suffering from an autoimmune disease or a skin disease, hi particular, an expression cassette of the invention may be used in the treatment of disorders such as psoriasis.
  • a therapeutically effective amount of an expression cassette or vector is an amount which enables the expression of an effective amount of a therapeutic protein.
  • An effective amount of a therapeutic protein is an amount which allieviates the condition of the subject, for example by reducing inflammation.
  • therapeutically effective amount of an expression cassette or vector is an amount which enables the expression of an antigen on macrophages or other antigen presenting cells such that an immune response sufficient to-induce -tolerance to -self antigen.
  • the expression cassettes of the invention may be administered directly as naked nucleic acid constructs. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents include cationic agents, for example calcium phosphate, DEAE dextran, Polyethylenimine (PEI), dendrimers, and lipofectants, for example lipofectam and transfectam. Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition. Viral vectors may also be used. Examples of suitable viral vectors include retroviruses, lentiviruses (e.g. HIV viruses), adenoviruses, alphaviruses, adeno-associated viruses, herpes simplex viral vectors.
  • retroviruses e.g. HIV viruses
  • adenoviruses e.g. HIV viruses
  • alphaviruses adeno-associated viruses
  • herpes simplex viral vectors simplex
  • Nucleic acid may be delivered directly, for example, by injection, preferably intradermally, subcutaneously or intramuscularly, or topically, orally or intranasally, or by aerosol, or for example, using a particle bombardment or patch transdermal delivery devices. More prefereably, the nucleic acid constructs of the invention are administered to the skin.
  • the nucleic acids are administered in a manner compatible with the dosage formulation and in such amount as will be prophylactically or therapeutically effective.
  • the quantity to be administered which is generally in the range of a lpg to 10 mg, preferably 1 pg to 10 ⁇ g for particle-mediated delivery, and preferably l ⁇ g to 10 mg for other routes of nucleic acid per dose, depends on the admimstration route, subject to be treated, capacity of the subject's immune system to produce an immune response and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner who may be familiar with each subject.
  • the antigen or therapeutic protein may be delivered by delivering an expression- cassette -or .vector to macrophag ⁇ s-or other cells ex vivo and administering the cells to a human or animal subject in need thereof.
  • a vaccine may be given in a single dose schedule or in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1- 10 separate doses, followed by other doses given at subsequent time intervals required to maintain or reinforce the immune response, for example at 1-4 months for a second dose, and if needed a subsequent dose after several months.
  • the dosage regime will also at least in part be determined by the need of the individual and be dependent upon the judgement of the practitioner.
  • Additional immunomodulators may be administered with the nucleic acid vaccine of the invention.
  • Such immunomodulators may be administered as nucleic acid encoding the immunomodulators.
  • nucleic acid may be expressed as a separate protein, but incorporated within the same vector as the heterologous antigen. Expression of the immunomodulator may be driven by additional control sequences or by the same control sequence as the antigen, for example by utilising IRES elements.
  • the immunomodulator may be expressed as a fusion protein with the antigen with or without a linker that may be cleavable by proteases.
  • a separate nucleic acid construct could be provided for expression of the additional immunomodulator or expression of additional antigens.
  • Such constructs may be administered together with the nucleic acid encoding heterologous antigen or may be administered separately.
  • Chemical immunomodulators may also be administered.
  • additional antigens or immunomodulators may be administered at the same time as nucleic acid encoding antigen, after or prior to such administration.
  • Recombinant PI bacteriophage containing the murine Emrl gene were obtained from. Genome S.ystems. (now IncyteGenomics),. restriction fragments encompassing . -the ⁇ 5' end of the gene were subcloned into the vector pBluescript SK " and the sequence of the promoter determined by standard methods. The major transcription start sites of the Emrl gene were determined by 5'. rapid amplification of cDNA ends (5' RACE, Clontech) using RAW cell mRNA as a template for cDNA synthesis. Nucleotide positions in the Emrl promoter are numbered taking the ATG of the translation initiation codon as position +1.
  • Emrl promoter A 5' deletion series of the Emrl promoter was generated by cloning PCR products between the Kpn I and Xho I sites of the vector pGL3 Basic (Promega). After confirmatory DNA sequencing, supercoiled plasmid DNAs were purified from E.coli TOP10F' (InVitrogen) by NaOH/SDS lysis followed by ethidium bromide CsCl ultracentifugation (Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual, 2nd Ed., Cold Spring Harbour Laboratory, Cold Spring Harbour, NY).
  • CHO and RAW cells were cultured as described previously (Greaves et al. Genomics. 1998; 54: 165-168).
  • RAW cells were transfected by electroporation using 20 ⁇ g of luciferase reporter plasmid and 2 ⁇ g of a ⁇ -galactosidase reporter plasmid.
  • CHO cells were transfected using a total of 5 ⁇ g of plasmid DNA and 50 ⁇ g of the cationic Lipofectamine (InVitrogen) as described previously (Greaves et al. Genomics. 1998; 54: 165-168).
  • Transfected cell lysates were prepared in lx RLB (Promega) and assayed for luciferase and ⁇ -galactosidase enzyme activity, for transactivation analyses cell lysate total protein concentration was measured using a BCA Assay Kit (Pierce).
  • Nuclear extracts and ETS factors prepared by coupled transcription translation (Promega) used in EMSA experiments were prepared as described previously (Greaves et al. Genomics. 1998; 54: 165-168).
  • the -277/-156 Emrl probe was prepared by 5' labelling a PCR fragment generated ' using' he primers
  • ChIP experiments were performed using published protocols (Hecht and Grunstein, Methods Enzymol 1999, 304: 399-414 and Wells et al, Mol. Cell Biol. 2000, 20: 5797-5807) and the following antibodies; anti PU.l anti NERF, anti Elf 1, anti Fli-1 (Santa Cruz).
  • the primers 5'GGTACAGAGGAAACTGAGGTTGG (SEQ TD NO: 7) and 5'GTCAGGGTTGCTCAACAAAGCC (SEQ ID NO: 8) were used to amplify the Emrl promoter and primers 5'CAGAGCCAGGCCTCGCGCCTCATTG (SEQ ID NO: 9) and 5' TCAGGACTCAGGCTGCTCGAGCCGC (SEQ ID NO: 10) used to PCR amplify the b-myb promoter.
  • Primary macrophages were prepared from C57BL6J mice by peritoneal lavage 4 days after injection of 1ml of 4% thioglycollate broth.
  • the sequence of the Emrl promoter was determined from subcloned fragments of a recombinant PI bacteriophage containing the 5' end of the Emrl gene. Multiple transcription start sites were mapped using 5' RACE and SI Nuclease mapping to the region immediately 5' of the ATG translation initiation codon. We have denoted the A of the ATG initiation codon position +1. Analysis of the Emrl promoter sequence (Fig. 1 A) showed that, in common with most macrophage-expressed genes, the murine Emrl promoter contains no recognisable TATA box sequence. The region between positions - 277 and -195 of the Emrl promoter consists of 73 consecutive purine residues containing 9 GGAA sequences. GGAA is the core of the recognition sequence for ETS family transcription factors r including..PU.
  • luciferase reporter gene plasmids were constructed that contained increasing lengths of Emrl 5' flanking sequence (Fig. 2A). These plasmids were transfected into the murine macrophage cell line RAW 264.7 and luciferase enzyme activities determined 24 hours later. The data of Fig. 2B show that important sequences for expression in RAW cells lie between positions —277 and position -187.
  • Previously characterised macrophage promoter fragments cloned in the same luciferase reporter plasmid were included in the same transfection experiment to allow a direct comparison of the Emrl promoter with the human CD68 promoter (Greaves et al. Genomics.
  • the -277 Emrl promoter fragment is ⁇ 3-fold more active that the -150 CD68 promoter and ⁇ 12-fold more active that the -510 human lysozyme promoter fragment (Fig. 2B).
  • the -277 Emrl promoter was also assayed in transiently transfected non- macrophage cell lines.
  • the -277 Emrl promoter fragment is at least 60 fold more active in RAW cells then in epithelial cell lines (Table 1).
  • s.e.m. is standard error of the mean for at least two independent transfection experiments.
  • Emrl promoter purine-rich sequence could confer high-level macrophage expression on a heterologous promoter.
  • the -110 CD68 promoter contains the basal promoter elements and displays less than 10% of maximal CD68 promoter activity in transfected RAW cells.
  • the Emrl PuRS was cloned in both orientations upstream of the CD68 -110 promoter fragment and these plasmids were designated PuRSb-110CD68 and PuRSa-110CD68 (Fig. 3A).
  • the plasmids were transfected into RAW cells and their activity compared with the -110 CD68 promoter and the -277 Emrl promoter.
  • the data of Fig. 3B demonstrate that a 122bp fragment of the Emrl promoter can confer high-level macrophage expression on a heterologous promoter independent of orientation.
  • the chimeric Emrl/CD68 promoters are more active than the native -277 Emrl promoter in RAW cells.
  • Emrl PuRS has been cloned the same Emrl PuRS fragment 5' of the -69 EIF4A1 promoter, which contains an SP1 site and a TATA box, and showed that this Emrl fragment stimulated EIF4A1 promoter activity either 8- fold or 90- fold depending upon the orientation of the Emrl sequences (Fig. 4).
  • the promoter activity of the Emrl/EIF4A1 chimeric promoter PuRSb-110 is ⁇ 50% that of the -277 Emrl promoter analysed in the same experiment. It is interesting that the Emrl PuRS does not act in an orientation-independent manner on the TATA box-containing EIF4A1 promoter. We interpret these observations as evidence that the Emrl PuRS needs to interact with specific sequences in the proximal promoter to be maximally active as a macrophage enhancer element.
  • Emrl PuRS nuclear proteins that can interact with the Emrl PuRS
  • EMSA experiments with a 32 P labelled Emrl — 278/-156 probe and in vitro translated ETS factors.
  • the Emrl PuRS binds PU.l with high affinity and can also bind in vitro translated Elf-1 and MEF proteins weakly.
  • the EMSA experiment performed with RAW nuclear extract gave a complex that co-migrates with PU.l.
  • Chromatin of RAW cells, peritoneal macrophages of C57BL/6S mice or NIH 3T3 fibroblast cells was cross-linked by incubation with formaldehyde and chromatin immunoprecipitation experiments performed as described above. Precipitated promoter fragments were detected following 30 cycles of PCR (shown to be within the linear range of amplification using primers specific for the Emrl PuRS or b-myb promoter (product sizes 323 bp and 412 bp respectively).
  • Emrl promoter is precipitated with an antibody specific for PU.l and is not precipitated by antibodies that recognise the ETS factors Fli-1, Elf-1 or NERF. Similar results were obtained in peritoneal macrophages and the specificity of PU.l binding to the Emrl promoter is shown by the lack of precipitation of the b-myb promoter. The macrophage- specific nature of the interaction between the Emrl promoter and PU.l is shown by ChIP experiments performed in NIH3T3 fibroblast cells.
  • Emrl proximal promoter fragment can be significantly transactivated by co-expression of the transcription factor C/EBP ⁇ in a dose-dependent manner (Fig.5).
  • Other promoters of macrophage-expressed genes that are up-regulated by C/EBP ⁇ include the encoding cyclo-oxygenase-2 (COX-2) (Wadleigh et al. J Biol Chem. 2000; 275: 6259-6266), Interleukin-6 and Monocyte Chemo attractive Protein- 1 (MCP-1) (Hu et al. J Immunol. 1998; 160: 2334-2342).
  • Emrl and Elf-1 bind to the Emrl PuRS between positions -277 and -156 and that C/EBP ⁇ can transactivate the -71 Emrl proximal promoter we looked for evidence that these transcription factors could co-operate to enhance expression of the Emrl promoter.
  • the -277 Emrl promoter was fransfected into CHO cells in the presence of limited amounts of expression vectors encoding PU.l, Elf-1 and C/EBP ⁇ .
  • the Mel promoter sequence beginning at position -881 contains a 128bp sequence containing 121 purines and only 7 pyrimidines including a stretch of 51 consecutive purines but only one GGAA sequence ( Figure 7).
  • Figure 7 We cloned a 166bp purine-rich fragment of the murine Mel gene promoter that extends from position -902 to position -737 into the -110CD68 promoter luciferase vector in both orientations ( Figure 8).
  • the PuRsx2 Emrl plasmid, the -278 Emrl plasmid and a -150 CD68 promoter plasmid were used to transiently transfect murine RAW cells in the presence of a beta- galactosidase expression vector.
  • Cell lysates prepared 16 hours later were assayed for luciferase and beta-galactosidase activity (relative light units (RLU) normalised for beta galactosidase expression). Results are expressed as fold luciferase activity obtained with the plasmid pGL3 Basic in the same transfection experiment.
  • the Mel PuRS enhanced expression from the CD68 minimal promoter in RAW cells only 3.7- or 2.1- fold (see Table 2 below).
  • COS7 cells COS7 cells RAW cells RAW cells
  • Emrl PuRS was synthesised as a series of four overlapping double stranded oligonucleotides for use in EMSA experiments as follows;
  • Emrl-A 5' GAGAGGAAGGGAAAGGGAAAGAG (SEQ IDNO: 12),
  • Emrl-D 5' AGAGGGAGAAATGTGGAC (SEQ ID- NO: 15).
  • EMSA competition • experiments the two double stranded oligonucleotides Emrl-B and Emrl-C were shown to bind PU.l with high affinity while the a 100-fold molar excess of the oligonucleotides Emrl-A and Emr 1-D were unable to compete for PU.l bound to a labelled Emrl PuRS probe (data not shown).

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Abstract

L'invention concerne une cassette d'expression qui comprend: (a) une séquence de commande contenant un fragment riche en purine d'un promoteur Emr 1 qui présente une activité de renforcement et un promoteur; et (b) une séquence codant une protéine hétérologue fonctionnellement liée à la séquence de commande. L'invention concerne également des cellules comprenant ladite cassette et des procédés d'expression génique au moyen de ladite cassette.
PCT/GB2002/004849 2001-10-30 2002-10-29 Expression genique Ceased WO2003038101A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2006122141A3 (fr) * 2005-05-10 2007-01-11 Biogen Idec Inc Methodes et produits permettant de determiner l'expression du gene f4/80 dans des cellules microgliales
WO2023203269A1 (fr) * 2022-04-19 2023-10-26 Instituto De Salud Carlos Iii Acide nucléicque et son utilisation dans le traitement du sarcome d'ewing
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