WO1999031260A2 - Sequences regulatrices intervenant dans l'expression du gene specifique du pancreas - Google Patents

Sequences regulatrices intervenant dans l'expression du gene specifique du pancreas Download PDF

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WO1999031260A2
WO1999031260A2 PCT/EP1998/008215 EP9808215W WO9931260A2 WO 1999031260 A2 WO1999031260 A2 WO 1999031260A2 EP 9808215 W EP9808215 W EP 9808215W WO 9931260 A2 WO9931260 A2 WO 9931260A2
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expression
recombinant dna
regulatory sequence
sequence
cell
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Peter Gruss
Birgitta Kammandel
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to regulatory sequences of the promoter of the Pax6 gene or a gene homologous to the ax6 gene, being capable of conferring expression in pancreatic cells.
  • the present invention also relates to recombinant DNA molecules and vectors comprising said regulatory sequences as well as to host cells transformed therewith.
  • the present invention additionally relates to pharmaceutical and diagnostic compositions comprising such regulatory sequences, recombinant DNA molecules and vectors.
  • the present invention relates desto transgenic non-human animals, comprising the afore-mentioned recombinant DNA molecules or vectors stably integrated into their genome.
  • the present invention also relates to the use of the before described recombinant DNA molecules and vectors for the preparation of pharmaceutical compositions for treating, preventing and/or delaying a disease related to the pancreas in a subject.
  • the regulatory sequences, the recombinant DNA molecules and vectors of the invention can be used for the preparation of pharmaceutical compositions for inducing a pancreatic disease in a non-human animal.
  • the present invention relates to a method for identifying agonists/activators or antagonists/inhibitors of genes or gene products involved in pancreatic diseases employing the above mentioned regulatory sequences, recombinant DNA molecules, vectors, cells, and transgenic non-human animals, to compounds identifiable by said methods, to antibodies directed to said compounds as well as to pharmaceutical and diagnostic compositions comprising said agonists/activators, antagonists/inhibitors and/or antibodies.
  • pancreas is a particularly important organ from the point of view of human medicine because it suffers from two important diseases: diabetes mellitus and pancreatic cancer. Diabetes effects at least 30 million people world wide and despite the availability of insulin remains a major problem. Pancreatic cancer causes about 6,500 death per anum in the UK and is virtually incurable. Despite this medical importance, research in the developmental biology of the pancreas has produced only a small number of clinically usable data in recent years; see Slack, Development 121 (1995), 1569-1580. In general, all patients suffering from type I diabetes and a large proportion of patients suffering from type II diabetes require daily insulin injections. This tedious and unpleasant treatment requires a high degree of discipline. However, although much effort has been spent in developing approaches to cure pancreatic diseases until now, there is no established treatment of such or related diseases.
  • the vertebrate Pax6 gene is related to the Drosophila pair-rule gene, paired (Walther and Gruss, Development 113 (1991 ), 1435-1449) and encodes two DNA binding domains, a paired- (Bopp et al., Cell 47 (1986), 1033-1040; Treisman et al., Genes Dev. 5 (1991 ), 594-604) and a pa/red-like homeo domain (Frigerio et al., Cell 47 (1989), 735-746).
  • the Pax6 gene shows a complex spatio-temporal expression, exclusively confined to the developing eye, the central nervous system and the pancreas (Macdonald and Wilson, Curr. Opin.
  • Pax6 plays, inter alia, a key role in the eye morphogenesis. The most striking consequence from homozygosity for mutations of Pax6 homologues in Drosophila (eyeless, Quiring et al., Science 265 (1994), 785-789), mice (Small eye mouse, Sey, Hogan et al., J. Embryo. Exp. Morph.
  • Pax6 is involved in the lens-specific transcription of the ⁇ A-crystallin gene, ⁇ 1-crystallin gene and ⁇ -crystallin gene (reviewed in Cvekl and Piatigorsky, Bioessays 18 (1996), 62-I-630).
  • Pax6 has an important function for the development of the brain and spinal cord. Recent evidence indicates that the Pax6-loss-of-fu notion causes distortion of the cortical plate (Schmahl et al., Acta-Neuropathol-Berl. 86 (1993), 126- 135) and migration defects of the cortical neurons (Caric et al., Development 124 (1997), 5087-5096) that are most probably due to a failure of the radial glia cell differentiation.
  • Pax6 is required for correct forebrain patterning, as indicated by the defects in the establishment of morphological and expression boundaries, axonal pathfinding and differentiation of diencephalon in the Small eye mutant (Stoykova et al., Development 122 (1996), 3453-3465; Stoykova et al., Development 124 (1997), 3765-3777; Mastick et al., Development 124 (1997), 1985-1997; Grindley et al., Mech. Dev. 64 (1997), 111-126; Warren and Price, Development 124 (1997), 1573- 1582) and in human probands of aniridia (Glaser et al., loc. cit. 1994).
  • the technical problem of tne present invention is to provide means and methods for the treatment or modulation of pancreatic and related diseases as well as methods for the identification of substances suitable for such purposes.
  • the solution to this technical problem is achieved by providing the embodiments characterized in the claims. Accordingly, the invention relates to a regulatory sequence of the promoter of the Pax6 gene or of a promoter of a gene homologous to the Pax6 gene being capable of conferring expression in pancreatic cells.
  • regulatory sequence refers to sequences which influence the specificity and/or level of expression, for example in the sense that they confer cell and/or tissue specificity. Such regions can be located upstream of the transcription initiation site, but can also be located downstream of it, e.g., in transcribed but not translated leader sequences.
  • promoter within the meaning of the present invention refers to nucleotide sequences necessary for transcription initiation, i.e. RNA polymerase binding, and may also include, for example, the TATA box.
  • promoter region of a gene homologous to the Pax6 gene includes promoter regions and regulatory sequences of genes from other species, for example, human which are homologous to the murine Pax6 gene and which display substantially the same expression pattern. Such promoters are characterized by their capability of conferring expression of a heterologous DNA sequence in substantially all pancreatic cells.
  • regulatory sequences from other species can be used that are functionally homologous to the regulatory sequences of the promoter of the murine Pax6 gene, or promoters of genes that display an identical or similar pattern of expression, in the sense of being expressed in pancreatic cells.
  • pancreas specific element(s) is/are located at the 5' of (the) exon 0.
  • This result is particularly interesting since the prior art (Xu and Saunders, The Journal of Biological Chemistry 272 (1997), 3430-3436; Plaza et al., Molecular and Cellular Biology 15 (1995), 3344- 3353) only identified exon 1 related regulatory sequences that direct expression of Pax6 in particular in neuronal differentiation and retina development.
  • genomic sequences of Pax genes and putative promoters have been reported, the regulatory sequences which are capable of expressing a heteroiogous DNA- sequence in specific tissues, i.e. the pancreas, have not been described in the art.
  • a 1.5 kb 5' UTR genomic fragment of exon 0 of the quail Pax6 gene published by Plaza et al. (Cell Growth Differ. 4 (1993), 1041-1050) does not comprise any pancreas specific sequences.
  • the pancreas is made up of two different tissues, the exocrine cells which are responsible for the production of the digestive enzymes and the endocrine cells, forming Islets of Langerhans which lie as cell clusters in the exocrine pancreas tissue. At least four different cell types can be distinguished in the islets: the glucagon producing ⁇ -cells, the insulin producing ⁇ -cells, the somatostatin producing ⁇ -cells and cells that produce the pancreatic polypeptide, referred to as PP-cells. Both, exocrine and endocrine tissue are derived from a endodermal primordium, see Slack, Development 121 (1995), 1569-1580.
  • Pax6 expression was not only detected at the beginning of pancreas development in the endocrine progenitor cells but also in the major ⁇ -, ⁇ - and ⁇ -cells (Walther, Development 113 (1991 ), 1435-1449; St. Onge, Nature 387 (1997), 406-409).
  • the regulatory region of the Pax6 gene depicted in Fig. 4 contains a pancreas specific element that alone is sufficient and necessary for conferring expression in pancreatic cells. Therefore, several regulatory sequences are identified which are useful to specifically express heterologous DNA sequences in pancreatic cells and/or tissue.
  • 5' upstream genomic fragments were cloned in front of a ⁇ -galactosidase reporter gene and the resulting chimeric genes were introduced by means of microinjection into the pronucleus of fertilized mouse egg cells.
  • pancreas element lies 5 kb upstream of the transcription start point of exon 0 (see Figure 2) and is restricted to a region of 1 ,100 base pairs.
  • the expression of the chimeric gene containing said pancreas element corresponds to the endogenous Pax6 expression in the pancreas.
  • the regulatory sequence comprising the pancreas element of the promoter of the Pax6 gene of mouse or the promoter of a gene homologous to the Pax6 gene can be used to drive the expression of heterologous DNA sequences specifically in pancreatic cells.
  • the present invention also relates to the use of regulatory sequences of promoter regions which are substantially identical to that of the murine Pax6 promoter or to a promoter of a homologous gene or to parts thereof and which are able to confer specific expression in pancreatic cells.
  • Such regulatory sequences differ at one or more positions from the above-mentioned regulatory sequences but still have the same specificity, namely they comprise the same or similar sequence motifs responsible for the above described expression pattern to which the one underlined in Fig. 4 is expected to belong.
  • Preferably such regulatory sequences hybridize to one of the above-mentioned regulatory sequences, most preferably under stringent conditions.
  • Particularly preferred are regulatory sequences which share at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above-mentioned regulatory sequences and have the same specificity.
  • Such regulatory sequences also comprise those which are altered, for example by nucleotide-deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison with the above- described nucleotide sequence.
  • Methods for introducing such modifications in the nucleotide sequence of the promoter of the invention are well known to the person skilled in the art and are described, e.g. in the examples.
  • the nucleotide sequences of the invention can be compared as appropriate computer programs known in the art such as BLAST, which stands for Basic Local Alignment Search Tool (Altschul, 1997; Altschul, J. Mol. Evol.
  • BLAST produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologues. With such means it is possible to identify conserved nucleotide sequences that may play a role in pancreas specific expression (see also the appended examples). Such conserved sequences will be described in more detail below. It is also immediately evident to the person skilled in the art that further regulatory sequences may be added to the promoter of the invention.
  • transcriptional enhancers and/or sequences which allow for induced expression of the regulatory sequences of the invention may be employed.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
  • the regulatory sequence of the invention comprises a nucleotide sequence selected from the group consisting of
  • nucleotide sequences comprising nucleotides 1 to 1150 or nucleotides 42 to 1151 of the nucleotide sequence as depicted in Fig. 4;
  • nucleotide sequences hybridizing with a nucleotide sequence as defined in (a) or (b) under stringent conditions;
  • nucleotide sequences comprising nucleotide sequences which are conserved in (a), (b) and (c).
  • the nucleotide sequence as depicted in Fig. 4 is part of a construct, 406/Sal, containing 8 kb of the promoter region of the murine Pax6 gene. From deletion experiments of the construct the position of the regulatory sequence was deduced and found to be restricted to an area of 2 kb as shown in Fig.1 , corresponding to nucleotides 1 to 2443 of the nucleotide sequence of Fig. 4.
  • the regulatory sequence used to confer pancreas specific gene expression comprises a region of 1.100 bp, between the restriction enzyme sides Spel and Hindi (see Fig.2), corresponding to nucleotides 1 to 1100 of , Fig. 4.
  • pancreas specific regulatory element is located in a region of exactly 1109 bp between the Spel-site (position 42 bp) and Hindi (1151 bp) as shown in Fig. 4.
  • said regulatory sequence comprises a nucleotide sequence comprising nucleotides 969 to 1086 of Fig. 4 (underlined sequence) or a corresponding nucleotide sequence of a Pax promoter or a Pax6 promoter other than the mouse promoter corresponding to said fragment.
  • Sources of Pax promoters are known to the person skilled in the art and originate from different species like, for example, human, quail or fugu (pufferfish) and can be obtained by hybridization experiments.
  • a particular importance for pancreas tissue specificity is assigned to the above referenced nucleotide sequence.
  • the nucleotide sequence comprising the element which confers pancreas specific gene expression comprises any one of the sequence motifs designated as motif C (CATTATTGT), motif D (TTTAATCCAATTATA) or Pbxl (ATCAATCA) either alone or in combination. If all of these motifs are present, they are preferably in the order as shown in Fig. 11. Motifs C and D represent hereby homeodomain DNA binding sites and the Pbxl consensus sequence might regulate Pax6 expression by direct binding of the transcription factor Pbxl (Lu, Mol. Cell Biol. 15 (1995), 3786- 3795).
  • nucleotide sequence comprising nucleotides 1354 to 1460 of the nucleotide sequence shown in Fig. 4 (the Bglll/Accl sequence as shown in Fig. 10) is deleted from said regulatory sequences of the invention.
  • the Bglll/Accl element of the promoter region of Pax6 confers eye lense and cornea specific gene expression and deletion of this element is expected to enhance pancreas specific gene expression. 10
  • said regulatory sequence is derived from the Pax6 gene of mouse, human or fugu (pufferfish).
  • the genomic sequence of the mouse Pax6 gene (Fig. 4) is derived from a ⁇ EMBL3A genomic library (phage clone gp52) generated from the mouse strain C57BI/6. Construct 1 was produced J->y subcloning a 3.7 kb EcoRI fragment and linearising it by a BglH partial digest therefore allowing the insertion of a ⁇ -galactosidase reporter gene within the first exon (EO) of Pax6.
  • Construct 1 produced little or no ⁇ -galactosidase in transgenic mice and was therefore elongated at the 5' end by introducing a 7 kb Sail fragment containing further upstream regulatory sequences (construct 2, 406/SalI deposited with the DSMZ under accession No. DSM 11998). Construct 2 was then digested with the restriction enzymes KpnI/NotI to remove the prokaryotic vector sequence and microinjected into oocytes to generate transgenic mice.
  • the regulatory sequence of the present invention was originally obtained from mouse strain C57BI/6 by screening a genomic Lambda phage library ( ⁇ EMBL3A) using a Pax6 cDNA probe described by Walther, Development 113 (1991 ), 1435.
  • Phage clone gp52 contained the most upstream genomic sequence and was used for the promoter analysis experiments.
  • said regulatory sequence is part of a recombinant DNA molecule.
  • DNA molecules may further comprise further genes such as insulin, glucagon, Pax4, Pax6, Isl1 , Pdx1 or other pancreatic genes suitable for example for expression or co-expression of the aforementioned genes in a specific and restricted manner in the pancreas.
  • the recombinant DNA molecule of the invention comprises a minimal promoter.
  • a minimal promoter is for example:
  • said minimal promoter is a Pax6 derived minimal promoter, for example, comprising the motifs C, D and Pbxl as shown in Figure 11 and/or a TATA or CAAT- box.
  • the regulatory sequencers operatively linked to a heterologous DNA sequence.
  • heterologous with respect to the DNA sequence being operatively linked to the regulatory sequence of the invention means that said DNA sequence is not naturally linked to the regulatory sequence of the invention.
  • Expression of said heterologous DNA sequence comprises transcription of the DNA sequence, preferably into a translatable mRNA.
  • Regulatory elements additionally required for ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They optionally comprise poly-A signals ensuring termination of transcription and stabilization of the transcript.
  • Additional regulatory elements may include transcriptional as well as translational enhancers.
  • Preferably said promoter is a minimal promoter, as indicated above. These promoters can be combined with the regulatory sequences of the invention in order to confer expression in pancreatic cells.
  • the heterologous DNA sequence of the above- described recombinant DNA molecules encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme.
  • the recombinant DNA molecule of the invention can be used alone or as a part of a vector to express heterologous DNA sequences, which, e.g., encode proteins other than Pax6, in pancreatic cells for, e.g., gene therapy or as diagnostics of diseases related to the pancreas.
  • heterologous DNA sequences which, e.g., encode proteins other than Pax6, in pancreatic cells for, e.g., gene therapy or as diagnostics of diseases related to the pancreas.
  • the recombinant DNA molecule or vector containing the DNA sequence encoding a protein of interest is introduced into the ceils which in turn produce the protein of interest.
  • the recombinant DNA molecule of the invention can be used for "gene targeting” and/or “gene replacement”, for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; Anderson, Nature 392 Supp.
  • nucleic acids Delivery of nucleic acids to a specific site in the body for gene therapy or antisense therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729).
  • Standard methods for transfecting cells with recombinant DNA are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469.
  • Gene therapy and antisense therapy to pancreatic diseases may be carried out by directly administering the recombinant DNA molecule or vector of the invention to a patient or by transfecting pancreatic cells with the recombinant DNA molecule or vector of the invention ex vivo and infusing the transfected cells into the patient.
  • research pertaining to gene transfer into cells of the germ line is one of the fastest growing fields in reproductive biology.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., WO94/29469, WO 97/00957, Anderson (1998) loc. cit. or Schaper (1996), loc. cit. and references cited therein. It is to be understood that the introduced recombinant DNA molecules and vectors of the invention express the heterologous DNA sequence after introduction into said cell and preferably remain in this status during the lifetime of said cell.
  • cell lines which stably express the heterologous DNA under the control of the regulatory sequence of the invention may be engineered according to methods well known to those skilled in the art.
  • host cells can be transformed with the recombinant DNA molecule or vector of the invention and a selectable marker, either on the same or separate vectors.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows for the selection of cells having stably integrated the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the heterologous DNA sequence under the control of the regulatory sequence of the invention.
  • Such engineered cell lines are particularly useful in screening compounds capable of modulating gene expression in pancreatic cells. Such compounds can be for example small molecules as described in Gottesfeld, Nature 387 (1997), 202-205.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), hypoxanthine-guanine phosphoribosyitransferase (Szybalska, Proc. Natl. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyitransferase (Lowy, Cell 22 (1980), 817) in tk, hgprt or aprt cells, respectively.
  • antimetaboiite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl. Acad.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC omithine decarboxylase
  • the person skilled in the art may also use the regulatory sequences of the invention to "knock out" an endogenous gene comprising identical or similar regulatory sequences, for example, by gene targeting, cosuppression, triple helix, antisense or ribozyme technology.
  • the regulatory sequences, recombinant DNA molecules and vectors of the invention may be designed for direct introduction or for introduction via iiposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • said cell is a germ line cell, embryonic cell, or egg ceil or derived therefrom.
  • the regulatory sequences of the invention may also be used in gene therapy to treat diseases such as for example type I and II diabetes, insulinomas and glucagonomas.
  • the regulatory sequences of the invention can be operatively linked to sequences encoding cellular growth factors capable of stimulating or inducing differentiation and proliferation of normal ⁇ -cells in the case of diabetes or cell death initiating factors capable of inhibiting the growth of tumorous cells present in pancreatic cancers.
  • said protein is selected from the group consisting of insulin, glucagon, Pax4, Pax6; Pdx1 , Isl1 and other proteins present in the pancreas.
  • sequences encoding the human insulin gene, Pax4, Pax6, Pdx1, IsM and other genes are advantageously operatively linked to the regulatory sequences of the invention and expressed in the cells.
  • Other heterologous proteins indicated above, e.g., proteins which may be specifically expressed in pancreatic cells to ensure the delivery of therapeutic peptides to the insulin producing ⁇ -cells may also be operatively linked to the regulatory sequences of the invention.
  • said protein is a scorable marker, preferably luciferase, green fluorescent protein or ⁇ -galactosidase (lacZ).
  • a scorable marker preferably luciferase, green fluorescent protein or ⁇ -galactosidase (lacZ).
  • lacZ ⁇ -galactosidase
  • pancreatic cells can be cultured in the presence and absence of a candidate compound in order to determine whether the compound affects the expression of genes which are under the control of regulatory sequences of the invention, which can be measured, e.g., by monitoring the expression of the above-mentioned marker.
  • telomeres may be used as well, encoding, for example, a selectable marker which provides for the direct selection of compounds which induce or inhibit the expression of said marker.
  • the regulatory sequences of the invention may also be used in methods of antisense therapy. Antisense therapy may be carried out by administering to an animal or a human patient, a recombinant DNA containing the regulatory sequences of the invention operably linked to a DNA sequence, i.e., an antisense template which is transcribed into an antisense RNA.
  • the antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and optionally up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to a portion of a specific mRNA sequence and/or DNA sequence of the gene of interest. Standard methods relating to antisense technology have been described (Melani, Cancer Res. 51 (1991), 2897-2901). Following transcription of the DNA sequence into antisense RNA, the antisense RNA binds to its target sequence within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein encoded by the mRNA. Such antisense therapy may be used to treat pancreatic diseases that are, for example, the result of Pax6 overexpression.
  • said antisense RNA or said ribozyme is directed against a gene involved in the development of the pancreas.
  • the invention relates to nucleic acid molecules of at least 15 nucleotides in length hybridizing specifically with a regulatory sequence as described above or with a complementary strand thereof.
  • Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences having no or substantially different regulatory properties.
  • the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65°.
  • Said nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length.
  • nucleic acid probes of 17 to 35 nucleotides in length are preferred. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length.
  • the nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of regulatory sequences according to the invention. Another application is the use as a hybridization probe to identify regulatory sequences hybridizing to the regulatory sequences of the invention by homology screening of genomic DNA libraries.
  • Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a regulatory sequence as described above may also be used for repression of expression of a gene comprising such regulatory sequences, for example due to an antisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201 , EP-A2 0 360 257) which specifically cleave the (pre)- mRNA of a gene comprising a regulatory sequence of the invention. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al.
  • nucleic acid probe with an appropriate marker for specific applications, such as for the detection of the presence of a regulatory sequence or recombinant DNA molecule of the invention in a sample derived from an organism.
  • nucleic acid molecules may either be DNA or RNA or a hybrid thereof.
  • said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
  • Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
  • Such nucleic acid molecules may further contain ribozyme sequences which specifically cleave the (pre)-mRNA comprising the regulatory sequence of the invention.
  • the present invention also relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a regulatory sequence or a recombinant DNA molecule of the invention.
  • said vector is an expression vector' and/or a targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the recombinant DNA molecule or vector of the invention into targeted cell populations.
  • the present invention furthermore relates to host cells transformed with a regulatory sequence, a DNA molecule or vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the regulatory sequence, vector or recombinant DNA molecule of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • Suitable mammalian cell lines comprise Saos-2 human osteosarcoma cells (ATCC HTB-85), HeLa human epidermoid carcinoma cells (ATCC CRL-7923), HepG2 human hepatoma cells (ATCC HB-8065), human fibroblasts (ATCC CRL-1634), U937 human histiocytic lymphoma cells (ATCC CRL-7939), RD human embryonic rhabdomyosarcoma cells (ATCC CCL-136), MCF7 human breast adenocarcinoma cells (ATCC HTB-22), JEG-3 human choriocarcinoma cells (ATCC HB36), A7r5 fetal rat aortic smooth muscle cells (ATCC CRL-1444), NIH 3T3 mouse fibroblasts (ATCC CRL-1658) HEP 3B (ATCC HB 8064), C6 (ATCC CCL 107) and GS 9L obtainable from the American Type Culture Collection.
  • Primary-culture HUVEC may be obtained from Clonetics Corp. (San Diego, CA) and can be grown in EGM medium containing 2% fetal calf serum (Clonetics). Said host cell can also be a pancreatic cell or a cell derived therefrom, or a primary cell, tumor cell, spheroid cell, aggregate cell, stem cell or a differentiated cell although any other animal, preferably mammalian cell may be appropriate as well.
  • the present invention relates to a composition, preferably a pharmaceutical composition comprising at least one of the aforementioned regulatory sequences, recombinant DNA molecules or vectors of the invention, either alone or in combination, and optionally a pharmaceutically acceptable carrier or excipient.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods.
  • Suitable excipients are equally well known and include water, gelatin, starch, magnesium stearate, talc, vegetable oils and the like.
  • compositions can be administered to the subject at a suitable dose.
  • Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day.
  • the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 22 copies of the DNA molecule.
  • the compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
  • the various recombinant DNA molecules and vectors of the invention are administered either alone or in any combination using e.g. appropriate gene delivery systems, and optionally together with an appropriate compound, for example insulin, and/or together with a pharmaceutically acceptable carrier or excipient.
  • said recombinant DNA molecules may be stably integrated ' into the genome of the mammal.
  • viral vectors may be used which are specific for certain cells or tissues, preferably for pancreatic cells and persist in said cells.
  • the pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delaying of different kinds of diseases, which are related to the expression or overexpression of genes in pancreatic cells.
  • a pharmaceutical composition of the invention which comprises a regulatory sequence, recombinant DNA molecule or vector of the invention in gene therapy.
  • Gene therapy approaches in connection with the recombinant DNA molecule of the invention have already been discussed herein above.
  • suitable pharmaceutical compositions may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others.
  • the pharmaceutical compositions according to the invention can be used for the treatment of diseases hitherto unknown as being related to pancreas related gene expression.
  • An embryonic cell can be for example an embryonic stem cell as described in, e.g., Nagy, Proc. Natl. Acad. Sci. 90 (1993) 8424-8428. Further applications of the pharmaceutical composition of the invention as well as general and specific methods of producing the active ingredients thereof, in particular for gene therapy purposes, have been described herein above.
  • the present invention also relates to diagnostic compositions comprising at least one of the aforementioned nucleic acid molecules, recombinant DNA molecules or vectors, and, optionally suitable means for detection.
  • Said compositions may further contain compounds such as further plasmids, antibiotics and the like for screening transgenic animals and/or animal cells useful for the genetic engineering of non- human animals, preferably mammals and most preferably mouse.
  • the diagnostic compositions of the invention may be used for methods of detecting and isolating regulatory sequences which are a functionally equivalent to regulatory sequences of the invention capable of modulating gene expression in pancreatic cells.
  • the present invention also relates to a method for the production of a transgenic non- human animal, preferably a transgenic mouse, comprising the introduction of a recombinant DNA molecule or vector of the invention into a germ cell, an embryonic cell or an egg or a cell derived therefrom.
  • the non-human animal to be used as a source for the production of the transgenic animal in the method of the invention may be a non-transgenic healthy animal, or may have a disease or disorder, preferably a pancreatic disease, such as types I and II diabetes, insulonomas and glucagonomas.
  • Said disease or disorder may be an inborn insufficiency or naturally developed or caused by genetic engineering, for instance by the expression of a DNA sequence encoding a protein involved in a pancreatic disease, preferably under the control of the regulatory sequence of the invention.
  • the invention also relates to transgenic non-human animals such as transgenic mice, rats, hamsters, dogs, monkeys, rabbits or pigs comprising a recombinant DNA molecule or vector of the invention or obtained by the method described above, preferably wherein said recombinant DNA molecule is stably integrated into the genome of said non-human animal, preferably such that the presence of said recombinant DNA molecule or vector leads to the transcription and/or expression of the heterologous DNA sequence by the regulatory sequence of the invention.
  • transgenic non-human animals such as transgenic mice, rats, hamsters, dogs, monkeys, rabbits or pigs comprising a recombinant DNA molecule or vector of the invention or obtained by the method described above, preferably wherein said recombinant DNA molecule is stably integrated into the genome of said non-human animal, preferably such that the presence of said recombinant DNA molecule or vector leads to the transcription and/or expression of the heterologous DNA sequence by the regulatory sequence of the
  • pancreas specific gene expression since panreatic cell specific gene expression has different patterns in different stages of physiological and pathological conditions, it is now possible to determine further regulatory sequences which may be important for the up- or down-regulation of pancreatic cell gene expression, for example in specific tumors. In addition, it is now possible to in vivo study mutations which affect different functional or regulatory aspects of specific gene expression in pancreatic cells.
  • pancreatic diseases The in vivo studies referred to above will be suitable to further broaden the knowledge on the mechanisms involved in pancreatic diseases. To date, it is known that type I diabetes is the result of partial or total destruction of the insulin-producing ⁇ -cells by immunological, chemical or viral factors. Nonetheless, a small population of cells within the pancreas are thought capable of regenerating the ⁇ -cell population. Certain genes have been shown to play a major role in ⁇ -cell neogenesis (Jonsson, Nature 371 (1994), 606; Ahlgren, Nature 385 (1997), 257; Sosa-Pineda, Nature 386 (1997), 399; St-Onge, Nature 387 (1997), 406).
  • the present invention further relates to a method for the identification of an agonist/activator and/or an antagonist/inhibitor of genes or gene products involved in pancreatic diseases comprising the steps of:
  • read out system in context with the present invention means a DNA sequence which upon transcription and/or expression in a cell, tissue or organism provides for a scorable and/or selectable phenotype.
  • read out systems are well known to those skilled in the art and comprise, for example, recombinant DNA molecules as described above.
  • plurality of compounds as used to describe the method of the invention is to be understood as a plurality of substances which may or may not be identical.
  • Said plurality of compounds may be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms.
  • said compounds may be known in the art but hitherto not known to be capable of suppressing or activating and/or enhancing the transcription of a pancreas-specific expressed gene.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into or fed to the animals.
  • the method of the invention further comprises the step of
  • the method of the invention further comprises the step of
  • step (f) subdividing the samples identified in step (c) and repeating steps (a) to (c) one or more times.
  • a sample containing a plurality of compounds is identified in the method of the invention, then it is in a further step (d) as described herein above either possible to isolate the compound from the original sample identified as containing the compound capable of suppressing or activating and/or enhancing the transcription of the read out system, e.g. a panreatic cell-specific expressed gene in an animal or human cell, or tissue or non-human animal. It can then be, in step (e) as described above, determined whether said sample or compound mimics or suppresses the cellular effects of the Pax6 protein, for example the differentiation of glucagon-producing ⁇ - cells in pancreas of the mouse as described in St. Onge, Nature 387 (1997), 406- 409.
  • the steps described above can be performed several times preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s). Accordingly, one can further as step (f) subdive the samples identified in step (c) and repeat steps (a) to (c) one or more times. Therefore, one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample.
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the method of the invention further comprises, optionally, the step of
  • Compounds which can be tested for in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, hormones, compounds produced by peptidomimetics, PNAs and the like. Said compounds can also be functional derivatives or analogues of known inhibitors or activators. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A.
  • Nucleic acids useful in the method of the invention comprise DNA or RNA or hybrids thereof.
  • said nucleic acid may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. These modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell.
  • PNA peptide nucleic acid
  • PNA peptide nucleic acid
  • PNAs binding of PNAs to complementary as well as various single stranded RNA and DNA nucleic acid molecules can be systematically investigated using, e.g., thermal turation and BIAcore surface-interaction techniques (Jensen, Biochemistry 36 (1997), 5072-5077).
  • the synthesis of PNAs can be performed according to methods known in the art, for example, as described in Koch, J. Pept. Res. 49 (1997), 80-88; Finn, Nucleic Acids Research 24 (1996), 3357-3363.
  • folding simulations and computer redesign of structural motifs of target proteins involved in pancreatic diseases can be performed using appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci.
  • agonists/activators or inhibitors/antagonists can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive amide alkylation and testing the resulting compounds, e.g., according to the methods described herein and in the appended examples. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Domer, Bioorg. Med. Chem. 4 (1996), 709-715.
  • a three-dimensional and/or crystallographic structure of inhibitors of the target proteins involved in pancreatic diseases can be used for the design of peptidomimetic inhibitors target proteins (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545- 1558).
  • antibodies sr ifically recognizing target proteins involved in pancreatic .; v-r ⁇ es or parts, i . occidental ⁇ ecific fragments or epitopes, of such target proteins and thereb> inactivating said protein may be employed.
  • These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
  • Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988 or EP-B1 0 451 216 and references cited therein.
  • the method of the invention (a) allows the identification (or verification) of compounds having the above referenced activating/enhancing or suppressing/inhibitory activities, and (b) optionally comprise the further step of improving the referenced feature(s) by, for example, peptidomimetics or computer programs.
  • said recombinant DNA molecule comprising said read out system is a recombinant DNA molecule of the invention or a vector of the invention as described in the embodiments hereinbefore.
  • said animal or human cell, tissue or non-human animal is a cell, tissue or transgenic non-human animal of the invention described in the embodiments hereinbefore.
  • said recombinant DNA molecule comprised in said animal or human cell, tissue or non- human transgenic animal is introduced into the genome by transfection, transformation, electroporation, infection or particle bombardment.
  • Determining whether a compound is capable of suppressing or activating and/or enhancing the transcription of a pancreas-specific regulated gene can be done, for example, in mice by monitoring the reporter gene. It can further be done by monitoring the behavior or the health status of the transgenic non-human animals of the invention contacted with the compounds and compare it to that of wild-type animals. In an additional embodiment, said behavior or health status may be compared to that of a transgenic non-human animal contacted with a compound which is either known to be capable or incapable of suppressing or activating and/or enhancing Pax6 gene expression and/or function of said transgenic non-human animal of the invention.
  • the compounds identified according to the method of the invention are expected to be very beneficial since treatment of pancreatic diseases such as diabetes is restricted to daily injections of insulin and, most importantly gene therapy that has been used so far is only limited due to the non-tissue specificity of the regulatory sequences used in the targeting vectors so far available.
  • the present invention provides methods for identifying compounds which modulate pancreas specific gene expression.
  • activators or compounds found to enhance Pax6 expression may be used in the processes of regenerating, increasing or enhancing the insulin producing ⁇ -cell population in patients suffering from diabetes.
  • antagonists or compounds found to downregulate Pax6 expression may be used in the treatment of insulinomas or glucagonomas where overexpression of endogenous Pax6 may induce normal cells to become tumorigenic.
  • the above-mentioned compounds can also be used to treat patients who have, or have had acute pancreatitis or surgi-sal pancreatectomy where the ⁇ -cell population has been destroyed.
  • the invention also comprises a method wherein the compound identified in the method described hereinbefore or a compound whose desired features have been further improved as described above is formulated in a pharmaceutical composition. Therefore, the present invention furthermore relates to a method for the preparation of an agonist/activator and/or an antagonist/inhibitor of genes or gene products involved in pancreatic diseases, comprising the steps (a), (b) and (c) as described herein above, wherein the method further optionally comprises any of the steps of (d), (e), (f) and/or (g) as described herein above and formulating the compound identified in any one of these later steps into a pharmaceutical composition.
  • the compounds identified obtained or improved according to the method of the present invention are expected to be very useful in diagnostic and therapeutic applications.
  • the invention relates to a compound obtained identified or improved according to the method of the invention said compound being an agonist/activator of Pax6 gene expression and/or function or an antagonist/inhibitor of Pax6 gene expression and/or function.
  • the therapeutically useful compounds identified or improved according to the method of the invention may be administered to a patient by any appropriate method for the particular compound, e.g., orally, intravenously, parenterally, transdermally, transmucosally, or by surgery or implantation (e.g., with the compound being in the form of a solid or semi-solid biologically compatible and resorbable matrix) at or near the site where the effect of the compound is desired.
  • This method would apply most generally to patients suffering from diabetes where Pax6 activation by the compound would lead to a regeneration of insulin producing cells.
  • Therapeutic doses are determined to be appropriate by one skilled in the art.
  • Such therapeutically useful compounds can be for example transacting factors which bind to the regulatory sequence of the invention. Identification of transacting factors is carried out using standard methods in the art (see, e.g., Sambrook, supra, and Ausubel, supra) or methods as described in the appended examples. To determine whether a protein binds to the regulatory sequences of the invention, standard DNA footprinting and/or native gel-shift analyses can be carried out. In order to identify a transacting factor which binds to the regulatory sequence of the invention, the regulatory sequence can be used as an affinity reagent in standard protein purification methods, or as a probe for screening an expression library.
  • transacting factor modulation of its binding to the regulatory sequences of the invention can be pursued, beginning with, for example, screening for inhibitors against the binding of the transacting factor to the regulatory sequences of the present invention or by applying mutagenesis techniques that would also effect the active site or a site involved in the regulation of the factor.
  • Activation or repression in connection with the goals of the present invention could then be achieved in a patient by administration of the transacting factor (or its inhibitor) or the gene encoding it, e.g. in a vector for gene therapy.
  • the active form of the transacting factor is a dimer, dominant-negative mutants of the transacting factor could be made in order to inhibit its activity.
  • transacting factor upon identification of the transacting factor, further components in the pathway leading to activation (e.g. signal transduction) or repression of a gene under the control of the regulatory sequences of the present invention can then be identified. Modulation of the activities of these components can then be pursued, in order to develop additional drugs and methods for modulating the expression of a gene under the control of the regulatory sequences of the present invention.
  • signal transduction e.g. signal transduction
  • the present invention also relates to an antibody specifically recognizing the compound of the present invention.
  • Monoclonal antibodies can be prepared, for example, based on the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981 ), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals.
  • antibodies or fragments thereof to the aforementioned pancreas-specific expressed gene products can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbour, 1988. These antibodies may be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv, or scFv fragments etc.
  • the present invention relates to pharmaceutical and diagnostic compositions comprising the above-described compounds which are agonists/activators or antagonists/inhibitors and/or antibodies and optionally a pharmaceutically acceptable carrier or suitable means for detection, respectively; see supra. Further, the present invention relates to the use of the regulatory sequence, the recombinant DNA molecule, vector, cell, pharmaceutical compositions, diagnostic compositions or a transgenic non-human animal of the invention for the identification of a chemical and/or biological substance capable of suppressing or activating and/or enhancing the transcription, expression and/or activity of pancreas-specific genes and/or its expression products.
  • the chemical or biological substance used in the methods and uses of the present invention is selected from the group consisting of peptides, proteins, nucleic acids, antibodies, small organic compounds, antibiotics, hormones, neural transmitters, compounds obtained by peptidomimimetics, and PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited, supra).
  • the present invention relates to the use of a regulatory sequence, a recombinant DNA molecule, vector, nucleic acid molecule of the invention, compound and/or antibody of the invention for the preparation of a composition for directing and/or preventing expression of genes specifically in the pancreas and/or for the preparation of a pharmaceutical composition for treating, preventing and/or delaying a pancreatic disease in a subject.
  • the present invention relates to the use of a recombinant DNA molecule, vector, nucleic acid molecule compound and/or antibody of the invention for the preparation of a composition for inducing a pancreatic disease in a non-human animal or in a transgenic non-human animal.
  • the regulatory sequences of the invention can be used for generating transgenic animals that display a pancreatic disease such as types I and II diabetes, insuionomas and glucagnonomas.
  • anti-sense RNA directed against Pax6 mRNA can be expressed under the control of said sequence, or the knock-out-animals can be generated via homologous recombinant with a promoter fragment of the invention.
  • compositions suitable for the introduction into animal cells preferably stem cells.
  • Said compositions may comprise pharmaceutically acceptable carriers such as described before.
  • pharmaceutically acceptable carriers such as described before.
  • compositions, uses, methods of the invention can be used for the treatment of all kinds of diseases hitherto unknown as being related to or dependent on the modulation of the pancreas.
  • the pharmaceutical compositions, methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.
  • Figure 1 Pax6 promoter/reporter gene constructs for the identification of decontrol elements for the transgene expression in lens, cornea and the pancreas.
  • the exact location of exon 0 (transcription start) on the restriction map is marked.
  • the arrow indicates the transcription start site in exon 0 (EO).
  • The' cloned regions have been marked by horizontal lines.
  • the identified 1100 nt cis-element for the pancreas is indicated by an box (A) while the 120 nt element for the lens and cornea element by a box (B).
  • restriction- enzymes were used:, Accl; B, Bglll; E, EcoRI; H, Hindlll; He, Hindi; N, Nsil; P, Pstl, S, Sail; Sp, Spel. ' x, Xbal
  • FIG. 3 Histological section of pancreas from line 406/Sal32.
  • A C, Transversal sections.
  • B Sagittal section.
  • D Transversal section stained with an antibody specific for glucagon.
  • E Transversal section stained with an antibody specific for insulin.
  • the earliest expression of the ⁇ - gaiactosidase reporter gene was detected in E9.0 embryos. This expression corresponded to the endogenous expression pattern of Pax6 (A), ⁇ -galactosidase activity was also detected in E12.5 embryos (B) and in newboms/adult animals (C) where ⁇ -galactosidase expression was restricted to cells expressing either glucagon (D) or insulin (E).
  • Figure 4 Nucleotide sequence of the Pax6 promoter. Underlined sequence: this region has a ' high sequence homology to the genomic sequence of the Pax6 phage-DNA of Fugu (pufferfish) rubripes. The location of the pancreas element is between the 5' Spe restriction side and the 3' Hindi restriction side.
  • Figure 5 Structure of the murine Pax6 transcripts. Arrows indicate the transcriptional start sites of the identified three transcripts, a, b, c. The translational start site (ATG, thin arrow) is located in exon 4. Exon 5, 6 and part of exon 7 contain the paired box, marked in black.
  • Figure 6 Sequence comparison between the mouse (M) and the quail (Q) exon sequences.
  • A, B represents mouse RT-PCR products for transcript a and transcript b;
  • C represents mouse genomic DNA sequence corresponding to exon ⁇ . 32
  • Figure 7 Developmental expression analysis of the lacZ reporter gene in a transgenic line carrying the construct 2 (406/Sal).
  • A-C, H-K) and (D-G, L-N) are views of embryos after whole-mount ⁇ -gal staining and after sectioning at the indicated stages respectively. * .,.
  • the arrowhead in C points to a stream of lacZ positive cells that extend from the head mesenchyme over the anterior edge of the first branchial arch.
  • the curved arrow in H points to the strom of the duct of the lacrimal gland.
  • C, L-M ).
  • M and N shows sections after ⁇ -gal whole mount staining and immunohistochemistry for detection of glucagon (M) or insulin (N).
  • the arrows in M and N point to colocalization of the lacZ reporter expression with endocrine cells of the islands producing glucagon or insulin, respectively.
  • FIG. 8 Transgene lacZ expression driven by the construct 12, (containing Fugu (pufferfish) control sequences) in mouse telencephaion (T), lens (L) and pancreas (P) at stage E 12.5.
  • Transgene lacZ expression driven by construct 14 (containing mouse control sequences) in dorsal telencephaion (T), hindbrain (Hb) and spinal cord (SC) at stage E 11.5.
  • Figure 9 Upper panel: Identification of the Pax6 c/s-elements responsible for the transgene expression in neural retina.
  • the arrow indicates the transcriptional start site in exon 0, exon 1 and exon ⁇ .
  • the reporter constructs carry the transcriptional start point from the Pax6 gene of exon ⁇ (construct 15), of exon 0 (constructs 17, 8, 19, 20) or from the TK-gene (construct 16).
  • the identified 530 nt cis- element controlling the transgene expression in the retina is indicated by yellow box.
  • Lower panel Transgene lacZ expression in developing neural retina.
  • FIGS. 1 and (F) are views after whole mount lacZ staining in transgenic mice, carrying the construct 17 (406/8) or the construct 18 (406/Fugu (pufferfish)), respectively.
  • the arrows in (A-C) and (F) point to a region within the dorsal neural retina that appears negative, observed in mice carying the mouse or Fugu (pufferfish) regulatory sequence, respectively.
  • (C) note the strong Pax6/ ⁇ acZ expression in the iris (tr) of the eye at postnatal stage (P1) and the lack of signal in the dorsal domain (arrowheads).
  • the curved arrow in (D, stage E 13.5) points to a thin layer of lacZ positive cells, connecting the strongly positive nasal and temporal retinal domains, observed in few transgenic embryos.
  • the arrowhead in (E) points to a strong ⁇ -gal staining in the inner nuclear layer of the neural retina.
  • Figure 10 Sequence comparison of the conserved elements in mouse, human and fugu (pufferfish) Pax6/genomic DNA that control the expression in the eye tissues of head surface ectodermal origin.
  • the minimal element which is necessary for driving the lacZ expression of the reporter gene into the lens and cornea is a 107 nt Bglll/Accl fragment (boxed). Two potential homeobox binding sites are shown as DNA motif A and B.
  • FIG 11 Sequence comparison of the mouse element controlling the expression in the pancreas with the human and the Fugu (pufferfish) Pax6 genomic DNA reveals a fragment of 124 nt with high percentage identitiy. This sequence contains potential binding sites for Pbxl. Two possible motifs (C and D) for homeobox binding sites are boxed.
  • Figure 12 Sequence comparison of the retina element between mouse, human and Fugu (pufferfish) genomic region. This sequence contains potential binding sites for the Msx1 and Pax2. and possible motifs for homeobox binding sites (motif E and motif I).
  • Figure 13 Scheme illustrating the localization of the identified control elements in the mouse Pax6 locus that control the expression of the gene in X e pancreas (box A); lens, cornea, lacrimal gland, conjunctiva (box B), telencephaion, spinal cord and hindbrain (box C) and neural retina (box D)
  • Figure 14 Table showing transgene expression in transient and founder embryos.
  • the regulatory sequence of the present invention was originally obtained from mouse strain C57BI/6 (commercially available at the Gottinstitut fur remediesstierzucht, Hannover) by screening of a genomic Lambda phage library ( ⁇ EMBL3A, obtainable from the laboratory of Prof. Sidney Brenner, MRC, Cambridge, UK), using a Pax6 cDNA probe described by Walther, Development 113 (1991).
  • Phage clone gp52 contained the most upstream genomic sequence and was used for the promoter analysis experiments.
  • a composite restriction map of part of the genomic region isolated is shown in Fig. 1.
  • construct 2 406/Sal.
  • DSMZ accession number DSM 11998; Deutsche Sammiung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1 b, D-38124 Braunschweig
  • Construct 2 originated from a 3,7 kb genomic EcoRI subclone which contains the first untranslated exon (exon 0, EO). This fragment was partially digested with Bglll, which cuts in Exon 0.
  • a lacZ-polyA-fragment (lacZ gene with its own ATG and Sv40 polyadenylation signal) was inserted into the Bglll-site of this construct via blunt-end-reaction (construct 1 , 406).
  • This construct did not show any Pax6 specific expression patterns.
  • a 7 kb Sall-subclone from murine genomic Pax6 gene with further 5' upstream genomic sequence was digested with Nsil and Kpnl and ligated to a 6.7 kb fragment of construct 406, obtained by linearisation with Kpnl and partial digestion with Nsil containing the minimal promoter of Pax6 and the lacZ-fragment.
  • construct 2 The deletions were produced by removing 5' and 3' genomic sequences of construct 2 (406/Sal). Subsequently, transgenic mice were produced with this construct (Fig.1). Therefor, construct 2 was linearised with Notl and Kpnl and the fragment was injected into the pronucleus of oocytes of FVB-mice. Production of transgenic embryos and whole-mount ⁇ -galactosidase staining was performed as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonal membranes of the embryos were analyzed using Southern blots with a lacZ probe.
  • the embryos were fixed overnight with 4% paraformaldehyd and after washing with PBS, the embryos were dehydrated with ethanol and embedded into paraffin. The cross and sagittal sections were counterstained with neutral-red and the transgene expression pattern was analyzed by light microscopy.
  • the earliest transgenic activity in the pancreas could be determined on day 9,0 p.c. (Fig. 3) with the promoter fragment (Fig. 1; construct 2 406/Sal), which mimics the endogenous Pax ⁇ expression, ⁇ -galactosidase activity could be observed in the pancreas throughout embryo development (Fig. 3). Strong expression of the transgene can also be demonstrated in newborn mice and in adult transgenic mice (6 weeks; Fig. 3).
  • pancreas element which lies at 5 kb upstream of the transcription start point of exon 0, is restricted to a region of 1100 bp defined by the deletion analysis described above.
  • transgenic activity corresponds to the endogenous Pax ⁇ expression in the pancreas
  • endocrine hormone production in the transgenic lines was examined.
  • specific antibodies against either insulin or glucagon ⁇ - galactosidase positive cells were shown to express either insulin or glucagon (Fig. 3).
  • No ectopic expression could be detected in the exocrine tissue of the pancreas.
  • the primary antibodies used in the analysis mouse anti-insulin (Sigma) and mouse anti- glucagon (Sigma), were applied on paraffin sections after ⁇ -galactosidase staining and detected with a secondary horseradish peroxidase antibody as described by Sosa-Pineda, (1997) Nature 386, 399-402.
  • Example 3 Further transgene construction for the identification of mouse Pax ⁇ regulatory elements
  • Construct 4 (TK-1 ) was generated by cloning a 2.4 kb Notl-Asp718 fragment fr-Q-m the 7 kb Sail subcione mentioned above (see construct 2), into the vector pax-L680 which contains a minimal TK promoter and a lacZ-gene SV40-polyA cassette.
  • Construct 5 (406/Hincll) was generated by removing the Sall/Hincll fragment respectively from construct 2.
  • Constructs 6 - 9 were generated by ligating the following blunt ended genomic DNA fragments into the construct 11 (construct 11 as described below): Hindi to EcoRI; (construct 6), EcoRI - EcoRI (construct 7), Accl - Accl (construct 8) Bglll - EcoRI (construct 9_ Construct 8 contains two copies of the fragment in 5' - 3' orientation, while construct 9 has three copies in 3' - 5' orientation.
  • construct 10 containing the Pax ⁇ minimal promoter PO was generated by deleting the Sall-Xbal fragment from construct 2. This construct only contains the Pax ⁇ minimal promoter PO and the lacZ-polyA cassette.
  • construct 11_the promoter PO was further shortened by deleting the upstream Xbal - BamHI fragment and subsequently the Hindi - EcoRI fragment from construct 6 was subcloned into it in ⁇ ' .to 3 ' orientation.
  • the construct 12/Fugu (pufferfish), (Fig. 8) carrying pufferfish control elements was generated as follows. A 12 kb genomic Sail fragment was subcloned into pBSKS+. A blunt ended IRES/lacZ/polyA-fragment was ligated into the blunt ended Kpnl- restriction-site in the polylinker of the 12 kb genomic subcione. The IRES (internal ribosome entry site) was added upstream to the lacZ gene to facilitate its cap- independent translation.
  • the lacZ-poly A cassette was inserted into the BamHI site of exon 4.
  • a total of 13 kb of upstream DNA sequence was added in multiple steps to generate the final construct 2118/14P (Fig. 8).
  • Construct 14 was generated by inserting the lacZ-polyA cassette into the Xbal site of the exon ⁇ present in the 1.2 kb EcoRI - Xbal clone (Fig. 9)
  • Construct 16 (TK-2, Fig. 9) was generated by cloning a 1.8 kb genomic Accl- fragment containing exon into the vector pax-L680 which contains a minimal TK- promoter and a lacZ-gene SV40-polyA cassette.
  • DNA sequences tested in the reporter transgene . s (construct 17, 19 and 20, Fig. 9) were isolated from the 1.8 kb Accl genomic- fragment, end-filled using the Klenow-fragment of DNA polymerase I and subcloned into the minimal-promoter oriented 5' to 3' with respect to the lacZ.
  • the upstream DNA sequences contained in the constructs 17, 19 and 20 are Accl- Accl (1.4 kb), Bglll - Xbal (0.6 kb) and Dralll - Xbal (0.29 kb), respectively.
  • Transgenic sequences were always purified from vector sequences by appropriate restriction enzymes prior to microinjection.
  • a genomic lambda-DASHII-library of the pufferfish (Fugu rubripes) was screened with a 320 bp EcoRI-fragment of the murine Pax ⁇ - cDNA.
  • Three Pax ⁇ - phage-clones were isolated and subcloned for sequence analysis.
  • the fugu (pufferfish) and mouse sequences were aligned with the program BESTFIT and FASTA of the GCG package.
  • Example 5 Localization of three transcription start sites and sequence analysis of the Pax ⁇ promoter regions in the mouse In order to delineate the cis-essential elements required for the spatial and temporal activity of the Pax ⁇ gene it was attempted to localize the transcriptional start sites assuming that at least some control elements are located 5' of these sites.
  • the mouse Pax ⁇ promoter region was identified using a combination of primer extension, RT-PCR and genomic DNA sequencing.
  • nt 113-91 complementary to the 5' end of the Pax ⁇ cDNA (Walther et al., Genomics 11 (1991 ), 424-434) was used and two primer extension products of 400 nt and 600 nt in length were obtained suggesting that the 5' end of the published Pax ⁇ cDNA does not contain the initiation site for mRNA transcription. This is compatible with the Pax ⁇ mRNA size of 3 kb (Walther, et al., loc. cit.).
  • RT-PCR experiments were performed.
  • the sequences of the RT-PCR-products for transcript a and transcript b (Fig. 6) in mouse matches with the 5' UTR-regions of the quail transcripts, indicating a conservation of the transcription start sites in the two species.
  • high homology has been detected for the mouse and the quail exon ⁇ after genomic DNA sequencing .
  • the upstream promoter region of exon 1 in mouse contains consensus sequences for various basal promoter elements, such as a conserved TATA-like sequence (AATATTT), three CCAAT boxes and consensus binding sites for Sp1 and Ap-2, which are also highly conserved in the Pax ⁇ - gene of the quail (Plaza et al., loc. cit.) and human (Xu and Saunders, J. Biol. Chem. 272 (1997), 3430-3436).
  • AATATTT conserved TATA-like sequence
  • CCAAT boxes consensus binding sites for Sp1 and Ap-2
  • Example 6 Expression of Pax ⁇ in ectodermal derivatives of the developing eye, in the pancreas and in the olfactory bulb is directed by a regulatory region located 5 ' from exon 0.
  • the first fusion construct 406 (construct 1 , Fig. 1 ), contains 3 kb sequences located upstream of exon 0. Injected embryos of generation 0 (Fo) were examined for the presence of the transgene by the expression of ⁇ - galactosidase ( ⁇ -gal) from embryonic day E10.5 to E12.5. Since the transgenic embryos either showed no or ectopic ⁇ -gal activity (Fig. 14, Table 1 ), this construct was elongated with a further 5 kb upstream fragment (construct 2, 406/Sall; Fig. 1).
  • the expression of the reporter gene driven by construct 2 in the developing eye is illustrated in Fig. 7.
  • the first expression of the transgene is detected at E9.0 in the surface ectoderm (Ec) over the presumptive eye region (Fig. 7A,D).
  • E9.5 -E9.75 ⁇ -gal activity increases within the area of the presumptive lens placode (c, Fig.7B) and at E 10.5 the expression becomes confined to the forming lens pit (LP, Fig. 7C,E), presumptive corneal ectoderm (Fig.7F) and a stream of cells that populates the anterior edge of the maxillary domain of the first branchial arch (arrowhead in Fig.7C).
  • a further domain of transgenic activity was detected within the temporal orbita in a duct that will later form the lacrimal gland (LGI, Fig.7l) and also has ectodermal origin.
  • the transgenic expression in the lacrimal gland (Fig.7J), and in the cornea (data not shown) was maintained one day after birth (P1). Consistent with the endogenous expression of Pax ⁇ in conjunctiva (Koroma et al., Investigative Olphathmology and visual science 38 (1997), 108-120), the conjunctival epithelium of the adult eye was also ⁇ -gal positive (data not shown).
  • Construct 2 (406/Sall) is also able to direct the Pax ⁇ reporter gene expression in the pancreas (Fig7. L-M).
  • endogenous Pax ⁇ is expressed in the developing pancreas (Walther and Gruss, loc. cit.) and Pax ⁇ transcripts are detected in all four cell types ( ⁇ , ⁇ , ⁇ , ⁇ ) of pancreatic islet cells, but not in the exocrine cell lines (Turque et al., Mol- Endocrinol. 8 (1994), 929-938).
  • ⁇ -gal staining appears in all transgenic lines in a subset of fore- and midgut cells (the pancreatic bud, P , Fig.
  • Example 7 Distinct regulatory elements are necessary for the expression of Pax ⁇ in eye ectodermal tissues and in the pancreas
  • transgenic mice carrying the 3 kb Pax ⁇ promoter/lacZ fusion construct 1 lack any ⁇ -gal activity, indicating that the regulatory regions for the lens, cornea and pancreas are located within a 2.4 kb fragment (Fig. 1 ).
  • insertion of this 2.4 kb fragment upstream to the lacZ driven by the minimal TK promoter failed to support any lacZ expression, indicating most probably that these regulatory elements are non-functional with the minimal TK promoter. Therefore, various overlapping fragments of the 2.4 kb regulatory region (construct 5 - 9, Fig. 1 , Fig. 14: Table 1) were placed upstream of the Pax ⁇ promoter PO.
  • construct 10 which contains only the minimal Pax ⁇ promoter PO provides no specific transgene activity.
  • construct 3 (406/Spe) was still sufficient to direct the reporter gene expression in both the surface ectoderm derivatives and the pancreas
  • construct 5 (406/Hincll) directs the lacZ expression only in the lens/cornea.
  • sequence comparison performed among corresponding mouse, human and fugu (pufferfish) DNAs revealed a 124 bp sequence of 74% homology (Fig. 11 ) in the Pax ⁇ regulatory region which is suggested to be responsible for controlling the expression of the gene in the pancreas.
  • construct 6 and construct 7 carrying construct 6 and construct 7 (see Fig. 1 ).
  • the construct 6 and construct 7 directed expression only in the developing lens and the cornea, while the construct 7 (406/E) containing a 280 bp fragment gave no ⁇ -gal staining.
  • construct 6 Further trimming of construct 6 to a 120 bp fragment (construct 8, 406/A) resulted in lacZ expression in the lens and additional ectopic patchy staining in retina (in 6 out of 11 transient assays, Fig. 14: Table 1 ). Furthermore, transgenic mice were created carrying the construct 9 (406/B) that contains an 130 bp Bglll/EcoRI fragment overlapping with construct 8, but in 3 '-5 Orientation. The reporter lacZ expression was detected in the lens and the cornea, indicating that this regulatory element can act as an independent enhancer. However, similar to construct 8, this construct also gave in addition to the correct lens and cornea specificity, additional ectopic expression in the retina, suggesting that a negative regulatory element might be missing on these 2 constructs.
  • Example 8 Identification of fugu (pufferfish) Pax ⁇ regulatory elements directing lacZ expression in the mouse telencephaion, lens and pancreas.
  • the tetraodontoid fish has a compact genome of approximately 400 Mb, which is nine times smaller than the mouse genome (Brenner et al., Nature 366 (1993), 265-268), thus making the analysis of regulatory sequences less time consuming.
  • Pax ⁇ is strongly conserved both structurally and functionally through evolution, the availability of information on the Pax ⁇ cis-reguiatory element in the fish would facilitate the identification of further regulatory elements within the large mouse Pax ⁇ locus.
  • the identification of enhancer regions using cross species comparison has already been successfully applied (Marshall et al., Nature 370 (1994), 567-571; Aparicio et al., Proc. Natl. Acad. Sci USA 92 (1995), 1684-1688; Kimura et al., Development 124 (1997), 3929-3941 ).
  • telencephaion a 13 kb fragment encompassing the region from exon 0 to exon 4 (thus lacking the lens, cornea and pancreas elements) was used to make construct 13 (2118/14P, Fig. 8).
  • the transgenic embryos exhibited a ⁇ -gal staining similar to the expression of the endogenous Pax ⁇ gene thus including ⁇ the regions of dorsal telencephaion, diencephalon, pretectum, hindbrain, spinal cord and nasal epithelium. Additional ectopic expression in the vertebrae and the kidney was also seen.
  • Example 9 Localization of conserved mouse- and fugu (pufferfish) regulatory elements directing transgenic expression in the neural retina
  • results from in vitro experiments with the quail Pax ⁇ gene revealed a region 7.5 kb downstream of the quail PO promoter acting as an enhancer in neural retina cells (Plaza et al., Mol. Cell. Biol. 15 (1995), 892-903).
  • the construct 17 (406/8, Fig. 9) contains a 1.8 kb Accl fragment upstream of the minimal promoter PO.
  • 13 exhibited ⁇ -gal staining only in the retina.
  • Construct 15 (Fig. 9) harboring a 1.2 kb EcoRI/Xbal fragment and a lacZ-polyA cassette as an insertion into exon ⁇ , directed similar expression pattern in the neural retina of transgenic embryos, indicating the location of the regulatory sequences in a 900 bp region (Fig. 9). Trimming the fragment to 530 bp included in construct 19 (406/BX, Fig. 9) resulted in a similar lacZ- activity in retina, while a further smaller 290 bp fragment in construct 20 (406/DX) failed to show a transgenic expression (Fig. 9). No ⁇ -gal staining could be detected when using a heterologous minimal TK- promoter, indicating that this specific minimal promoter is not sufficient to activate the Pax ⁇ regulatory sequences (construct 16, Fig. 9).
  • Example 10 Conservation of putative regulatory regions in the pufferfish Pax ⁇ - locus The nucleotide sequences of the identified regulatory elements reveal several DNA binding motifs of transcription factors which are highly conserved among mouse, human and fugu (pufferfish), suggesting that they may act as upstream regulators of the Pax ⁇ gene.
  • the 340 bp Hindl/EcoRI murine fragment (construct 6) responsive for the surface ectoderm expression shows a high sequence homology within 245 bp of human and fugu (pufferfish) genomic Pax ⁇ sequences (Fig. 10).
  • the 245 bp sequences contain two conserved TAAT-core motifs, critical components of many homeodomain DNA binding sites.
  • Motif A"CTTAATG” is located in position nt 56 - nt 62
  • Motif B “GCTAATGTCT” is located in position nt 210 - nt 220.
  • the 1100 bp fragment for the pancreas specific element revealed a sequence of 120 nt with high sequence identity to human and fugu (pufferfish) genomic Pax ⁇ DNA, containing two motifs for homeodomain DNA binding sites: motif C: "CATTATTGT” in position nt 60 - nt 68 and motif D "TTTAATCCAATTATA" in position nt. 156 - nt. 170, (Fig. 11 ).
  • AATCAATCA PBX-1 consensus binding-site
  • the sequence of the retina specific fragment shows a high conservation among mouse, human, fugu (pufferfish) and quail (Fig. 12).
  • Position nt 185 reveals a homeodomain binding site for the transcription factor MSX-1 "CAATTAG” (Catron et al., Mol. Cell. Biol. 13 (1993), 2357-2365).
  • Two further putative homeodomain binding sites "AAATTAAG” and “GTTTTATT” are located at positions nt 233 and nt 262 respectively.
  • the sequence at nt 199 reveals a binding motif for the transcription factor Pax2 (Czerny et al., Genes Dev. 7 (1993), 2048-2061 ; Epstein et al., J. Biol. Chem. 269 (1994), 8344-8361).

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Abstract

L'invention concerne: des séquences régulatrices du promoteur du gène Pax6 ou d'un gène homologue de Pax6, capable d'activer une expression de cellules pancréatiques; des molécules et vecteurs d'ADN de recombinaison comprenant ces séquences régulatrices et des cellules hôtes transformées par ces dernières; des compositions pharmaceutiques et de diagnostic comprenant ces séquences régulatrices, ainsi que les molécules et vecteurs d'ADN de recombinaison; des animaux non humains transgéniques comprenant les molécules ou vecteurs d'ADN de recombinaison précédemment cités intégrés de manière stable dans leur génome. L'invention concerne également l'utilisation des molécules et vecteurs d'ADN de recombinaison précédemment cités pour la préparation de compositions pharmaceutiques destinées au traitement, à la prévention et/ou au retardement d'une maladie liée au pancréas chez un sujet. En outre, l'invention concerne l'utilisation des séquences régulatrices et des molécules et vecteurs d'ADN de recombinaison de l'invention pour préparer des compositions pharmaceutiques destinées à activer une maladie du pancréas chez un animal non humain; ainsi qu'une méthode d'identification d'agonistes/activateurs ou d'agonistes/inhibiteurs de gènes ou de produits géniques intervenant dans les affections du pancréas grâce aux séquences régulatrices précédemment citées, aux molécules d'ADN de recombinaison, vecteurs, cellules et animaux transgéniques non humains. L'invention concerne enfin des composés pouvant être identifiés grâce à cette méthode, des anticorps contre ces composés et des compositions pharmaceutiques et de diagnostic comprenant ces agonistes/activateurs, antagonistes/inhibiteurs et/ou anticorps.
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WO1999057294A1 (fr) * 1998-05-07 1999-11-11 The Rockefeller University Elements de regulation transcriptionnelle du cristallin et procedes d'utilisation de ces elements
US6337392B1 (en) 1998-05-07 2002-01-08 The Rockefeller University Lens transcriptional control elements and methods of use thereof
EP1348768A2 (fr) * 1996-12-31 2003-10-01 Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. Procédé pour le traitement, diagnostic ou détection du diabète
EP1567644A2 (fr) * 2002-04-16 2005-08-31 Origene Technologies Inc. Genes et batteries de genes specifiques de tissus
US8524500B2 (en) 2003-08-08 2013-09-03 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US10669557B2 (en) 2003-08-08 2020-06-02 Sangamo Therapeutics, Inc. Targeted deletion of cellular DNA sequences
WO2021155825A1 (fr) * 2020-02-08 2021-08-12 北京大学第三医院（北京大学第三临床医学院） Utilisation d'un gène pax6 ou d'un produit d'expression de ce dernier dans la préparation d'un médicament destiné à inhiber la fibrose
US11311574B2 (en) 2003-08-08 2022-04-26 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1348768A2 (fr) * 1996-12-31 2003-10-01 Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. Procédé pour le traitement, diagnostic ou détection du diabète
EP1348768A3 (fr) * 1996-12-31 2003-12-10 Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. Procédé pour le traitement, diagnostic ou détection du diabète
WO1999057294A1 (fr) * 1998-05-07 1999-11-11 The Rockefeller University Elements de regulation transcriptionnelle du cristallin et procedes d'utilisation de ces elements
US6337392B1 (en) 1998-05-07 2002-01-08 The Rockefeller University Lens transcriptional control elements and methods of use thereof
EP1567644A2 (fr) * 2002-04-16 2005-08-31 Origene Technologies Inc. Genes et batteries de genes specifiques de tissus
EP1567644A4 (fr) * 2002-04-16 2006-04-05 Origene Technologies Inc Genes et batteries de genes specifiques de tissus
US8524500B2 (en) 2003-08-08 2013-09-03 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US9289451B2 (en) 2003-08-08 2016-03-22 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US9782437B2 (en) 2003-08-08 2017-10-10 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
US10669557B2 (en) 2003-08-08 2020-06-02 Sangamo Therapeutics, Inc. Targeted deletion of cellular DNA sequences
US10675302B2 (en) 2003-08-08 2020-06-09 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
US11311574B2 (en) 2003-08-08 2022-04-26 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
WO2021155825A1 (fr) * 2020-02-08 2021-08-12 北京大学第三医院（北京大学第三临床医学院） Utilisation d'un gène pax6 ou d'un produit d'expression de ce dernier dans la préparation d'un médicament destiné à inhiber la fibrose

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