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WO1999050284A2 - Molecule d'acide nucleique codant des proteines influant sur le developpement osseux - Google Patents

Molecule d'acide nucleique codant des proteines influant sur le developpement osseux Download PDF

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Publication number
WO1999050284A2
WO1999050284A2 PCT/EP1999/002055 EP9902055W WO9950284A2 WO 1999050284 A2 WO1999050284 A2 WO 1999050284A2 EP 9902055 W EP9902055 W EP 9902055W WO 9950284 A2 WO9950284 A2 WO 9950284A2
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Prior art keywords
nucleic acid
acid molecule
protein
lobo
gene
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PCT/EP1999/002055
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German (de)
English (en)
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WO1999050284A3 (fr
Inventor
André ROSENTHAL
Thomas Wirth
Andreas Rump
Jochen Hess
Thomas Aigner
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Rosenthal Andre
Andreas Rump
Jochen Hess
Thomas Aigner
Thomas Wirth
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Application filed by Rosenthal Andre, Andreas Rump, Jochen Hess, Thomas Aigner, Thomas Wirth filed Critical Rosenthal Andre
Priority to EP99913304A priority Critical patent/EP1066318A1/fr
Priority to AU31476/99A priority patent/AU3147699A/en
Publication of WO1999050284A2 publication Critical patent/WO1999050284A2/fr
Publication of WO1999050284A3 publication Critical patent/WO1999050284A3/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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

Definitions

  • the present invention relates to nucleic acid molecules encoding proteins that influence bone development in mammals, the encoded proteins, and diagnostic and pharmaceutical compositions containing such nucleic acid molecules or proteins.
  • the invention further relates to transgenic non-human mammals which have been transformed with the described nucleic acid molecules or which have an altered expression of the described proteins.
  • hereditary diseases are known in humans, which lead to bone growth and development disorders. These include, for example, spondyloepiphyseal dysplasia and achondroplasia. The exact genetic causes of such disorders are usually unclear and therapeutic Approaches or diagnostic procedures for early detection are largely not available.
  • the object of the present invention is therefore to provide nucleic acid molecules whose expression product influences growth and development processes, in particular in connection with bones, in animals and humans.
  • nucleic acid molecules that comprise a nucleotide sequence that is defined in Seq ID No. 9 or in Seq ID No. 14 encoded amino acid sequence shown, as well as nucleic acid molecules that the in Seq ID No. 8 or Seq ID No. 13 nucleotide sequence shown, in particular the coding region.
  • nucleic acid molecules can contain the corresponding coding regions in a coherent form or can also be interrupted by non-coding regions.
  • Such molecules can therefore also be genomic sequences in which the coding regions (exons) are interrupted by non-coding regions (introns).
  • the protein encoded by such a nucleic acid molecule is a protein whose inactivation in mammals leads to an elongation of the bones with the exception of the cranial bones.
  • Such nucleic acid molecules have been found in connection with the generation of a so-called transgenic "donor" mouse, ie a mouse, which should serve as a donor for an artificial protein.
  • This artificial protein should be in certain tissues of the "donor” mouse can be expressed without having a function in this mouse. Only after crossing the donor mouse with a suitable transgenic recipient mouse should the protein take effect and activate certain genes of the recipient mouse. Transgenic donor mice have been produced several times.
  • the artificial gene usually do not show a phenotype because the artificial gene is simply injected into fertilized eggs and accidentally integrates into any area of the murine genome. Since only about 5% of the genome is coding, the probability that a defect is caused in an essential gene is correspondingly low.
  • the mammalian genome is diploid, ie all genes are duplicated. Therefore, most mutations are recessive, ie they are not expressed: the mutated gene is countered by a fully functional copy that can compensate for the defect that is generated.
  • the donor mouse produced showed an extremely striking phenotype: all bones (with the exception of the skull) are 1.3 to 1.5 times longer. As a result, the transgenic mouse is about 1.5 times longer than the corresponding wild type (see FIG. 1). This phenotype is dominant and is passed on stably, i.e. when a transgenic mutane is crossed with a healthy wild-type mouse, 50% of the offspring show the phenotype described above.
  • LOBO gene (“ngng bones ”) in the following the transgenic mouse and first 87 kb (SEQ ID NO: 5 and 6) and then a total of 138 kb (SEQ ID NO: 10 to 12) from the corresponding homologous region of the wild-type mouse.
  • Detailed computer analysis of the sequence data led to the identification of a gene which consists of at least 13 coding sections ("exons") and is at least 110,000 bases long, but probably much longer.
  • the coding region of the murine genomic sequence initially identified carries the information for 393 amino acids (see Seq ID No. 2).
  • a DNA probe was constructed which was used to isolate a human PI clone which carries the human LOBO homologous gene.
  • the sequence of the 13.3 kb long region initially sequenced is in Seq ID NO. 7 shown.
  • the sequence of the isolated and identified coding regions (exons) of this gene is given in Seq ID No. 3, as well as the amino acid sequence derived therefrom.
  • the sequence of the 311 kb long region subsequently sequenced is in Seq ID No. 15 to 21 shown.
  • the sequence of the coding regions (exons) identified therein is in SEQ ID NO. 13, the amino acid sequence derived therefrom in SEQ ID No. 14.
  • genomic sequence information a complete, 3100 bp long cDNA of the murine LOBO gene could then be isolated (SEQ ID NO: 8). Of these 3100 bp, 1857 bases from the 3 'end are covered by genomic sequencing.
  • the exon / intron structure is therefore also known for this section: there are 12 exons that are numbered in ascending order from the 3 'end, ie the furthest 3' exon has the number 1, the outermost, previously identified exon has that Number 12.
  • sequence data provided by the present invention, it is possible, using standard methods, for example chromosomal walking, to isolate and characterize the regions of the gene which are still missing.
  • the murine cDNA carries the information for a protein of 870 amino acids in length (SEQ ID NO: 9).
  • a sequence comparison of the amino acid sequence derived from the murine cDNA sequence with known sequences showed that the encoded Protein has a certain homology to a protein from C. elegans (database accession number Q09568), as well as homologies to the Dis3 protein family and to the RNAsell protein family.
  • nucleic acid molecules according to the invention encode a protein whose change, in particular reduction and / or inactivation in animals, preferably in vertebrates, preferably in mammals and particularly preferably in mice, leads to an elongation of the bones with the exception of the cranial bones.
  • An extension preferably means an extension by a factor of at least 1.2, preferably by a factor of 1.3 and particularly preferably by a factor in the range from 1.3 to 1.5.
  • the term “change”, in particular reduction and / or “inactivation” means on the one hand on a quantitative level that the expression of the protein is reduced in comparison to the wild type, preferably by at least 50% and particularly preferably totally repressed.
  • Analysis of the mutation in the genome of the donor mouse described above showed that the insertion of the artificial gene was within an intron of the LOBO gene and resulted in the deletion of 11 base pairs. The latter should not be a problem in the intron, since this area is not coded anyway. It can therefore be assumed that the artificial DNA insertion leads to a disturbance in the maturation of the mRNA ("splicing"), since the artificially introduced gene contains splicing signals. This presumably leads to so-called "aberrant splicing".
  • change in particular reduction and / or “inactivation”, therefore preferably means in the context of the present invention that the amount of transcripts which encode the protein described in the cells compared to cells of corresponding wild-type animals by at least 50% is reduced, preferably by at least 70%, particularly preferably by at least 90%.
  • “change”, in particular reduction and / or inactivation means that it is no longer possible to detect any transcripts which code for the protein described.
  • the amount of transcripts can be detected using techniques known to those skilled in the art, for example by Northern blot analysis.
  • the term "change”, in particular reduction and / or inactivation, means in qualitative terms that a LOBO protein which has been changed in the amino acid sequence is expressed, in particular a protein which has completely or largely lost its biological function.
  • These can be shortened forms, forms that have deletions or insertions, forms that have one or more point mutations, or forms that have a combination of one or more forms of this change.
  • transgene insertion in the horrd LOBO mouse does not affect the expression signals (promoter, enhancer, etc.), it could be assumed that at least a shortened and, in addition, chimeric LOBO mRNA is produced, from the natural start of transcription to to the splice signal in the inserted sequence.
  • the long-bone phenotype can therefore have two causes: (a) the amount of transcripts encoding the complete LOBO protein falls below a critical value ("loss of function" mutation) due to the transgene insertion and / or ( b) a shortened, chimeric LOBO protein is produced which has only partial functions of the LOBO protein or modified functions compared to the LOBO protein ("gain of function" mutation).
  • the “change”, in particular reduction and / or inactivation, of the protein encoded by the nucleic acid molecules according to the invention in mice preferably also leads to at least one of the following changes:
  • the bones show significantly thickened growth zones at the histological level (see FIG. 4). This is preferably based on a significant increase in the number of cells in the growth zone (chondrocytes). Furthermore, these chondrocytes are significantly larger than those of corresponding wild-type mice;
  • the function of the proteins encoded by the nucleic acid molecules according to the invention can be estimated. It is assumed that these proteins also have similar functions due to their structural similarity to the two other protein groups mentioned. On this basis, the following functions can be postulated for the LOBO proteins:
  • the LOBO protein most likely has the ability to bind RNA (previously proven for the LOBO-like SSDI protein from S. cerevisiae and for the VACB and RNAse type II proteins); and or (d) the LOBO protein has at least one protein binding partner.
  • This is probably a G-protein or a G-protein-controlling protein (detected for Dis3 from S. pombe, which binds to the G-protein regulator RCC1 and controls its activity).
  • the provision of the nucleic acid molecules according to the invention is of great importance both scientifically and clinically. On the one hand, his further research could help to better understand cell cycle control. This is particularly important for cancer research.
  • the nucleic acid molecules according to the invention could be responsible for human growth disorders which are not due to diet or hormones.
  • the present invention also relates to nucleic acid molecules whose complementary strand hybridizes with one of the nucleic acid molecules according to the invention described above and which encode a protein with the abovementioned properties.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrock et al. , Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • “Stringent conditions” mean that hybridization only takes place if a sequence identity of at least 90%, preferably of at least 95% and particularly preferably of at least 97% is present over the entire length of the molecule hybridizing with the molecule according to the invention.
  • stringent and non-stringent hybridization conditions are published, for example, in Harnes and Higgins (ed.), "Nucleic acid hybridization: A practical approach", IRL Press, Oxford-Washington DC, 1985.
  • An example of stringent hybridization conditions is, for example, filter hybridization Polynucleotide samples with the filter washed for 20 min in 0.1 x SET buffer and 0.1% SDS solution at 68 ° C.
  • An example of non-stringent hybridization conditions is, for example, filter hybridization with polynucleotide samples, the filter being washed for 20 min in 2 ⁇ SET buffer and 0.1% SDS solution at 50 ° C.
  • nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention can originate from any animal organism which expresses such a protein. It is preferably molecules that code corresponding proteins from higher animal organisms, preferably from vertebrates, particularly preferably from mammals and in particular from mice or humans.
  • Nucleic acid molecules that hybridize with the molecules of the invention can e.g. can be isolated from genomic or from cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, e.g. by means of hybridization according to standard methods (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification by means of PCR.
  • nucleic acid molecules can be used as the hybridization sample that exactly or essentially the ones under Seq ID No. Have 8 or 13 indicated nucleotide sequence or parts of this sequence.
  • the fragments used as the hybridization sample can also be synthetic fragments which have been produced with the aid of the common synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention. If genes which hybridize with the nucleic acid sequences according to the invention have been identified and isolated, the sequence should be determined and the properties of the proteins encoded by this sequence should be analyzed.
  • the molecules hybridizing with the nucleic acid molecules according to the invention include, in particular, fragments, derivatives and allelic variants of the nucleic acid molecules described above, which encode a protein with the properties described above.
  • the term derivative in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions and have a high degree of homology to these sequences.
  • Homology means a sequence identity at the amino acid level over the entire length of at least 70%, in particular an identity of at least 80%, preferably over 90%, particularly preferably over 95% and in particular at least 97%.
  • Homology preferably also means a sequence identity at the nucleic acid sequence level of at least 60%, preferably at least 70%, particularly preferably at least 85% and particularly preferably at least 95%.
  • the deviations from the nucleic acid molecules described above may have arisen, for example, through deletion, addition, substitution, insertion or recombination.
  • nucleic acid molecules which are homologous to the molecules described above and which are derivatives of these molecules are generally variations of these molecules which are modifications which have the same biological function. These can be both naturally occurring variations, for example sequences from other animal species, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis. Furthermore, the variations can be synthetically produced sequences.
  • allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques.
  • the proteins encoded by the different variants of the nucleic acid molecules according to the invention have certain common characteristics. For example, biological
  • the proteins encoded by the nucleic acid molecules of the invention preferably have the same biological function or activity as described above for the murine protein, i.e. in the event of change, in particular reduction and / or
  • Nucleic acid molecule encoded protein at least one of the following two consensus sequences.
  • the present invention further relates to nucleic acid molecules whose sequence deviates from the sequence of a nucleic acid molecule described above due to the degeneration of the genetic code.
  • the nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or molecules produced by genetic engineering or chemical synthesis processes. Examples of mouse and human genomic sequences are given in Seq ID No. 5, 6, 7, 10 to 12 and 15 to 21 shown. With the help of "fluorescent in situ hybridization" (Fish) on complete murine metaphase chromosomes, the murine gene was located in band ID on chromosome 1 of the mouse. This Band is synthetic with band 2q35, especially with region 2q35-37 on human chromosome 2. This section also contains an alkaline phosphatase gene, the position of which is well known in the literature.
  • Fish fluorescent in situ hybridization
  • the invention further relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above.
  • the vectors are preferably suitable for gene therapy.
  • the nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure expression in prokaryotic or eukaryotic cells.
  • expression can mean transcription as well as transcription and translation.
  • Regulatory elements include promoters in particular.
  • a number of promoters are available for the expression of a nucleic acid molecule according to the invention in prokaryotic cells, for example the E. coli lac or trp promoter, the PR or Pj _, promoter of the lambda phage, lad, lacZ, T3, T7, gpt, etc.
  • Eukaryotic promoters are, for example, the CMV immediate early promoter, the HSV promoter, the thymidine kinase promoter, the SV40 promoter, LTRs of retroviruses and the mouse metallothionine I promoter.
  • a large number of expression vectors for expression in prokaryotic or eukaryotic cells have already been described, for example for eukaryotes pKK223-3 (Pharmcia Fine Chemicals, Uppsala, Sweden) or GEM1 (Promega Biotec, Madison, WI, USA), pSV2CAT, pOG44 and for prokaryotes pQE70, pQE60, pBluescript SK, etc.
  • vectors according to the invention can also contain elements for contain a further increase in transcription, such as so-called transcription enhancers.
  • transcription enhancers include the SV40 enhancer, the polyoma enhancer, the cytomegalovirus early promoter enhancer and adenovirus enhancer.
  • the present invention further relates to host cells, in particular prokaryotic or eukaryotic host cells, which are transformed with a nucleic acid molecule or vector according to the invention.
  • host cells in particular prokaryotic or eukaryotic host cells, which are transformed with a nucleic acid molecule or vector according to the invention.
  • examples of such cells are bacterial cells, e.g. E. coli, Streptomyces, Bacillus, Salmonella typhimurium; Fungal cells, such as yeast cells, in particular Saccharomyces cerevisiae; Insect cells, e.g. Drosophila or SF9 cells; animal cells such as CHO or COS cells; Plant cells etc.
  • the present invention further relates to a method for producing a protein which is encoded by a nucleic acid molecule according to the invention, a host cell according to the invention being cultivated under conditions which allow expression of the protein and the protein subsequently being obtained from the cells and / or the culture medium .
  • Methods for the expression of foreign proteins in different types of host cells and for the production of the protein produced are known to the person skilled in the art.
  • the invention further relates to a protein which is encoded by a nucleic acid molecule according to the invention or which is obtainable by a method according to the invention.
  • the present invention further relates to antibodies which are directed against the proteins according to the invention.
  • Such antibodies preferably recognize a specific one according to the invention Protein, ie they show no significant cross-reaction with other proteins.
  • the term “antibody” here includes both monoclonal and polyclonal antibodies, as well as fragments of antibodies, these fragments recognizing a protein according to the invention, for example Fab fragments.
  • the term antibody also includes chimeric antibodies as well as humanized antibodies. Methods for producing monoclonal or polyclonal antibodies are known to the person skilled in the art and are described.
  • the hybridoma technique Koehler and Milstein, Nature 256 (1975), 495-497
  • the trioma technique the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4 (1983 ), 72) or the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).
  • the present invention relates to nucleic acid molecules of at least 15, preferably more than 50 and particularly preferably more than 200 nucleotides in length, which hybridize specifically with a strand of a nucleic acid molecule according to the invention.
  • Hybridizing specifically here means that these molecules hybridize with nucleic acid molecules which code for a protein according to the invention, but not with nucleic acid molecules which code for other proteins.
  • Hybridization preferably means hybridization under stringent conditions (see above).
  • Such nucleic acid molecules can be used, for example, as primers for amplification by means of PCR or as hybridization samples.
  • the invention relates to those nucleic acid molecules which hybridize with transcripts of nucleic acid molecules according to the invention and can thereby prevent their translation.
  • Such nucleic acid molecules can, for example, be components of antisense constructs or ribozymes.
  • the present invention further relates to diagnostic compositions containing a nucleic acid according to the invention. remolecule or vector, a protein according to the invention and / or an antibody according to the invention.
  • the nucleic acid molecules according to the invention can be used, for example, to determine the location of the corresponding gene on a chromosome. This can provide information about the correlation with genes that are associated with certain diseases.
  • One method for determining the localization is, for example, the “fluorescent in situ hybridization” (Fish) described in Verma et al. (Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988)).
  • nucleic acid molecules according to the invention can be used to determine whether certain individuals have mutations in the corresponding sequences.
  • Antibodies can also be used as detection reagents for the presence of a protein according to the invention in a sample.
  • the present invention furthermore relates to pharmaceutical compositions which contain a nucleic acid molecule according to the invention, a vector according to the invention, a protein according to the invention and / or an antibody according to the invention, optionally in combination with a pharmaceutically acceptable carrier.
  • nucleic acid molecules or vectors according to the invention can be used in the context of gene therapy to treat disease states which are due to a dysfunction of the corresponding gene, for example to an expression of the protein according to the invention which is too low or too high in an individual.
  • nucleic acid molecules according to the invention can be used in connection with "gene targeting” and / or “gene replacement” in order to convert a mutated gene back into a functional form or to generate a mutated gene by homologous recombination (see, for example, Mouellic, Proc. Natl. Acad. Sci. USA 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press).
  • a protein according to the invention or an antibody according to the invention can also be used, if appropriate regulate the amount of appropriate protein in an individual.
  • suitable pharmaceutically acceptable carriers include, for example, phosphate-buffered salt solutions, water, emulsions, such as, for example, oil / water emulsions, sterile solutions, etc.
  • Compositions which contain such carriers can be formulated by customary processes.
  • the pharmaceutical compositions can be administered to the subject concerned in a suitable dose.
  • Types of administration are, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.
  • the dosage depends on many factors, for example on the size, gender, weight, age of the patient, as well as the type of compound administered, the type of administration etc. In general, the daily dose is 1 ⁇ g to 10mg units a day.
  • compositions can be administered locally or systemically. In general, administration will be parenteral, for example intravenously. DNA can also be administered directly at the target site, for example by biolistic administration.
  • the present invention further relates to a method for producing a transgenic non-human animal, preferably a transgenic mouse, which comprises introducing a nucleic acid molecule or vector according to the invention into a germ cell, embryonic cell, an egg cell or a cell derived therefrom.
  • the non-human animal used in such a method as a donor of the cells can be, for example, a healthy non-transgenic animal or an animal which has a disease or disorder, in particular one which has a growth disorder, preferably a bone growth disorder.
  • a disease or disorder can be congenital or natural it can be caused by genetic manipulation, for example by the introduction and / or expression of a foreign DNA.
  • the present invention furthermore relates to transgenic non-human animals which have been transformed with a nucleic acid molecule or vector according to the invention or which are obtainable by the process described above.
  • the nucleic acid molecule according to the invention is preferably stably integrated into the genome.
  • transgenic animals are transgenic rats, hamsters, dogs, monkeys, rabbits or pigs. Transgenic mice are preferred.
  • the present invention also relates to transgenic non-human animals, in particular mice, in which the expression of the protein according to the invention is reduced.
  • a reduction can be achieved, for example, by genetically modifying the cells of the animals so that they express an antisense RNA, a ribozyme or a cosuppression RNA, which leads to a reduction in the expression of the proteins according to the invention in the cells.
  • a reduction of the expression can be achieved by the proteins of the invention also, that at least one, preferably all copies of the corresponding gene are inactivated in the genome of cells of a OF INVENTION ⁇ to the invention the molecule.
  • Such inactivation can be achieved, for example, by inserting foreign DNA into coding or non-coding regions of the corresponding gene. It is also possible to inactivate the regulatory regions of the gene. Deletion of regions of the gene is also possible.
  • the present invention also relates to the possibility of activating nucleic acid molecules according to the invention in vivo, ie in cells, cell cultures or organisms (“gene activation”). This can be done, for example, by inserting a promoter, which is, for example, constitutive and a very high one, into the genome of a cell that contains a nucleic acid molecule according to the invention, before the nucleic acid molecule according to the invention Expression guaranteed, or a promoter that is inducible and guarantees a very high expression upon induction.
  • a promoter which is, for example, constitutive and a very high one
  • HSL Homo sapiens OBO
  • FIG. 1 shows a heterozygous LOBO mouse with an insertion in the LOBO gene (top) in comparison to a wild-type mouse.
  • the two animals are siblings and are about 6 weeks old.
  • FIG. 2 shows the sequencing strategy initially pursued for the sequencing of the murine and human LOBO gene. Since initially only the 3 'end of the gene was sequenced, the exons were numbered consecutively from the 3' end with 1,2,3 etc. Three murine wild-type cosmid clones (middle), two plasmid clones from the transgenic LOBO mouse (top) and a human PI clone (bottom) were sequenced. The arrows indicate the exons known at first. Seven exons were on the genomic sequence, the eighth exon initially only existed on one EST clone. The plasmid clones from the transgenic LOBO mouse (top) contain the introduced artificial gene and the adjacent murine sequences. These murine sequences are identical to the corresponding sequences of the wild-type mouse except for 10 base pairs, which have been replaced by the artificial gene in the transgenic mouse.
  • FIG. 3 shows a sequence comparison between the LOBO protein from human (HS) and mouse (MM) with eukaryotic Dis3 homologous and Dis3-like proteins.
  • FIG. 4 shows a histological thin section through a bone growth zone of the LOBO mouse (right) compared to the wild type (left). The excessive bone growth of the LOBO mouse is also reflected at the histological level: in comparison to the wild type, the growth zone (proliferative zone) of the LOBO bone is significantly thickened. In addition, the number of hypertrophic chondrocytes in the growth zone is significantly increased. Furthermore, the chondrocytes of the LOBO mutant are significantly larger than those of the wild-type mouse.
  • FIG. 5 shows a Norther blot with RNA from human tumor tissues.
  • a commercially available Northern blot (from Clontech), which contains RNA from 8 different human tumor tissues, was hybridized with a radioactively labeled LOBO probe. This probe was made by PCR amplification of a human LOBO EST clone.
  • LOBO is overexpressed in chronic myelogenous leukemia (lane 3) and in melanoma (lane 8). In contrast, it does not appear to be expressed at all in Burkitt's lymphoma.
  • FIG. 6 shows a relationship analysis of LOBO with similar proteins.
  • the analysis was carried out with the program PHYLIP 3.5 ("Neighbor Joining Method"). How from The family tree shows that the LOBO proteins from mouse and human are a separate group, which is related to the eukaryotic Dis3 proteins and the proteins of the RNAse II type. Although some of the invertebrate organisms listed have been completely or at least largely sequenced, there is no real LOBO homolog among them.
  • FIG. 7 shows an X-ray image of the leg of a LOBO mouse (right) compared to the wild type (left). Each individual bone of the LOBO leg is 1.5 times longer than the wild type.
  • Figure 8 shows the phenotype of an adult, heeterozygous LOBO mouse.
  • the inceimpuls bone growth leads to a pronounced deformation of the entire animal, the ability to move is severely restricted. Due to the malformation, female LOBO mice can only be mated in exceptional cases, so that homozygous offspring can rarely be obtained. The LOBO males are reproductive.
  • FIG. 9 shows a clone map and a gene model of the murine LOBO gene on chromosome 1, band D. 7 overlapping cosmid clones were sequenced (A), which give a coherent genomic sequence of 138,884 base pairs. Sequence comparison with the murine LOBO cDNA has so far identified 12 LOBO exons (B). The position of the artificially integrated DNA segment ("cassette”) could be localized by parallel sequencing of the LOBO gene of the transgenic mouse and of the wild-type mouse. It is located in the intron between exons 8 and 7.
  • FIG. 10 shows a clone map and a gene model of the human LOBO region on chromosome 2q37.
  • B BAC / PAC clones sequenced
  • 11 human LOBO exons have so far been identified (A).
  • 6 additional genes were identified in the 3 'region of the LOBO gene, 5 of which were known at the cDNA level.
  • the sixth gene is new.
  • STS marker WI-9864 which has been mapped to 8q24, the chromosomal position of the LOBO gene is clearly verified.
  • transgenic mouse In connection with the investigation of a specific artificial protein, a transgenic mouse was created which was to serve as a donor mouse, ie as a donor for the artificial protein. This protein should be in certain tissues of the "donor" mouse can be expressed without having a function in this mouse. Only after crossing the donor mouse with a suitable transgenic recipient mouse should the protein take effect and activate certain genes of the recipient mouse.
  • the donor mouse was produced by insertion mutagenesis as part of the implementation of a transgenic mouse project.
  • the actual goal of the project was to establish transgenic mice that express the tetracycline-regulated transactivator (tTA) in lymphoid cells.
  • the expression cassette used for microinjection in Pronuclei comprised the following elements in the 5 1 - 3 'direction: ⁇ E: enhancer from the intron of the heavy chain of the mouse immunoglobulin genes (700 bp); a synthetic promoter consisting of an octamer oligonucleotide and the minimal promoter of the mouse ⁇ -globin gene (Wirth et al., Nature 329 (1987), 174-178) and a Tet-R / VP16 construct.
  • the enhancer / promoter combination was described in Annweiler et al. (Nucl. Acids. Res. 20 (1990), 1503-1509).
  • the Tet-R / VP16 construct is described in Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551).
  • the total size of the DNA fragment is approximately 3 kb.
  • 1-2 picoliters of a DNA solution containing the expression cassette described above were injected into the male nucleus of a fertilized egg cell of an NMRI mouse.
  • the egg was then transplanted into the fallopian tube of a sham pregnant female nurse mouse and carried away by it for delivery.
  • Transgenic donor mice usually do not show a phenotype because the artificial gene is simply injected into fertilized eggs and randomly integrated into any area of the murine genome.
  • the probability that a defect is caused in an essential gene is correspondingly low.
  • the mammalian genome is diploid, ie all genes are duplicated. Because a possibly mutated gene usually When there is a fully functional copy that can compensate for the defect in the mutated version, most mutations are recessive, ie they do not occur if only one copy of the gene is affected.
  • the transgenic mouse is a total of approximately 1.5 times longer than a corresponding wild-type mouse (see FIG. 1). Because of the greatly elongated bones (see FIG. 7), the transgenic mouse was referred to as the LOBO mouse (for LQng B ⁇ nes).
  • mice bone growth usually comes to a standstill in the course of individual development. In LOBO mice, it seems that the bones keep growing until the animals die. In adult animals, this leads to a deformation of the entire individual (see FIG. 8), which goes so far that the animals can no longer move and female mutants - with very few exceptions - can no longer be mated.
  • LOBO mice show an increased mortality starting at about 6 weeks after birth, and after a year, all mice from are currently unknown Reasons died. Homozygous mice are viable. Although only two litters with homozygous animals have been received so far, the expected number of homozygous animals are born. Like the heterozygous animals, they show increased bone growth, which can be clearly seen on the longer fingers.
  • this smaller transcript a) represents a splice variant of the gene
  • b) the use of an alternative promoter or c) represents the cross-reaction with a related gene
  • the mutated region from the transgenic mouse was subcloned into bacteria.
  • the localization of the mutated region in the mouse genome and the subsequent subcloning were possible because the nucleotide sequence of the aforementioned artificial gene was known and this information could be used in corresponding molecular biological experiments.
  • the gene which is referred to below as the "LOBO gene”
  • the initially sequenced region of the mouse genomic DNA clones is in Seq ID No. 5 and 6.
  • the sequenced area comprised a total of 86902 base pairs. For technical reasons, this area was divided into two areas, with the first 49999 base pairs in Seq ID No. 5 and comprise an exon and the remaining 36901 base pairs in Seq ID No. 6 are shown.
  • the exons are located at the following positions: Seq ID No. 5: 8520-8753
  • the open reading frame begins at position 8520 in Seq ID No. 5.
  • the stop codon is located at position 18202 in Seq ID No. 6.
  • the coding area codes the in Seq ID No. 2 amino acid sequence shown.
  • a detailed computer analysis of the sequence data initially obtained led to the identification of a gene which consists of at least 8 coding sections (“exons”).
  • the coding area initially identified, which is shown in Seq ID No. 1 shows the information for 393 amino acids.
  • An overview of the murine clones obtained and sequenced in the subsequent sequencing of the 138 kb region is shown schematically in FIG. 10.
  • the sequenced area comprises a total of 138884 base pairs (see Seq ID No. 12 to 15) and contains 12 exons.
  • the exons are located in the following positions:
  • the open reading frame begins at position 1118 in SEQ ID NO: 10.
  • the stop codon is at position 120185.
  • a detailed computer analysis of the genomic sequence data led to the identification of a gene which consists of at least 13 coding sections ("exons") and at least 120 kb long, but probably much longer.
  • a complete cDNA was isolated using the exons identified by genomic sequencing. This is in Seq ID No. 8 and has a length of 3100 bp.
  • the Polyadenylation signal begins at base 3067, the poly-A tail begins at position 3083.
  • the coding region of the cDNA is 2610 base pairs long. It starts in Seq ID No. 8 at position 125 and ends at position 2734. The stop codon begins at position 2735.
  • the coding region generates an 870 amino acid long protein, the sequence of which is shown in SEQ ID NO: 9. From the cDNA in Seq ID NO. 8 so far, only the area from position 1243 to position 3083 (beginning of the poly-A tail) has been covered genomically by the 12 exons listed in the table above.
  • the cDNA sequence from positions 1 to 1242 has not yet been genomically sequenced, ie the intron / exon structure of the gene and its regulatory signals are hitherto unknown.
  • the first nucleotide of the open reading frame is at position 2.
  • the stop codon is at position 6759.
  • the amino acid sequence represented by the coding region is in Seq ID No. 4 shown.
  • a clone containing the human genomic sequence was deposited under DSM 12073. The sequence data initially available showed that the human gene has also only been partially cloned to date. An overview of the initially obtained and sequenced mouse and human clones is shown schematically in FIG. In order to be able to sequence the rest of the human gene, the sequence of the human PI clone was used to identify two further human clones, one of which overlaps in the 5 'region and the other in the 3' region with the clone already present .
  • the first nucleotide of the open reading frame is at genomic position 2703.
  • the stop codon is at position 101273.
  • the human genomic LOBO sequence contains 4 gaps, each of which is a maximum of 100 base pairs in size. These gaps are in the following positions: Gap 1: 11805 to 11836 Gap 2: 35184 to 35199 Gap 3: 191949 to 191975 Gap 4: 251627 to 251646
  • Insertion comes to disruption in the maturation of the mRNA.
  • Adenylation signal which leads to a non-poly-adenylated RNA, which significantly reduced one compared to the normal mRNA
  • this chimeric RNA should be quite low and below the Northern blot detection limit. In fact, this chimeric RNA is in the
  • LOBO protein causes, which possibly still performs partial functions of the complete LOBO protein or with it
  • Binding partner or substrate competes.
  • the sequence of the LOBO protein derived from the human cDNA shows high homology to the human Dis3 gene.
  • this gene was from a Japanese working group was shown that its expression rate in tumor tissues was significantly changed compared to the corresponding normal tissues.
  • a commercially available Northern blot which was loaded with RNAs from various tumor tissues, was hybridized with a human LOBO probe. Indeed, significant differences in expression between the various types of tumors can be observed (FIG. 5). However, the biological interpretation of this data is difficult. However, it is conceivable that the LOBO gene plays a role in cancer development.
  • the mouse and human amino acid sequences derived from the LOBO cDNAs were compared with known proteins. It was found that the amino acid sequence has areas that are highly conserved, from mammals (mouse and human) to invertebrates (Caenorhabditis elegans) and unicellular eukaryotes (Saccharomyces cerevisiae, Schizosaccharomyces pombe) to the prokaryotes.
  • a relationship analysis of these proteins shows that the LOBO proteins from mouse and human are a separate group (see FIG. 6), but which is related to two further protein groups.
  • VacB RNAse type II proteins from bacteria
  • RNAse type II proteins from bacteria
  • Dis3 homologous proteins from various eukaryotes, from mammals to unicellular yeasts.
  • the LOBO protein most likely has the ability to bind RNA (demonstrated for the LOBO-like SSDI protein from S. cerevisiae and for the VACB and RNAse type II proteins).
  • the LOBO protein has at least one protein binding partner. This is probably a G protein or a G protein controlling protein (detected for Dis3 from S. pombe, which binds to the G protein regulator RCC1 and controls its activity).
  • AHO Albright hereditary osteodystrophy
  • LOBO is the candidate gene for "albright hereditary osteodystrophy" is.
  • the type of mutation as it exists in the mouse (insertion of an artificial gene) is artificial and certainly not given in the AHO patients.
  • large deletions that will likely delete the entire LOBO gene, the predominant type of mutation.
  • a gene can cause both short and tall stature depending on the type of mutation.
  • the same mutation of the same gene in mouse and human can lead to different phenotypes, since these organisms are different in many ways.

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Abstract

L'invention concerne une molécule d'acide nucléique codant des protéines qui influent sur le développement osseux de mammifères, ainsi que les protéines ainsi codées, des anticorps et des compositions d'ordre pharmaceutique et diagnostique. L'invention concerne en outre des animaux transgéniques qui expriment les protéines décrites, ainsi que des animaux chez lesquels le gène correspondant est inactivé et qui présentent un allongement des os.
PCT/EP1999/002055 1998-03-27 1999-03-26 Molecule d'acide nucleique codant des proteines influant sur le developpement osseux WO1999050284A2 (fr)

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EP99913304A EP1066318A1 (fr) 1998-03-27 1999-03-26 Molecule d'acide nucleique codant des proteines influant sur le developpement osseux
AU31476/99A AU3147699A (en) 1998-03-27 1999-03-26 Nucleic acid molecules which code proteins influencing bone development

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EP2036924A2 (fr) 2001-10-04 2009-03-18 Biotempt B.V. Peptides régulant des gènes

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EP0917469A1 (fr) * 1996-04-19 1999-05-26 ZymoGenetics, Inc. Procedes permettant d'induire la formation osseuse
US6008013A (en) * 1996-07-05 1999-12-28 University Of Rochester Chondrocyte proteins

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2036924A2 (fr) 2001-10-04 2009-03-18 Biotempt B.V. Peptides régulant des gènes
EP2060585A2 (fr) 2001-10-04 2009-05-20 Biotempt B.V. Peptides régulant des gènes

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