WO2003048196A2 - Moyens et procedes permettant de diagnostiquer et de traiter des malformations cranio-faciales - Google Patents

Moyens et procedes permettant de diagnostiquer et de traiter des malformations cranio-faciales

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Publication number
WO2003048196A2
WO2003048196A2 PCT/EP2002/013936 EP0213936W WO03048196A2 WO 2003048196 A2 WO2003048196 A2 WO 2003048196A2 EP 0213936 W EP0213936 W EP 0213936W WO 03048196 A2 WO03048196 A2 WO 03048196A2
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WO
WIPO (PCT)
Prior art keywords
foxd3
gene
polynucleotide
zebrafish
polypeptide
Prior art date
Application number
PCT/EP2002/013936
Other languages
English (en)
Inventor
Elzbieta Knapik
Sherri Sachdev
Original Assignee
Gsf-Forschungzentrum Für Umwelt Und Gesundheit Gmbh
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Filing date
Publication date
Application filed by Gsf-Forschungzentrum Für Umwelt Und Gesundheit Gmbh filed Critical Gsf-Forschungzentrum Für Umwelt Und Gesundheit Gmbh
Priority to AU2002361031A priority Critical patent/AU2002361031A1/en
Publication of WO2003048196A2 publication Critical patent/WO2003048196A2/fr

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Classifications

    • 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
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a variant foxd3 polynucleotide, a gene comprising said polynucleotide, and a vector comprising the polynucleotide or the said gene.
  • the present invention encompasses a host cell genetically engineered with the polynucleotide, the gene or the vector of the invention.
  • the present invention relates to a method for producing a molecular variant ⁇ foxd3 polypeptide or fragment.
  • the invention relates to an antibody which binds specifically to the polypeptide of the invention.
  • the present invention also encompasses a solid support comprising one or a plurality of the polynucleotide, the gene, the vector, the polypeptide, the antibody or the host cell of the invention in immobilized form.
  • the present invention relates to a method for obtaining and/or identifying an inhibitor or antagonist or an activator or agonist of foxd3 activity.
  • the present invention relates also to a method for the production of a pharmaceutical composition.
  • the present invention relates to a method for obtaining and/or identifying a polynucleotide which is associated with craniofacial malformation. Diagnostic and pharmaceutical composition are also encompassed by the present invention.
  • the present invention relates to the use of the polynucleotide, the gene, the polypeptide, the antibodv or the solid support of the invention for the preparation of a diagnostic or pharmaceutical composition for diagnosing, treating and/or preventing craniofacial malformations.
  • the present invention further encompasses the use of an antagonist or inhibitor obtainable by any one methods for the preparation of a pharmaceutical composition for treating and/or preventing diseases or disorders which are caused by or related to foxd3 overexpression.
  • the present invention relates to a kit.
  • Vertebrate craniofacial cartilage development is a complex process of morphogenesis governed by a network of evolutionarily conserved genes.
  • Elegant embryologica! experiments in chick and frog have demonstrated that neural crest cells give rise to the majority of the facial skeleton, several types of neuronal cells and part of the connective tissues (Garcia-Castro et al., 1999; Mayor et al., 1999).
  • neural crest cells are induced from the ectodermal germ layer at the inter-phase between future neuronal and epidermal tissues (Liem et al., 1995; Nieto et al., 2001 ) but the precise molecular mechanisms controlling neural crest cell specification, migration and terminal differentiation are not understood.
  • Craniofacial malformations originate from deficiencies during embryonic development. A pivotal role among these embryonic deficiencies play those involving improper cell fate specification. Craniofacial malformations have been reported in different species, including humans, mice, rat, and zebrafish.
  • Mutants which exhibit phenotypes comparable to some of the known human syndromes are also known for the zebrafish as model organism.
  • large-scale ENU-mutagenesis screens have been conducted to systematically untangle the developmental gene networks (Driever et al., 1996 and reviewed in Knapik, 2000).
  • the collection of isolated mutants includes zebrafish with defects in skeletogenesis and other neural crest derived tissues.
  • the mother superior (mos m188 ) mutation dramatically reduces the facial skeleton in developing zebrafish larvae and is often accompanied by cardiac edema.
  • mos mutants the most dorsal elements of the first two pharyngeal arches are reduced and all posterior branchial arches are deleted.
  • the technical problem underlying the present invention is to provide suitable means and methods for diagnosing, preventing and/or treating of the craniofacial malformations and diseases or disorders related thereto.
  • the present invention relates to a polynucleotide comprising a polynucleotide selected from the group consisting of:
  • polypeptide (e) a polynucleotide encoding an foxd3 polypeptide or fragment thereof, wherein said polypeptide comprises an amino acid substitution of Ser to Cys at position 62 of the foxd3 polypeptide Accession No: AAC06366).
  • polynueleotides refers to nucleic acid molecules, preferably to DNA or RNA. Most preferably, the polynueleotides comprise a nucleic acid sequence as shown in SEQ ID No: 1 or 5. Polypeptides referred to in the context of this invention, preferably, comprise an amino acid sequence as shown in SEQ ID No: 2 or 8. Reference or wild type sequences for the polynueleotides are Accession No: AF052249. Reference or wild type sequence for the polypeptides of the invention is Accession No: AAC06366.
  • variants of polynueleotides or polypeptides which differ from the wild type or reference sequence by structure or composition are also encompassed by this invention
  • the differences in structure or composition occur by way of nucleotide or amino acid substitution(s), addition(s) and/or deletion(s).
  • said nucleotide substitution(s), addition(s) or deletion(s) comprised by the present invention result(s) in one or more changes of the corresponding amino acid(s) of the polypeptides of the invention.
  • the variant polynueleotides and polypeptides also comprise fragments of said polynueleotides or polypeptides of the invention.
  • polynueleotides and polypeptides as well as the aforementioned fragments thereof are characterized as being associated with foxd3-related diseases or disorders.
  • said disease or disorder are craniofacial malformations or prevalences therefor as described in detail below.
  • Said association preferably, is accompanied by the presence of a T at a position corresponding to position 380 of the foxd3 nucleic acid sequence or the presence of a Cys at a position corresponding to position 62 of the amino acid sequence as described above in detail.
  • hybridizing refers to polynueleotides which are capable of hybridizing to the polynueleotides of the invention or parts thereof which are associated with a foxd3-related disease or disorder.
  • said hybridizing polynueleotides encompassed by the invention are also associated with the foxd3 related diseases and disorders.
  • said polynueleotides capable of hybridizing to the polynueleotides of the invention or parts thereof which are associated with foxd3-related disease or disorder are at least 70%, at least 80%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%, identical to the polynueleotides of the invention or parts thereof which are associated with foxd3-related disease or disorder. Therefore, said polynueleotides may be useful as probes in Northern or Southern Blot analysis of RNA or DNA preparations, respectively, or can be used as oligonucleotide primers in PCR analysis dependent on their respective size.
  • said hybridizing polynueleotides comprise at least 10, more preferably at least 15 nucleotides in length while a hybridizing polynucleotide of the present invention to be used as a probe preferably comprises at least 100, more preferably at least 200, or most preferably at least 500 nucleotides in length.
  • hybridization conditions are referred to in standard text books such as Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • Preferred in accordance with the present inventions are polynueleotides which are capable of hybridizing to the polynueleotides of the invention or parts thereof which are associated with a foxd3- related disease or disorder under stringent hybridization conditions, i.e.
  • said stringent hybridization conditions are overnight hybridization in 20 x SSC, 0,1 % SDS at 65 °C followed by several washes in 1 x SSC with decreasing SDS conditions starting with 1 % SDS down to 0,1 % SDS.
  • nucleotides or amino acids may differ in the indicated number but may still have similar neighboring nucleotides or amino acids.
  • Said nucleotides or amino acids which may be exchanged, deleted or comprise additional nucleotides or amino acids are also comprised by the term "corresponding position".
  • Said nucleotides or amino acids may for instance together with their neighbors form sequences which may be involved in the regulation of gene expression, stability of the corresponding RNA or RNA editing, as well as encode functional domains or motifs of the protein of the invention.
  • the mutations in the variant genes of the invention result in an amino acid substitution. It is of course also possible to genetically engineer such mutations in wild type genes or other mutant forms. Methods for introducing such modifications in the DNA sequence of said genes are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • foxd3 plays a critical role in craniofacial development.
  • foxd3 othologues could be identified in a variety of species other than zebrafish, including human HFH2, genesis (Genbank accession number: AF197560; SEQ ID NO: 11 ), mouse HFH2, genesis (Labosky et al., 1998; Genbank accession number: NM_010425; SEQ ID NO: 12), chick CWH3 (Kos et al., 2001 ; Genbank accession number U37274 corrected by Kos; SEQ ID NO: 13) and frog XFD6 (Pohl et al., 2001 ; Genbank AJ298865 or AJ298866; SEQ ID NO: 14).
  • the human ortholog of foxd3 maps to chromosome 1 p31 , a genomic region known to be associated with several translocations known to be involved in germ cell neoplasia, testicular carcinomas, neuroblastomas, and lymphomas, as well as the Niikawa-Kuroki syndrome which has phenotypic characteristics essentially identical to those of the mos zebrafish mutants.
  • the developmental role of FoxD3 has remained until now somewhat elusive.
  • polynueleotides of the present invention it is possible to diagnose and/or treat or prevent craniofacial malformations and diseases or disorders related thereto comprising those metioned below by using standard assays for detection of polynueleotides.
  • the polynueleotides may also be useful for the development of modulators of the biological activities of foxd3.
  • the mos Ser ⁇ Cys mutation lies within an acidic domain close to the N-terminus with so far unknown function.
  • acidic domains of transcription factors are important for interacting with mediator proteins that are part of the RNA polymerase II complex (Ptashne et al., 1990; Tjian et al., 1994).
  • the results obtained in accordance with the present invention demonstrate that a proper acidic domain of foxd3 polypeptides is in deed required for biological activity. This is a surprising result, since previous studies aiming to analyze the function of winged helix transcription factors concentrated exclusively on the fork head DNA binding domain (Marsden et al., 1998).
  • said polynucleotide is associated with a craniofacial malformation.
  • craniofacial malformation refers to those malformations of the cranifacial entity which are caused or whose formation are influenced by genetic parameters, such as the proper foxd3 function.
  • craniofacial malformations also comprise diseases or disorders which are caused by abnormal craniofacial embryonic development.
  • the specification and migration of neural crest cells during embryonic development is critical for the proper development of a variety of organs and tissues including the craniofacial cartilage, the heart and the nervous system.
  • embryonic craniofacial malformations caused by impaired neural crest specification or migration may also effect these organs and tissues.
  • craniofacial malformations encompass but are not limited to the cleft palate syndrome, loss of middle ear ossicles, Congenital Heart Disease (CHD), Niikawa- Kuroki syndrome (NKS), cancer, preferably germ cell neoplasia, testicular carcinomas, neuroblastomas, and lymphomas, most preferably, lymphoblastic leukemia, and mental retardation.
  • CHD Congenital Heart Disease
  • NKS Niikawa- Kuroki syndrome
  • cancer preferably germ cell neoplasia, testicular carcinomas, neuroblastomas, and lymphomas, most preferably, lymphoblastic leukemia, and mental retardation.
  • the clinical characteristics of said diseases and disorders are well known in the art and described in detail in standard text books of medicine, such as Pschyrembl or Stadmen.
  • said polynucleotide is DNA or RNA.
  • the polynucleotide of the invention may be, as also mentioned supra, DNA, cDNA, genomic DNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynueleotides either alone or in combination.
  • said polynucleotide is part of a vector, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the vectors and plasmids encompassed by the present invention are discussed infra in detail.
  • the invention also relates to a gene comprising the polynucleotide of the invention.
  • genes comprise structural elements which encode an amino acid sequence as well as regulatory elements which are involved in the regulation of the expression of said genes.
  • Structural elements are represented by exons which may either encode an amino acid sequence or which may encode for RNA which is not encoding an amino acid sequence but is nevertheless involved in RNA function, e.g. by regulating the stability of the RNA or the nuclear export of the RNA.
  • Regulatory elements of a gene may comprise promoter elements or enhancer elements both of which could be involved in transcriptional control of gene expression. It is very well known in the art that a promoter is to be found upstream of the structural elements of a gene. Regulatory elements such as enhancer elements, however, can be found distributed over the entire locus of a gene. Said elements could be reside, e.g., in introns, regions of genomic DNA which separate the exons of a gene. Promoter or enhancer elements correspond to polynucleotide fragments which are capable of attracting or binding polypeptides involved in the regulation of the gene comprising said promoter or enhancer elements. For example, polypeptides involved in regulation of said gene comprise the so called transcription factors.
  • Said introns may comprise further regulatory elements which are required for proper gene expression.
  • Introns are usually transcribed together with the exons of a gene resulting in a nascent RNA transcript which contains both, exon and intron sequences.
  • the intron encoded RNA sequences are usually removed by a process known as RNA splicing. However, said process also requires regulatory sequences present on a RNA transcript said regulatory sequences may be encoded by the introns.
  • regulatory elements of a gene could be also involved in the control of genetic stability of a gene locus. Said elements control, e.g., recombination events or serve to maintain a certain structure of the DNA or the arrangement of DNA in a chromosome. Therefore, single nucleotide polymorphisms can occur in exons of a gene which encode an amino acid sequence as discussed supra as well as in regulatory regions which are involved in the above discussed process.
  • the analysis of the nucleotide sequence of a gene locus in its entirety including, e.g., introns is in light of the above desirable.
  • the present invention moreover, relates to a vector comprising the polynucleotide or the gene of the invention.
  • Said vector may be, for example, a phage, plasmid, viral or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
  • the polynueleotides or genes of the invention may be joined to a vector containing selectable markers for propagation in a host.
  • a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate, or in a complex with a charged lipid or in carbon-based clusters. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
  • the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
  • Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E.
  • regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI , pcDNA3 (In-vitrogene), pSPORTI (GIBCO BRL).
  • said vector is an expression vector and/or a gene transfer or 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 polynueleotides or vector of the invention into targeted cell population.
  • the polynueleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • isolated fractions thereof refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector of the invention.
  • Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide.
  • Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes.
  • the present invention encompasses a host cell genetically engineered with the polynucleotide, the gene or the vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell; see supra.
  • the polynucleotide or vector 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 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.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • a polynucleotide coding for a mutant form of variant polypeptides of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, supra). The genetic constructs and methods described therein can be utilized for expression of variant polypeptides of the invention in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • the transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the proteins of the invention can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions.
  • the isolation and purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
  • the present invention relates to a method for producing a molecular variant foxd3 polypeptide or fragment thereof comprising
  • culture encompasses suitable means for maintaining and propagating the host cells. Moreover, said conditions must allow that the molecular variant foxd3 protein is synthesized.
  • suitable means encompass, e.g., culture media. It is well known by the person skilled in the art how to compose a culture medium in order to provide the aforementioned means.
  • recovering refers to means and methods for providing the molecular variant foxd3 polypeptide either in pure or in substantially pure form.
  • purification methods known in the art can be applied.
  • the term “recovering” also encompasses culture medium containing the produced polypeptide, which has not been purified, or fractions of such a culture medium.
  • polypeptide or fragment thereof encoded by the polynucleotide, the gene or which is obtainable by the method of the invention.
  • variant polypeptide of the invention can be further modified by conventional methods known in the art.
  • variant proteins according to the present invention it is also possible to determine the portions relevant for their biological activity or inhibition of the same.
  • polypeptide and protein as used herein are exchangeable. Moreover, what is comprised by said terms is standard textbook knowledge.
  • the invention relates to an antibody which binds specifically to the polypeptide of the invention.
  • the antibody specifically recognizes or binds an epitope containing one or more amino acid substitution(s) being associated with craniofacial malformations as defined above.
  • the antibody is, inter alia, useful for therapeutic or diagnostic applications including those described in detail below.
  • Antibodies against the variant polypeptides of the invention can be prepared by well known methods using a purified protein according to the invention or a (synthetic) fragment derived therefrom as an antigen.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol.
  • said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, human or humanized antibody, primatized, chimerized or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these.
  • antibodies or fragments thereof to the aforementioned polypeptides 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. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the variant polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the antibody of the invention is monoclonal or polyclonal.
  • the present invention also encompasses a solid support comprising one or a plurality of the polynucleotide, the gene, the vector, the polypeptide, the antibody or the host cell of the invention in immobilized form.
  • solid support refers to a flexible or non-flexible support that is suitable for carrying said immobilized targets.
  • Said solid support may be homogenous or inhomogeneous.
  • said solid support may consist of different materials having the same or different properties with respect to flexibility and immobilization, for instance, or said solid support may consist of one material exhibiting a plurality of properties also comprising flexibility and immobilization properties.
  • Said solid support may comprise glass-, polypropylene- or silicon-chips, membranes oligonucleotide-conjugated beads or bead arrays.
  • immobilized means that the molecular species of interest is fixed to a solid support, preferably covalently linked thereto.
  • This covalent linkage can be achieved by different means depending on the molecular nature of the molecular species.
  • the molecular species may be also fixed on the solid support by electrostatic forces, hydrophobic or hydrophilic interactions or Van-der-Waals forces.
  • the above described physico-chemical interactions typically occur in interactions between molecules.
  • biotinylated polypeptides may be fixed on a avidin- coated solid support due to interactions of the above described types.
  • polypeptides such as antibodies, may be fixed on an antibody coated solid support.
  • the immobilization is dependent on the chemical properties of the solid support.
  • the nucleic acid molecules can be immobilized on a membrane by standard techniques such as UV-crosslinking or heat.
  • said solid support is a membrane, a glass chip, a polypropylene ship, a silicon ship, a bead or an optical filter substrate.
  • the present invention relates to a method for obtaining and/or identifying an inhibitor or antagonist of foxd3 activity comprising the steps of:
  • foxd3 activity encompasses all biological functions of the foxd3 polypeptide and all immunological properties thereof. Biological functions and immunological properties of foxd3 as well as suitable assays to determine said functions or properties are well known in the art and have been described in detail elsewhere in this specification. Moreover, the foxd3 activity is also dependent on the rate of transcription of the foxd3 gene and the rate of translation of the corresponding RNA as well as on the stability of said RNA. Thus, foxd3 activity also encompasses the level of transcripts and the level of protein present in a cell. The term “inhibitor or antagonist” refers to compounds which are capable of either inhibit or reduce the foxd3 activity.
  • said inhibitors or antagonists at least reduce the foxd3 activity significantly.
  • Suitable tests to detect whether a compound is an inhibitor antagonist comprise those described herein and the accompanied examples. Whether the activity is reduced significantly can be determined by well known statistical tests, such as Student ' s T-test, Chi 2 -test or the U-test of Mann and Whitney. Moreover, the person skilled in the art is capable of adopting these statistical tests without further ado.
  • the term "contacting" encompasses means which allow the physical interaction of a compound suspected to be an inhibitor with the foxd3 protein, the foxd3 RNA or the foxd3 gene. Moreover, also encompassed are means which allow physical interaction with proteins which are either specifically involved in the regulation of foxd3 transcription or translation.
  • the compounds suspected to be inhibitors or antagonists may be dissolved in a suitable solvent which can be used as culture medium for the embryos or as additive for said culture medium. Thereby, the compounds can diffuse into the cells of the embryo. This technique is particularly well suited for compounds which are capable of permeating cell membranes.
  • the compound may be introduced into the embryo via transfection techniques known in the art, such as calcium phosphate precipitation, via microinjection, or via retroviral transfection.
  • embryo refers to developmental stages of the zebrafish starting from the zygote to the larvae stage, preferably to embryos of the zygote stage to the prior primordial stage.
  • compound in a method of the invention includes a single substance or a plurality of substances which may or may not be identical.
  • Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compounds may be known in the art but hitherto not known to be useful as an inhibitor, respectively.
  • the plurality of compounds may be, e.g., added to the culture medium or injected into a cell or non-human animal of the invention.
  • a sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or 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. It can then be determined whether said sample or compound displays the desired properties, for example, by the methods described herein or in the literature (Spector et al., Cells manual; see supra). Depending on the complexity of the samples, 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).
  • said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical.
  • the methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art or by using and modifying the methods as described herein.
  • the person skilled in the art will readily recognize which further compounds may be used in order to perform the methods of the invention, for example, enzymes, if necessary, that convert a certain compound into a precursor.
  • Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
  • Compounds which can be used in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds may act as agonists or antagonists of the inveniton. Preferably, said compounds are oligonucleotides, RNA, most preferably, antisense RNA, or antibodies. Said compounds can also be functional derivatives or analogues of known drugs. 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.
  • Appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptides of the invention by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above- described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known inhibitors, analogs, antagonists or agonists.
  • peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. 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 Dorner, Bioorg. Med. Chem. 4 (1996), 709- 715.
  • the three-dimensional and/or crystallographic structure of said compounds and the polypeptides of the invention can be used for the design of peptidomimetic drugs (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). It is very well known how to obtain said compounds, e.g. by chemical or biochemical standard techniques. Thus, also comprised by the method of the invention are means of making or producing said compounds. In summary, the present invention provides methods for identifying and obtaining compounds which can be used in specific doses for the treatment of specific forms of foxd3-related diseases, such as craniofacial malfunctions and the diseases referred to in accordance with this invention.
  • Gastrulation refers to the developmental stages where the mesoderm is formed in the developing embryo. Gastrulation in the zebrafish is accompanied by the epibolic cell movements of the cells of the blastoderm.
  • determining the phenotype encompasses the determination of the morphology including morphometric means as well as the determination of the expression of genes which are known to be indicative for the craniofacial malformations which are accompanied with the impaired foxd3 function in mos mutants.
  • the present invention it is now possible to obtain or identify inhibitors or antagonists of foxd3 and thereby to provide compouns which are useful as drugs for the therapy of diseases and disorders related to, e.g., an enhanced foxd3 expression.
  • the compounds identified and obtained in accordance with the method of the present invention have, due to the characteristics of the method, proven their usefulness in vivo rather than in cell culture studies or in cell free assays which generally have the disadvantage that results obtained thereby are not in compliance with the results obtained for the same compounds in vivo.
  • the present invention relates to a method for obtaining and/or identifying an activator or agonist of foxd3 activity comprising the steps of:
  • activator or agonist refers to compounds which are capable of either enhancing or mimicing the foxd3 activity.
  • said compounds activate the foxd3 activity at least significantly.
  • Suitable tests to detect whether a compound is an activator or agonist comprise those described herein and the accompanied examples. Whether the activity is enhanced significantly can be determined by well known statistical tests, such as Student ' s T-test, Chi 2 -test or the U-test of Mann and Whitney. Moreover, the person skilled in the art is capable of adopting these statistical tests without further ado.
  • said determining the phenotype comprises morphological evaluation.
  • said morphological evaluation comprises evaluation of the tail morphology, evaluation of the heart morphology or evaluation of the craniofacial morphology.
  • the defects observed in the mos mutant are caused by a defect in the foxd3 protein. Therefore, the morphological alterations specified before which are characteristic for the mos mutant are suitable to determine altered foxd3 function in vivo.
  • said determining the phenotype comprises detection of gene expression.
  • the altered foxd3 function can be determined by analyzing the expression of genes whose expression is reduced, enhanced, present or absent in mos mutants. Suitable marker genes are known in the art.
  • said gene is snaiH or snail2.
  • the nucleic acid sequence of snaiH can be obtained from GenBank as Accession Number: S 68 799.
  • the nucleic acid sequence of snail2 is provided under GenBank Accession Number: U 24 225.
  • said mother superior zebrafish mutant embryo and said wild type zebrafish embryo comprise a reporter gene in their genomes.
  • reporter gene refers to genes encoding products which can be easily determined due to their chemical or physical properties.
  • a suitable reporter gene may exhibit bioluminescence or enzymatic properties which allow a reliable detection of the said product.
  • the said reporter gene is most preferably comprised by genome of the mother superior zebrafish mutant embryo and the wild type zebrafish embryo. Methods and techniques for integrating a reporter gene into the genome of a zebrafish are well known in the art and are described, e. g., in Long et al. 1997.
  • reporter gene is operably linked to an expression control sequence which is recognized by the foxd3 protein.
  • the expression "operably linked to an expression control sequence which is recognized by the foxd3 protein” refers to a nucleic acid sequence which specifically allows the regulation of the expression of the said reporter gene by foxd3 protein, i. e. such an expression control sequence comprises one or more transcription factor binding site for foxd3.
  • said expression control sequence which is recognized by the foxd3 protein is derived from snaih , snail2 or foxd3.
  • said reporter gene is selected from the group consisting of green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), firefly luciferase, remilla luciferase or chloramphenicol tranferase (CAT).
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • YFP yellow fluorescent protein
  • CAT chloramphenicol tranferase
  • the invention relates also to a method for obtaining and/or identifying a target gene of foxd3 comprising:
  • a nucleic acid sequence which is comprised by gene suspected to be a target gene encompasses those sequences of a putative target gene which are involved in the regulation of the expression of the said gene.
  • said nucleic acid sequences comprise transcription factor binding sites for the foxd3 protein.
  • an oligonucleotide comprising the said nucleic acid sequence is brought into contact with foxd3 protein or a fragment thereof comprising at least the DNA binding domain as specified above under conditions which allow the interaction of the oligonucleotide and the foxd3 protein or a fragment thereof. Such conditions are well known in the art and are described in standard text books, for example in Sambrook et al.
  • determining binding of said oligonucleotide refers to means which are suitable for the detection of the complex of the said oligonucleotide and foxd3 protein or fragment thereof.
  • suitable means are for example the use of radioactively labeled oligonucleotide, a antibody which specifically recognizes foxd3 protein or a fragment thereof.
  • determining binding of said oligonucleotide is carried out after the complex has been resolved by Gel-electrophoresis.
  • the person skilled in the art is familiar with carrying out assays as the method of the present invention which are also described in the literature as electro-mobility shift assay (EMSA).
  • ESA electro-mobility shift assay
  • the method of the present invention allows the identification of target genes due to the physical interaction of the DNA binding domain specified herein with an oligonucleotide comprising a sequence relevant for the expression of a gene suspected to be a target gene. Said physical interactions is an indicator for the said gene to be, indeed, a target gene of foxd3.
  • the invention relates to the method for obtaining and/or identifying a target gene of foxd3 comprising:
  • step (c) determining the nucleic acid sequence of the oligonucleotide of step (b),
  • step (d) comparing the nucleic acid sequence of the oligonucleotide determined in step (c) with the nucleic acid sequences of genes suspected to be target genes, wherein a target gene can be identified due to its sequence identity with the sequence of the oligonucleotide determined in step (c).
  • oligonucleotides refers to a collection of oligonucleotides having different nucleic acid sequences.
  • oligonucleotides which have a sequence corresponding to a expression control sequence of genes suspected to be target gene and sequences which have no relation thereto.
  • such a collection of oligonucleotide is produced by randomized oligonucleotides synthesis.
  • a nucleic acid sequence which corresponds to an expression control sequence of a foxd3 target gene can be identified due to the physical interaction of foxd3 of a fragment thereof comprising at least a DNA binding domain with a particular oligonucleotide of the said plurality of oligonucleotides.
  • the present invention relates to a method for obtaining and/or identifying a target gene of foxd3 comprising :
  • step (c) identifying and/or obtaining mRNA species which are present in the mRNA preparation from wildtype zebrafish embryos of step (b) but which are absent in the mRNA preparation form mother superior mutant zebrafish embryos of step (a), wherein said mRNA species are transcripts of foxd3 target genes.
  • identifying and obtaining mRNAs which are present in the mRNA preparation of wildtype zebrafish embryos of step (b) and which are absent in the mRNA preparation from mother superior mutant zebrafish embryos of step (a) refers to suitable techniques for determining differentially expressed mRNA species. Such techniques comprise but are not limited to the preparation of subtractive libraries or differential display assay. The person skilled in the art, furthermore, knows how to carry out such assays. Adoptions can be carried out without further ado. Moreover, in another aspect the structure of the mRNAs prepared in step (a) and (b) may be determined, e. g., by sequencing of the corresponding cDNAs.
  • nucleic acid sequences thereof thus obtained can be compared by a suitable computer program with known sequences of putative target genes.
  • the person skilled in the art is also in the position to obtain genomic sequences o unknown target genes without further ado based on the provision of the corresponding cDNAs as described above.
  • the said cDNAs may be used, e.g., as proteins for screening genomic libraries.
  • foxd3 target genes will also have a therapeutic and diagnostic value for foxd3 associated diseases and dissorders including craniofacial malformations. Thanks to this invention, their identification has become possible.
  • the present invention also relates to a method for identifying and/or obtaining a foxd3 binding partner comprising:
  • a polypeptide comprising at least the acidic domain of the foxd3 protein encompasses polypeptides which comprise the acidic domain as set forth above and may in addition also comprise further or minor efforts of foxd3 or another protein.
  • the said polypeptide comprising at least the acidic domain of the foxd3 encompasses polypeptides which comprise the acidic domain as set forth above and may in addition also comprise further or minor efforts of foxd3 or another protein.
  • the said polypeptide comprising at least the acidic domain of the foxd3 encompasses polypeptides which comprise the acidic domain as set forth above and may in addition also comprise further or minor efforts of foxd3 or another protein.
  • the said polypeptide comprising at least the acidic domain of the foxd3 encompasses polypeptides which comprise the acidic domain as set forth above and may in addition also comprise further or minor efforts of foxd3 or another protein.
  • the said polypeptide comprising at least the acidic domain of the foxd3 encompasses polypeptide
  • Such a hetero logons polypeptide is particularly suitable for carrying out, e.g., yeast two-hybrid screens.
  • the term "compound” as referred to herein preferably means polypeptides or fragments thereof which are suspected to be binding partners of foxd3. Moreover, the said polypeptide or fragments thereof may also be fused to polypeptides sequences of other proteins or domains thereof, such as DNA binding or transactivated domains.
  • the term "determining binding of said compound to the acidic domain” refers to suitable means which allow detection of that binding including detection of the complex of the acidic domain and the compound by applying antibody based techniques or by applying functional assays such as the activation of a suitable reporter system. Suitable reporter system have been described in the art with respect to yeast two-hybrid assays. The person skilled in the art can adapt this method accordingly without further do.
  • the present invention relates also to a method for the production of a pharmaceutical composition
  • a method for the production of a pharmaceutical composition comprising the steps of the method of the invention referred to herein above and the further step of formulating the antagonist or inhibitor identified and/or obtained in a pharmaceutical acceptable form.
  • the therapeutically useful compounds identified according to the methods of the invention can be formulated and administered to a patient as discussed above.
  • a pharmaceutical composition comprising the steps of the above-described methods; and formulating a drug or pro-drug in the form suitable for therapeutic application and preventing or ameliorating the disorder of the subject diagnosed in the method of the invention.
  • Drugs or pro-drugs after their in vivo administration are metabolized in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459).
  • a corresponding formulation as a pro-drug can be used which is converted into its active in the patient.
  • Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329).
  • steps of formulation for a pharmaceutical composition are well known in the art and depend, inter alia, on the way of administration for said pharmaceutical composition as will be discussed infra in detail.
  • the present invention also encompasses a oligonucleotide having the sequence shown in SEQ ID No: 3.
  • oligonucleotide encompasses polynueleotides of short length.
  • said oligonucleotide is capable of specifically interacting with the foxd3 gene referred to in accordance with the present invention in vivo. More preferably, said oligonucleotide comprises at least 10, at least 15, at least 20, or at least 25 nucleotides in length.
  • the oligonucleotide of the invention may comprise nucleotide modifications, such as chemical modifications.
  • an oligonucleotide as shown in SEQ ID No: 3 efficiently inhibits foxd3 activity in vivo. Therefore, said oligonucleotide may be used as the basis for the development of a drug.
  • the present invention relates to a method for obtaining and/or identifying a polynucleotide which is associated with craniofacial malformation comprising the steps of:
  • step (d) identifying at least a part of the nucleic acid sequence determined in step (b), which comprises at least one nucleotide which is absent in the foxd3 reference sequence (Accession Number: AF052249) wherein said at least part of the nucleic acid sequence determined in step (b) is capable of hybridizing to the foxd3 gene (Accession Number: AF052249).
  • complement refers to the capability of an intact functional allele of a gene to compensate for the deficiencies of a defect allele in a genome.
  • the complementation test is well known in the art and can be carried out, e.g., by crossing a heterozygous mos mutant with a heterozygous mutant suspected to carry a defect in the foxd3 gene and by determining the phenotypes of the offspring whereby phenotypes exhibiting the morphological characteristics of the mos mutant , when present in a representative number, are indicative for non-complementation.
  • Non-complementing mutants carry another defective foxd3 allele.
  • sequence of the non-complementing alleles of foxd3 can be determined by routine methods comprising, e.g., PCR followed by sequencing of the amplification products derived from the mutant foxd3 gene. Sequence comparisons can be carried out by the well known computer programs available, such as the BLAST algorithm, or by visual inspection of the sequences.
  • novel alleles of foxd3 encoding a defective foxd3 protein can be easily determined. Thereby, further domains and regions which are important for the function of foxd3 in vivo can be identified. Those, alleles will serve as the basis for the development of further diagnostic tools.
  • a diagnostic composition is encompassed by the present invention comprising the polynucleotide, the gene, the polypeptide, the antibody or the solid support of the invention.
  • a diagnostic composition can be applied in a method of testing the status of a disorder or susceptibility to such a disorder by using a polynucleotide gene or nucleic acid of the invention, e.g., in the form of a Southern or Northern blot or in situ analysis.
  • Said nucleic acid sequence may hybridize to a coding region of either of the genes or to a non-coding region, e.g. intron.
  • said nucleic acid molecule can again be used in Northern blots.
  • said testing can be done in conjunction with an actual blocking, e.g., of the transcription of the gene and thus is expected to have therapeutic relevance.
  • a primer or oligonucleotide can also be used for hybridizing to one of the above mentioned foxd3 gene or corresponding mRNAs.
  • the nucleic acids used for hybridization can, of course, be conveniently labeled by incorporating or attaching, e.g., a radioactive or other marker. Such markers are well known in the art.
  • the labeling of said nucleic acid molecules can be effected by conventional methods.
  • variant foxd3 gene can be monitored by using a primer pair that specifically hybridizes to either of the corresponding nucleic acid sequences and by carrying out a PCR reaction according to standard procedures.
  • Specific hybridization of the above mentioned probes or primers preferably occurs at stringent hybridization conditions.
  • stringent hybridization conditions is well known in the art; see, for example, Sambrook et al., "Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridisation, A Practical Approach", Hames and Higgins eds., IRL Press, Oxford, 1985.
  • RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase T ⁇ , RNase T 2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments as described above.
  • An additional embodiment of the present invention relates to a method wherein said determination is effected by employing an antibody of the invention or fragment thereof.
  • the antibody used in the method of the invention may be labeled with detectable tags such as a histidine flags or a biotin molecule.
  • the invention furthermore encompasses a pharmaceutical composition
  • a pharmaceutical composition comprising an antagonist or inhibitor obtainable by any one of the aforementioned methods, an agonist or activator obtainable by any one of the aforementioned methods , the oligonucleotide of the present invention, a foxd3 polynucleotide having a nucleic acid sequence as shown in Genbank accession number AF197560, Labosky et al., 1998 (Genbank accession number: NM_010425), Kos et al., 2001 (Genbank accession number: U37274) or Pohl et al., 2001 (Genbank accession number: AJ298865 or AJ298866) or a foxd3 polypeptide having amino acid sequence as shown in Genbank accession number AF197560, Labosky et al., 1998 (Genbank accession number: NM_010425), Kos et al., 2001 (Genbank accession number: U37274) or Pohl
  • composition as used herein comprises the substances of the present invention and optionally one or more pharmaceutically acceptable carrier.
  • the substances of the present invention may be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, methylester, HCI, sulfate, chloride and * e like.
  • the pharmaceutical compositions can be conveniently administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
  • the substances may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like.
  • the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • the substance according to the present invention can be administered in various manners to achieve the desired effect. Said substance can be administered either alone or in the formulated as pharmaceutical preparations to the subject being treated either orally, topically, parenterally or by inhalation. Moreover, the substance can be administered in combination with other substances either in a common pharmaceutical composition or as separated pharmaceutical compositions.
  • the diluent is selected so as not to affect the biological activity of the combination.
  • examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • a therapeutically effective dose refers to that amount of the substance according to the invention which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. 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. Progress can be monitored by periodic assessment.
  • a typical dose can be, for example, in the range of 1 to 1000 ⁇ g; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day. If 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. However, depending on the subject and the mode of administration, the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 10 mg per kg body mass.
  • compositions and formulations referred to herein are administered at least once in accordance with the use of the present invention.
  • the said pharmaceutical compositions and formulations may be administered more than one time, for example from one to four times daily up to a non-limited number of days.
  • compositions of the substance according to the invention are prepared in a manner well known in the pharmaceutical art and usually comprise at least one active substance referred to herein above in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent thereof.
  • the active substance(s) will usually be mixed with a carrier or diluted by a diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper or other suitable containers or vehicles.
  • a carrier may be solid, semisolid, gel-based or liquid material which serves as a vehicle, excipient or medium for the active ingredients.
  • Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.
  • the formulations can be adopted to the mode of administration comprising the forms of tablets, capsules, suppositories, solutions, suspensions or the like.
  • the dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients at the wrong dose.
  • the present invention relates to the use of the polynucleotide, the gene, the polypeptide, the antibody or the solid support of the invention for the preparation of a diagnostic composition for diagnosing craniofacial malformations.
  • the present invention also relates to the use of a foxd3 polynucleotide having a nucleic acid sequence as shown in Genbank accession number AF197560, Labosky et al., 1998 (Genbank accession number: NM_010425), Kos et al., 2001 (Genbank accession number: U37274) or Pohl et al., 2001 (Genbank accession number: AJ298865 or AJ298866) or a foxd3 polypeptide having an amino acid sequence as shown in Genbank accession number AF197560, Labosky et al., 1998 (Genbank accession number: NM_010425), Kos et al., 2001 (Genbank accession number: U37274) or Pohl et al., 2001 (Genbank accession number: AJ298865 or AJ298866) or any one of SEQ ID Nos: 11 to 14 or an agonist or activator obtainable by any one the aforementioned methods for the
  • a polynucleotide encoding functional and expressible foxd3 protein or said protein itself can be used to treat and/or prevent craniofacial malformations, preferably when the treatment is applied during embryonic development of a subject.
  • agonists or activators of foxd3 which can be identified or obtained by the methods described supra.
  • said craniofacial malformation is selected from the group consisting of: cleft palate syndrome, loss of middle ear ossicles, Congenital Heart Disease (CHD), Niikawa-Kuroki syndrome (NKS), cancer and mental retardation.
  • CHD Congenital Heart Disease
  • NBS Niikawa-Kuroki syndrome
  • the clinical characteristics of said diseases are well known in the art and are described in standard text books such as Pschyrembl and Stedman.
  • the present invention further encompasses the use of an antagonist or inhibitor obtainable by any one of the aforementioned methods or the oligonucleotide of the invention for the preparation of a pharmaceutical composition for treating and/or preventing diseases or disorders which are caused by or related to foxd3 overexpression.
  • foxd3 Overexpression or ectopic misexpression of foxd3 in embryonic development will cause defects due to the presence of an increased number or aberrant location of neural crest cells in the embryo. Said neural crest cells, as described above, will inter alia differentiate into cartilage and bone. Thus, overexpression or ectopic misexpression of foxd3 leads to the aberrant formation of cartilage and bone tissue in a subject resulting in severe disorders.
  • inhibitors or antagonists, including the oligonucleotide of the present invention, of either a foxd3 polynueleotides or proteins can be used to treat and/or prevent diseases or disorders which are caused by or related to foxd3 overexpression.
  • the treatment is applied during embryonic development of a subject.
  • Also within the scope of the present invention is a method for treating one or more of the diseases referred to in accordance with the uses of the present invention, wherein said method comprises at least the step of administering the pharmaceutical compositions of the invention in a pharmaceutically acceptable form to a subject.
  • the subject referred to in accordance with the present invention is an animal, more preferably a vertebrate, most preferably a human.
  • the present invention relates to a kit comprising the polynucleotide, the gene, the polypeptide, the antibody or the solid support of the invention.
  • the kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals.
  • the kit of the invention can be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool.
  • the parts of the kit of the invention can be packaged individually in vials or other appropriate means depending on the respective ingredient or in combination in suitable containers or multicontainer units.
  • kit Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art.
  • the kit may be used for methods for detecting expression of a mutant form of the polypeptides, genes or polynueleotides in accordance with any one of the above-described methods of the invention, employing, for example, immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples.
  • immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples.
  • Figure 1 Characterization of the mos phenotype. a, Comparison of live WT and mos zebrafish larvae at 3 days postfertilization (3d) and 5d shown from the lateral view. The mos phenotype is first visible at 2 - 2.5 days postfertilization (d) (not shown). By 3 d, the branchial arches region is clearly reduced, with ventral bending of the Meckel's cartilage and a gaping jaw (left panels, arrow). The phenotype progressively worsens and displays arrested jaw formation followed by cardiac edema at 5 d (right panels, arrows). The mos larvae do not inflate their swim bladder and die at 6-7 d (not shown).
  • b Alcian blue staining of craniofacial cartilage in WT and mos larvae shown from the lateral view (top panels) and ventral view (bottom panels). Sibling WT and mos embryos (5 d) were stained with Alcian blue to reveal specific craniofacial skeletal deficits.
  • c Schematic drawing of WT and mos craniofacial cartilage elements (Kimmel et al., 2001). The mos mandibular arch appears correctly shaped and the articulation between Meckel's cartilage (red) and the palatoquadrate (pink) is established. In most cases Meckel's cartilage is bent (dorsally or ventrally) and the palatoquadrate has a reduced or absent anterior process.
  • the second, hyoid arch is more severely affected.
  • the hyosymplectic (light blue) has a normal anterior extension but its most dorsal process is truncated.
  • the ceratohyal (dark blue) is predominantly absent.
  • the remaining posterior pharyngeal arches (green) and the basibranchial (turquoise) do not form.
  • the neurocranium (yellow) is the least affected by the mos mutation. In a small percentage of the mos embryos, fusion of the trabeculae is incomplete, resulting in a cleft ethmoid plate. Scale bar represents 250 ⁇ m within each figure.
  • Figure 2 Whole-mount in situ hybridization showing st7a/72 and snaiH gene expression in WT and mos embryos. Embryos are shown from the dorsal views. Scale bar represents 250 ⁇ m.
  • Figure 3 The genetic and physical map of the mother superior chromosomal region.
  • Figure 4 Sequence analysis of foxd3 and the mos mutation.
  • a Representative sequencing data from homozygote wild type, heterozygote wild type and homozygote mos embryos.
  • b Alignment of foxd3 nucleotide sequences (upper) and predicted amino acid sequences (lower) in wild type and mos mutants with the previously published zebrafish foxd3 sequence.
  • c Alignment of the predicted FoxD3 amino acid sequences of different species.
  • the red * denotes the position of the Ser ⁇ Cys amino acid substitution caused by the mos mutation.
  • FIG. 5 Analysis of foxd3 expression in WT and mos mutant embryos.
  • a, f At 5.25h, foxd3 expression can be seen in the organizer region (white arrowheads) and there is no apparent difference between mos and wild type.
  • b By 10h, foxd3 expression is seen in the tail bud (white arrow) and premigratory cranial neural crest cells (black arrowhead) in WT embryos (75% of the clutch).
  • g mos mutants (25% of the clutch) show decreased staining in the tail bud and no detectable expression in the neural crest cells.
  • foxd3 expression continues in premigratory as well as migratory neural crest cells while no neural crest staining is evident in the corresponding region of the mos mutants.
  • d i
  • foxd3 expression has intensified in somites, premigratory (trunk) and migratory (crania!) neural crest cells of WT embryos whereas only the somite expression is maintained in mos mutants.
  • FIG. 6 Partial rescue and phenocopy of the mos mutation.
  • a Partial rescue of the early mos phenotype shown by analysis of snaill expression.
  • WT embryos (20 h) the expression of snaill in the mandibular (m), hyoid (h), first and second branchial arches can be seen as clusters of positive cells. These clusters are absent in ADOS mutants.
  • Two examples of partially rescued embryos injected with 20 pg foxd3 mRNA at the 1-4 cell stage are shown.
  • the snaill expression level increases in the lateral rim of the neural tube (rescuel ) and is more intense in rescue 2, although the normal clustering of cells forming branchial primordia were not observed.
  • b Alcian blue staining of rescued and morphant mos larvae. Two examples of partially rescued mos mutants injected with 20 pg foxd3 mRNA are shown. An example of the morphant cartilage phenotype produced by injecting WT embryos with 10 nl foxd3 morpholino (0.25 mM). Arrows point to the remaining craniofacial elements that are relatively unaffected. c, Dose dependent foxd3 morphant phenotypes.
  • Zebrafish were maintained and staged as described (Kimmel et al., 1995). Heterozygous carriers of the mos 188 mutation (Neuhauss et al., 1996) within the AB strain were out crossed to wild-type zebrafish of the TL strain to facilitate genetic mapping based on polymorphic linkage analysis. Mos heterozygotes within the AB/TL background were identified by random intercross analysis and were used for the production of all embryos used in the current study.
  • Cartilage was stained with Alcian blue essentially as described (Neuhauss et al., 1996).
  • the zebrafish larvae were fixed overnight in phosphate-buffered saline (PBS) containing 4% paraformaldehyde, washed several times in PBS and bleached in 40 mM potassium hydroxide containing 10% hydrogen peroxide for 1 hour.
  • Specimens were then stained in Alcian blue solution (0.1 % Alcian blue (Sigma), 70% ethanol, 1 % concentrated hydrochloric acid) overnight and destained for at least 5 hours in acidic ethanol (5% concentrated hydrochloric acid, 70% ethanol).
  • the zebrafish foxd3 and snaill probes have been described (Kelsh et al., 2000; Thisse et al., 1993). Embryos were washed as described, gradually reducing the proportion of hybridization buffer and SSC and increasing the proportion of PBT (Thisse et al., 1993). Blocking was done in PBT containing 2% goat serum and 2mg/ml BSA (1 hour; room temperature). The embryos were then incubated with preabsorbed alkaline-phosphate-coupled anti-digoxigenin Fab fragments (Boehringer Mannheim) diluted 1/5000 in blocking solution (2 hours; room temperature).
  • NTMT 100 mM Tris-HCI pH 9.5, 50 mM Mg Cl 2 , 100 mM NaCl, 0.1 % Tween-20. Alkaline-phosphate was revealed in NTMT containing 225 ⁇ g/ml NBT and 175 ⁇ g/ml BCIP. When the appropriate color developed, the reaction was stopped in PBT containing 5 mM EDTA. For analysis and photography, the embryos were transferred to an 80% glycerol 20% PBT solution.
  • Zebrafish snail2 is a molecular marker for presumptive neural crest cells (Thisse et al., 1995). In mos mutants, snail2 expression is dramatically reduced at 12.5 hours postfertilization (12.5 h; upper panels). Zebrafish snaill is a molecular marker for early development of the branchial arch primordia (Thisse et al, 1993). In mos mutants, snaill expression is markedly reduced in the primordia region, although adjacent snaill expression in the paraxial mesoderm and in posterior somites is maintained normally. The results of the temporal expression analysis of snail 2 and snail 1 expression in wildtype embryos are shown in Figure 2.
  • PCR reaction conditions for genetic mapping analysis were 10 mM Tris pH 8.4, 50 mM KCI, 1.5 mM MgCI 2 , 0.001 % gelatin, 100 ⁇ M each dNTP, 0.1 mg/ml BSA, 100 nM each forward and reverse primer, 0.3 units Taq DNA polymerase (Roche). Cycling parameters were 3 minutes 96°C followed by 40 cycles (45 seconds 96°C, 45 seconds 55°C, 45 seconds 72°C), followed by 3 minutes 72°C. PCR products were separated by 3% agarose gel electrophoresis (1.5 % normal agarose, 1.5% NuSieve agarose (BioWhittaker Molecular Applications)).
  • PCR primers were end-labeled using adenosine 5'-[ ⁇ - 32 P] triphosphate (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (30 minutes, 37°C). Radiolabeled primers were then used for PCR reactions as described for SSLP analysis. The reaction products were denatured and analyzed using Mutation Detection Enhancement (MDE) gel solution (BioWhittaker Molecular Applications) according to manufacturer's protocol.
  • MDE Mutation Detection Enhancement
  • ZMAP integrated map of the zebrafish genome
  • a two-generation cross and SSLP markers were used to genetically map the mos mutation to zebrafish chromosome 6 (LG6).
  • markers were used to isolate individual clones: Z15259 (isolated clones: I0344, J0928, J1028, K0344, K0928, K1028, M0344), fa04h11 (C0126, D0126), fc48e01 (B03194), fb10d09 (E2143), fb64d12 (I0730), her ⁇ (B1630).
  • Markers: B03194F, B03194R and B1630R represent STSs developed from end sequences of the corresponding PAC clones (red boxes). New genetic markers were developed based on the sequences of ESTs, cloned genes and the ends of PAC clones and were tested by SSCP analysis.
  • foxd3 and her ⁇ genes were the only cloned genes mapped in the vicinity of mos.
  • the foxd3 polymorphism (shown in light blue) was confirmed by sequencing and shown to map to the mos locus with 0 recombination events in 3,134 meioses.
  • Three PAC clones containing foxd3 were also isolated (G2361 , E2361 , D2361 ) and analyzed by sequencing.
  • the foxd3 polymorphism identified by SSCP analysis was sequenced in 6 wild type and 6 mos siblings.
  • the mos mutation (A ⁇ T) at position 380 is predicted to result in the conversion of the zebrafish Foxd3 Ser62->Cys. Point mutations such as this are the expected result of ENU-induced DNA lesions (Knapik, 2000).
  • the foxd3 gene has been cloned in many species and contains a conserved fork head DNA-binding domain and a transactivation domain near the C-terminus of the protein (Sutton et al., 1996; Odenthal et al., 1998; Kos et al., 2001 ; Pohl et al., 2001 ).
  • the mos Ser ⁇ Cys mutation lies within an acidic domain close to the N-terminus but the function of this domain is not known.
  • FoxD3 has been referred to by different names prior to the unified nomenclature system (Kaestner et al., 2000): zebrafish (Fkd6 (Odenthal et al., 1998)), human (HFH2, genesis (genebank AF197560)), mouse (HFH2, genesis (Labosky et al., 1998)), chick (CWH3 (Kos et al., 2001 ) and frog (XFD6) (Pohl et al., 2001 ).
  • the zebrafish foxd3 contains an acidic domain (aa 21-113 of alignment), the fork head domain (aa 141-236 of alignment) and a transactivation domain (aa 380-398 of alignment).
  • the mos mutation might cause a detrimental structural change in the Foxd3 protein.
  • the substituted Cys residue could interfere with proper disulfide bond formation in the FoxD3 protein and/or lead to intermolecular cross-linking with other proteins.
  • Ser to Cys mutations or polymorphisms that have been shown to disrupt protein function or have been linked to a disease phenotype (Chen et al., 2001 ; MacMullen et al., 2001 ; Xing et al., 2001 ). So far, the majority of biochemical and structural analyses of the winged helix transcription factors has concentrated exclusively on the fork head DNA binding domain (Marsden et al., 1998).
  • Synthetic zebrafish foxd3 mRNA was synthesized using the mMessage Machine kit according to manufacturer's instructions (Ambion). The capped mRNA (20 pg) was injected into one- to four-cell stage embryos.
  • Partial rescue of the early mos phenotype was observed by whole-mount in situ hybridization with snaill.
  • the mos phenotype is partially rescued.
  • Two examples of partially rescued embryos are shown in Figure 6.
  • the snaill expression level increases in the lateral rim of the neural tube (rescuel ) and is more intense in rescue 2, although the normal clustering of cells forming branchial primordia were not observed.
  • Morpholino antisense zebrafish foxd3 oligonucleotides were synthesized by Gene Tools, LLC. The sequence was designed according to manufacturer's suggestions as follows: 5'-CACTGGTGCCTCCAGACAGGGTCAT-3' (SEQ ID No: 3). The oligonucleotides were diluted in 1x Danieau buffer at a range of concentrations (1 mM - 0.1 mM) and approximately 10 nl was injected into one- to four-cell stage embryos.
  • FIG. 6 An example of the morphant cartilage phenotype produced by injecting WT embryos with 10 nl foxd3 morpholino (0.25 mM) is shown in Figure 6.
  • the morphant shows unilateral loss of ceratobranchials and complete deletion of basibranchial cartilage. Arrows in said Figure point to the remaining craniofacial elements that are relatively unaffected.
  • the foxd3 morphant phenotypes could be induced in a dose dependent manner.
  • foxd3 morpholino antisense oligonucleotides (Gene Tools, LLC) were injected into 1-4 cell stage embryos in a range of concentrations (1 mM, 0.5 mM, 0.25 mM, 0.1 mM). The 1mM concentration was lethal for most injected embryos following epiboly. In embryos that survived through gastrulation, development of the craniofacial region and the somites was dramatically impaired, causing truncation of the tail. A severe heart edema develops very early ( ⁇ 2 d) but embryos can live as long as 5-6 d. The severity of the morphant phenotype decreases with decreasing doses of foxd3 morpholino.
  • results indicate the average (%) of injected embryos from one to three independent experiments that showed each phenotype classification.
  • the total number of injected embryos for each morpholino concentration is given (n). Live embryos were observed under the microscope and representative examples are shown in Figure 6c.
  • Class I Very mild craniofacial malformation with almost normal tails. Similar to
  • Class II Craniofacial malformation and cardiac edema most similar to mos mutants.
  • Class III Severe craniofacial reduction, cardiac edema and truncated tail. Similar to
  • Class IV Complete deletion of ventral facial region, very large amount of cardiac edema and complete truncation of the tail. Similar to Figure 6c (1 mM).
  • the winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 128, 1467-1479 (2001 ).
  • Thisse, C Thisse, B. & Postlethwait, J.H. Expression of snail2, a second member of the zebrafish snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos. Dev. Biol. 172, 86-99 (1995).
  • Thisse, C Thisse, B., Schilling, T.F. & Postlethwait, J.H. Structure of the zebrafish snaill gene and its expression in wild-type, spadetail and no tail mutant embryos. Development s, 1203-1215 (1993).

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Abstract

La présente invention concerne un polynucléotide variant foxd3, un gène comprenant ledit polynucléotide, et un vecteur comprenant ce polynucléotide ou ce gène, ainsi qu'une cellule hôte modifiée par génie génétique à l'aide du polynucléotide, du gène ou du vecteur selon la présente invention. Elle concerne également un procédé de production d'un polypeptide ou fragment foxd3 variant moléculaire. Elle concerne encore un polypeptide ou fragment dudit polypeptide codé par le polynucléotide ou le gène ou qui peut être obtenu par le procédé selon la présente invention, ainsi qu'un anticorps qui se lie spécifiquement au polypeptide selon la présente invention, et un support solide comportant un ou plusieurs polynucléotides, gènes, vecteurs, polypeptides, anticorps ou cellules hôtes selon la présente invention sous forme immobilisée. Elle concerne en outre un procédé permettant d'obtenir et / ou d'identifier un inhibiteur ou antagoniste ou un activateur ou agoniste de l'activité de foxd3, un procédé de production d'une composition pharmaceutique, et un procédé permettant d'obtenir et / ou d'identifier un polynucléotide qui est associé à une malformation cranio-faciale. La présente invention concerne enfin des compositions diagnostiques et pharmaceutiques, l'utilisation du polynucléotide, du gène, du polypeptide, de l'anticorps ou du support solide selon la présente invention pour la préparation d'une composition diagnostique ou pharmaceutique permettant de diagnostiquer, de traiter et / ou de prévenir des malformations cranio-faciales, et l'utilisation d'un antagoniste ou d'un inhibiteur pouvant être obtenu par n'importe quel procédé pour la préparation d'une composition pharmaceutique destinée à traiter et / ou à prévenir des maladies ou des états pathologiques qui sont provoqués par la surexpression de foxd3 ou liés à cette surexpression. La présente invention concerne en dernier lieu un kit.
PCT/EP2002/013936 2001-12-07 2002-12-09 Moyens et procedes permettant de diagnostiquer et de traiter des malformations cranio-faciales WO2003048196A2 (fr)

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