WO1998048829A1 - Cristallin de vertebre obtenu a partir d'une cellule ectodermique et procedes d'utilisation de celui-ci - Google Patents

Cristallin de vertebre obtenu a partir d'une cellule ectodermique et procedes d'utilisation de celui-ci Download PDF

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WO1998048829A1
WO1998048829A1 PCT/US1998/008592 US9808592W WO9848829A1 WO 1998048829 A1 WO1998048829 A1 WO 1998048829A1 US 9808592 W US9808592 W US 9808592W WO 9848829 A1 WO9848829 A1 WO 9848829A1
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Prior art keywords
lens
pax
vertebrate
expression
nucleic acid
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PCT/US1998/008592
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English (en)
Inventor
Richard A. Lang
Ali Hemmati-Brivanlou
Curtis R. Altmann
Robert L. Chow
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The Rockefeller University
New York University
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Priority to AU72645/98A priority Critical patent/AU7264598A/en
Publication of WO1998048829A1 publication Critical patent/WO1998048829A1/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
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids

Definitions

  • the invention relates generally to methods of producing a vertebrate lens, to the lenses themselves, and the use of such methods and lenses.
  • Extracapsular cataract extraction and implantation of intraocular lenses are being performed at ever increasing rates in the United States, as well as throughout the 20 industrial world. In the United States well over one million such procedures are performed every year. As the population of the United States and the rest of the industrial world continues to age, the incidence of such procedures is also expected to increase.
  • the vertebrate lens is derived from the ectodermal layer, the outermost of the three primary cell layers of the embryo.
  • the vertebrate eye, ears, nervous system, epidermis, fingernails, hair, and the mucous membranes of the mouth and anus also derive from the ectodermal layer.
  • Crystallins comprise approximately 90% of the water-soluble proteins of the lens. These proteins serve as structural components and contribute to the optical properties of the lens. Interestingly, some crystallins have a completely different role in other tissues.
  • pig ⁇ -crystallin is an NADPH.quinone oxidoreductase. Od er crystallins are encoded by genes which appear to have evolved through gene duplication of corresponding housekeeping genes. Still other crystallins appear to be expressed exclusively in the lens.
  • Pax-6 a member of the Pax gene family.
  • Pax genes encode proteins having a paired domain and a homeodomain, both of which bind to DNA.
  • Pax proteins play significant roles in tissue differentiation during embryogenesis.
  • Pax-6 is a transcription factor found in the eye and the nervous system during early development of these tissues.
  • mice a heterozygous mutation in the Pax-6 gene results in microphthalmia, whereas the corresponding homozygous mutation is lethal at birth, the result of severe craniofacial malformations.
  • the heterozygous Pax-6 phenotype results in a condition known as ANIRIDIA which is characterized by various ocular malformations.
  • Drosophila the ectopical expression of Pax-6 in imaginal disks can induce the formation of ectopic eyes [Haider et al. , Science 267: 1788-1792 (1995)].
  • the eye of the Drosophila is a typical insect compound eye, and therefore bears no anatomical resemblance to the eyes of vertebrates. More particularly, the Drosophila eye does not have a lens that is analogous to the vertebrate lens.
  • the present invention provides a vertebrate lens produced by selectively inducing lens development in vertebrate ectodermal cells.
  • the vertebrate lens of the present invention can be produced ectopically in a reliable manner, i.e. , greater than 50% , and in some cases greater than 70% of the time, thereby providing an abundant source of vertebrate lenses for use in lens transplantation, as well as other medical procedures.
  • One aspect of the present invention describes a method for artificially inducing the formation of a vertebrate lens in an ectodermal cell that comprises adding an amount of Pax-6 to the ectodermal cell that is sufficient to induce lens formation in the ectodermal cell.
  • a mature vertebrate lens is produced.
  • the vertebrate is a frog.
  • the vertebrate is a mammal.
  • the mammal is a human.
  • purified Pax-6 is added directly to the ectodermal cell.
  • a nucleic acid that encodes Pax-6 is added to the ectodermal cell, and adding the Pax-6 is performed by expressing the Pax-6 nucleic acid in the ectodermal cell.
  • the adding of the nucleic acid to the ectodermal cell is performed by
  • the Pax-6 nucleic acid is added by the addition of a vector containing the nucleic acid.
  • the expression of the Pax-6 nucleic acid is under the control of an inducible promotor, and adding the Pax-6 is performed through the induction of the promotor.
  • adding the Pax-6 is performed by expressing the Pax-6 naturally encoded in the genome of the ectodermal cell. In this case an agent is added to the ectodermal cell which selectively induces the expression of Pax-6, without inducing the formation of neural tissue.
  • the Pax-6 nucleic acid is a naturally occurring DNA encoding Pax-6.
  • nucleic acid is a Pax-6 cDNA. In yet another particular embodiment the nucleic acid is an RNA encoding Pax-6. In a preferred embodiment, adding the RNA is performed by injecting the RNA into the ectodermal cell.
  • Another aspect of the present invention describes a method of identifying an agent that selectively induces the formation of a lens without inducing the formation of neural tissue.
  • One embodiment of this aspect of the invention comprises the steps of adding an agent to an ectodermal cell, detecting the expression of Pax-6, detecting the expression of a lens marker, and detecting the expression of a neural marker.
  • the agent is identified when Pax-6 and the lens marker are detected, but the neural marker is not detected.
  • the identified agent induces die formation of the lens by inducing the expression of Pax-6 without inducing the formation of neural tissue.
  • the lens marker is ⁇ Bl-crystallin.
  • the neural marker is the general neural marker NCAM.
  • the lens marker is ⁇ Bl-crystallin and the neural marker is NCAM.
  • the vertebrate is a frog.
  • me vertebrate is a mammal. In the most preferred embodiment the mammal is a human.
  • the present invention also includes a lens produced by the med ods of the present invention.
  • me lens is a mature vertebrate lens.
  • the vertebrate is a frog.
  • the vertebrate is a mammal. In the most preferred embodiment the mammal is a human.
  • Another aspect of the present invention includes methods of using the lenses of the present invention for lens transplantation in a vertebrate.
  • One such embodiment comprises placing the lens in an eye of the vertebrate.
  • the vertebrate is a mammal.
  • the mammal is a human.
  • the present invention further includes a method of treating a vertebrate with a defect in its lens development pathway by administering a therapeutically effective amount of a nucleic acid encoding Pax-6.
  • the method of treating a vertebrate with a defect in the lens development pathway is performed by administering a therapeutically effective amount of Pax-6.
  • the present invention also includes pharmaceutical compositions for the treatment of a defect in the lens development pathway.
  • One such embodiment comprises a therapeutically sufficient amount of a nucleic acid encoding Pax-6 and a pharmaceutically acceptable carrier.
  • Another such embodiment comprises a therapeutically sufficient amount of Pax-6 and a pharmaceutically acceptable carrier.
  • Still another such embodiment comprises a therapeutically sufficient amount of an agent which selectively induces the expression of Pax-6, without inducing the formation of neural tissue. Accordingly, it is a principal object of the present invention to provide a vertebrate lens.
  • Figure 1A shows the anti-bovine ⁇ -crystallin antibody labeling of tadpole stage 38 embryo of the normal lens from an injected embryo;
  • Figure IB is a close up of Figure 1A.
  • Figure IC shows a transverse section of anti-bovine- ⁇ -crystallin antibody stained tadpole, stage 28.
  • Figure ID shows a whole mount in situ hybridization of a stage 28 embryo using a Xenopus ⁇ Bl-crystallin probe.
  • Figure IE depicts the results of an animal cap assay of Pax-6 injected Xenopus embryos assessed for the expression of tissue specific markers at stage 34 using RT-PCR.
  • the lens-specific marker BBl-crystallin was produced in a dose dependent manner (lanes 1-4, 1.0, 0.25, 0.06 and 0.5 ng/embryo respectively) in the absence of NCAM expression.
  • Injection of RNA encoding Noggin (lane 5, 2.0 ng) serves as a positive control for response of the explants and gives the expected expression of d e neural marker NCAM.
  • Muscle actin is used as a mesodermal marker and EFl ⁇ as a loading control.
  • Figure IF shows an uninjected animal cap stained with ⁇ - crystallin antibody.
  • Figures 1G and 1H show animal caps injected with 1.4 ng Pax- 6-FLAG RNA (sibling control at stage 33/34).
  • Figure 2 Pax-6 induces ectopic lenses in whole embryos in a cell autonomous fashion.
  • Figures 2A-2C show ⁇ -crystallin staining of embryos (stage 38) injected with RNA encoding Pax-6-FLAG (0.12-0.25 ng/embryo) which reveal the formation of ectopic lenses (brown) in whole mount;
  • Figures 2D-2G show the same in section.
  • Arrowheads (red) in Figures 2B, 2F and 2G indicate the border between lens epithelium and fiber cells; the large arrowhead in Figure 2B indicates endogenous lens.
  • the embryo in Figure 2A was cleared in benzyl benzoate and is shown at a higher magnification in Figure 2B.
  • Embryos shown in Figure 2C and 2D were co-stained (blue) with antibodies to NCAM. Some ectopic lenses were not associates with neural tissue (Figure 2C, black arrowhead; Figure 2D and 2E) while others were close to (Figure 2D, far left ectopic lens; Figure 2F) or tightly associated with neural tissue ( Figure 2C, white arrowhead; Figure 2G). Sections through endogenous (Figure 2H, left) and ectopic lenses ( Figure 2H, arrowheads and Figure 21) in embryo co-injected with ⁇ -gal and ⁇ -crystallin RNAs.
  • RNA encoding dominant-negative type I BMP receptor (lane 1 , 2.0 ng), follistatin (lane 2, 2.0 ng), noggin (lane 3,2.0 ng), chordin (lane 4, 2.0 ng), dissociated animal cap cells (lane 6) but not uninjected embryos (lane 5) led to the expression of NCAM, Pax-6 and ⁇ Bl-crystallin in animal caps at tailbud stage.
  • EF-l ⁇ was amplified as a loading control and the absence of muscle actin indicated mat mesoderm was not present.
  • RNA from tailbud embryo was used as a positive control while an amplification without RT (lane 7) controlled for absence of contaminating DNA.
  • the administration of Pax-6 to an ectodermal cell unexpectedly has a direct effect on lens development in the absence of any other extrinsic factor.
  • the presence of the transcription factor Pax-6 directs induction of the lens in an ectodermal cell and furthermore can induce the expression of a lens-specific marker without inducing a general neural marker.
  • Ectopic Pax-6 expression also results in the formation of ectopic lenses in whole embryos. Indeed, Pax-6 is sufficient to induce lens both from ectodermal explants and in the whole embryo.
  • the present invention provides the first evidence ever of a molecule having lens inducing ability.
  • an "ectopic lens” is an otherwise natural lens which is produced outside of d e location that it is normally is formed.
  • Pax-6 is meant to include all active forms of the highly conserved Pax-6 protein including genetically engineered and natural occurring mutants, fusion proteins thereof, and active fragments thereof, wherein an active form of Pax-6 can function as a transcription factor during lens development in an ectodermal cell.
  • the Pax-6 is a vertebrate Pax-6.
  • Pax-6 gene, or “Pax-6" nucleic acid, or “Pax-6” RNA encodes a Pax-6 protein as defined above.
  • the "lens development pathway" in a vertebrate encompasses all of the factors and steps involved in the development of a vertebrate lens starting with the initial induction of an ectodermal cell to begin the process up to the ultimate formation of the mature vertebrate lens.
  • Pax-6 can be administered directly as the purified protein; indirectly by administering a nucleic acid encoding Pax-6 eitiher alone e.g. , injecting naked mRNA, or as part of a vector; or by administering an agent that induces the expression of Pax-6 naturally encoded in the genome of the ectodermal cell. In the latter case, the agent selectively induces the expression of Pax-6, without inducing the formation of neural tissue.
  • the expression of the Pax-6 nucleic acid can be under the control of an inducible promotor, and the addition of Pax-6 can be performed through the induction of the promotor.
  • Pax-6 can be delivered in a vesicle, such as a liposome, or as naked protein.
  • the Pax-6 nucleic acid may be delivered as the naked nucleic acid, or inside an appropriate expression vector or vertebrate virion.
  • Vectors may be introduced into the desired host cells by methods known in the art, as exemplified below.
  • the present invention also discloses methods for identifying an agent that selectively induces me formation of a lens without inducing the formation of neural tissue.
  • One such method includes the steps of adding an agent to an ectodermal cell, detecting the expression of Pax-6, detecting the expression of a lens marker, and detecting me expression of a neural marker. The agent is identified when Pax-6 and the lens marker are detected, but the neural marker is not detected. The identified agent induces the formation of the lens by inducing the expression of Pax- 6 without inducing the formation of neural tissue.
  • Pax-6 and/or a neural marker can monitored by any number of means including expressing a tagged (FLAG-tag [Pricket, et al., Biotechniques, 7:580-589 (1989)]) version of Pax-6 as exemplified herein, expressing a Pax-6 fusion protein, or through the use of specific antibodies raised against Pax-6, or the neuronal marker as exemplified herein.
  • the expression can be indirectly monitored by the transcription of corresponding mRNAs through hybridization of the mRNAs with labeled probes. Appropriate labels for the nucleic acids and proteins, including antibodies are exemplified below.
  • the lenses produced by the methods of the present invention can be used in lens transplantation procedures in vertebrates.
  • the procedure comprises placing the lens in an eye of the vertebrate, preferably a mammal, and more preferably a human.
  • Detailed procedures for performing lens transplantation are exemplified below, with the understanding that a skilled ophthalmologist would be most able to use the lenses provided by the present invention in the appropriate manner depending on the circumstances of the particular situation.
  • Methods of treating a vertebrate with a defect in its lens development pathway are also provided by the present invention. Such methods are performed by administering a therapeutically effective amount of purified Pax-6, or a nucleic acid encoding Pax-6 to die vertebrate.
  • the present invention also includes pharmaceutical compositions for the treatment of a defect in the lens development pathway.
  • Such pharmaceutical compositions comprise a pharmaceutically acceptable carrier and a therapeutically sufficient amount of: purified Pax-6, or a nucleic acid encoding Pax-6, or an agent which selectively induces the expression of Pax-6, without inducing the formation of neural tissue.
  • Nucleic Acids Encoding Pax-6 Proteins The present invention contemplates obtaining a nucleic acid encoding a Pax-6 including a full length, or naturally occurring form of Pax-6, thereof from any vertebrate, particularly mammalian, and more particularly human, source.
  • the term "gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
  • a "vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e. , capable of replication under its own control.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • "Heterologous" DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to d e cell.
  • nucleic acid molecule refers to d e phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxy adenosine, deoxyguanosine, deoxythymidine, or deoxy cytidine; "DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and diioesters, in either single stranded form, or a double- stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary strucmre of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g. , restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e. , the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperamre and solution ionic strength (see Sambrook et al., supra).
  • the conditions of temperamre and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55°, can be used, e.g.
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g. , 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g. , 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of d e nucleic acids and d e degree of complementation, variables well known in the art.
  • RNA:RNA The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • T m The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in die following order: RNA:RNA,
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides; and most preferably 30 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65 °C.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g. , mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g. , the immunoglobulin superfamily) and homologous proteins from different species (e.g. , myosin light chain, etc.) [Reeck et al. , Cell 50:667 (1987)].
  • corresponding to is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • corresponding to refers to the sequence similarity, and not die numbering of the amino acid residues or nucleotide bases.
  • a gene encoding Pax-6 can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining Pax-6 gene are well known in the art, as described above (see, e.g. , Sambrook et al., 1989, supra). Accordingly, any vertebrate cell potentially can serve as the nucleic acid source for a Pax-6 gene.
  • the derivative or analog is functionally active, i.e. , capable of inducing lens formation in an ectodermal cell, in a manner roughly equal to that of the full-length, wild-type Pax-6.
  • Pax-6 derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity relative to native Pax-6 or are have increased stability. Such substitutions in turn lead to amino acid substitutions bestowing a particularly preferable property to Pax-6.
  • a Cys may be introduced at a potential site for disulfide bridges with another Cys.
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • the nucleic acids encoding Pax-6 derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned Pax-6 gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al. , 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • die Pax-6-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutations enhance the functional activity of the mutated Pax-6 gene product.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al. , 1978, J. Biol. Chem. 253:6551 ; Zoller and Smith, 1984, DNA 3:479-488;
  • the nucleotide sequence coding for Pax-6, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted directly into an ectodermal cell or alternatively into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter. "
  • the nucleic acid encoding Pax-6 can be operationally associated with a promoter in an expression vector of the invention.
  • Bom cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding Pax-6 and/or its flanking regions.
  • a recombinant Pax-6, derivative, chimeric construct, fusion protein or analog thereof, may be expressed in an ectodermal cell.
  • Expression of Pax-6 may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in die ectodermal cell. Some appropriate promoters and vectors may be selected from those listed below.
  • Vectors may be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al. , 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al. , Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g. , vaccinia virus, adenovirus, etc.); insect cell systems infected widi virus (e.g. , baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g. , vaccinia virus, adenovirus, etc.
  • insect cell systems infected widi virus e.g. , baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA e.g., bacteriophage, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on die host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant Pax-6, derivative, chimeric construct, or analog thereof may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • the cell containing die recombinant vector comprising the nucleic acid encoding Pax-6 is culmred in an appropriate cell culmre medium under conditions that provide for expression of Pax-6 by the cell.
  • Pax-6 protein expression may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control Pax-6 gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al. , 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981 , Proc. Natl. Acad. Sci. U.S.A.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al. , 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al. , 1985, Mol. Cell. Biol. 5:1639- 1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al. , 1985, Namre 315:338-340; Kollias et al. , 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al. , 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Namre 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al. , 1986, Science 234: 1372-1378).
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g. , E. coli plasmids col ⁇ l, pCRl, pBR322, pMal-C2, p ⁇ T, pG ⁇ X (Smith et al. , 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g.
  • phage ⁇ e.g., NM989, and other phage DNA
  • yeast plasmids such as me 2 ⁇ plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • both non-fusion transfer vectors such as but not limited to pVL941 (BamHl cloning site; Summers), pVL1393 (BamHl, Smal, Xbal, EcoRl, Notl, Xmalll, Bglll, and Pstl cloning site; Invitrogen), pVL1392 (Bglll, Pstl, Notl, Xmalll, EcoRI, Xbal, Smal, and BamHl cloning site; Summers and Invitrogen), and pBluefi ⁇ cIII (BamHl, Bglll, Pstl, Ncol, and Hindlll cloning site, widi blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (if ⁇ mHl and Kpnl cloning site, in which the BamHl recognition
  • Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g. , any expression vector with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector, such as p ⁇ D (Pstl, Sa l, Sbal, Smal, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991).
  • DHFR dihydrofolate reductase
  • a glutamine synthetase/methionine sulfoximine co-amplification vector such as p ⁇ 14 (Hindlll, Xbal, Smal, Sbal, EcoR , and Bell cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech).
  • a vector that directs episomal expression under control of Epstein Barr Virus can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, N&el, Hindlll, Nhel, Pvull, and Kpnl cloning site, constimtive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHl, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull, and Kpnl cloning site, constimtive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, Hindlll, Notl, Xhol, Sfil, BamHl cloning site, inducible methallothionein Ila gene promoter, hygromycin selectable marker: Invitrog
  • Selectable mammalian expression vectors for use in the invention include pRc/CMV (Hindlll, BstXl, Notl, Sbal, and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (Hindlll, Spel, BstXl, Notl, Xbal cloning site, G418 selection; Invitrogen), and others.
  • Vaccinia virus mammalian expression vectors for use according to the invention include but are not limited to pSCl l (Smal cloning site, TK- and ⁇ -gal selection), pMJ601 (Sail, Smal, Afil, Narl, BspMU, BamHl, Apal, Nhel, Sacll, Kpnl, and Hindlll cloning site; TK- and ⁇ -gal selection), and pTKgptFIS (EcoRI, Pstl, Sail. Accl, Hindll, Sbal, BamHl, and Hpa cloning site, TK or XPRT selection).
  • Yeast expression systems can also be used according to the invention to express the Pax-6 protein.
  • the vectors of the present invention can be introduced into the desired host cells by methods described above.
  • Purification of Pax-6 can be performed using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g. , ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • PAGE polyacrylamide gel electrophoresis
  • 2-dimensional gel electrophoresis e.g. , ion exchange, affinity, immunoaffinity, and sizing column chromatography
  • centrifugation e.g. , ion exchange, affinity, immunoaffinity, and sizing column chromatography
  • differential solubility e.g. a specific embodiment, a Pax-6 fusion protein can be expressed.
  • a Pax-6 fusion protein comprises at least a functionally active portion of a non-Pax-6 protein joined via a peptide bond to at least a functionally active portion of a Pax-6 polypeptide.
  • the non-Pax-6 sequences can be amino- or carboxy-terminal to the Pax-6 sequences. More preferably, for stable expression of a proteolytically inactive Pax- 6 fusion protein, the portion of the non-Pax-6 fusion protein is joined via a peptide bond to the amino terminus of the Pax-6 protein.
  • a recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a functionally active portion of a non-Pax-6 protein joined in-frame to the Pax-6 coding sequence, and preferably encodes a cleavage site for a specific protease, e.g. , thrombin or Factor Xa, preferably at the Pax-6-non-Pax-6 juncture.
  • e fusion protein is expressed in Escherichia coli.
  • Labels Pax-6 and nucleic acids encoding Pax-6, as well as assorted reagents and markers employed to monitor Pax-6, and/or lens development, and/or neural development etc. may be appropriately labeled.
  • Suitable labels include enzymes, fluorophores (e.g. , fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu 3 + , to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g. , biotin), and chemiluminescent agents.
  • FITC fluorescene isothiocyanate
  • PE phycoerythrin
  • TR Texas red
  • rhodamine free or chelated lanthanide series salts, especially Eu 3 + , to name a
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, sl Cr, 57 Co, 58 Co, 59 Fe, ⁇ Y, 125 I, 131 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectropho tome trie, amperometric or gasometric techniques known in the art.
  • Direct labels are one example oflabels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation. e.g.- U.V. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • a phosphorylation site can be created on an antibody of the invention for labeling with 32 P, e.g. , as described in European Patent No. 0372707 (application No. 89311108.8) by Sidney Pestka, or U.S. Patent No. 5,459,240, issued October 17, 1995 to Foxwell et al.
  • Proteins, including antibodies, can be labeled by metabolic labeling. Metabolic labeling occurs during in vitro incubation of the cells that express the protein in die presence of culture medium supplemented with a metabolic label, such as [ 35 S]- methionine or [ 32 P]-orthophosphate.
  • die invention further contemplates labeling with [ 14 C]-amino acids and [ 3 H]-amino acids (wid the tritium substituted at non-labile positions).
  • a protein can be labeled with a FLAG-tag, an eight amino acid epitope [Pricket, et al., Biotechniques, 7:580-589 (1989)] as exemplified herein.
  • the present invention contemplates screens for an agent that acts by inducing d e expression of Pax-6 to selectively induce the formation of a lens, without inducing the formation of neural tissue.
  • cDNA expression screening can be performed, wherein Pax-6 expression is determined.
  • Namral effectors found in the ectodermal cell can be fractionated and tested using standard effector assays as exemplified herein, for example.
  • namral products libraries can be screened using the assays of the present invention for screening such agents.
  • Lens Replacement There are number of procedures for removing and/or replacing a lens from a vertebrate eye that are currently practiced by ophthalmologists. The skilled artisan would be able to choose an appropriate methodology for the insertion of a lens of the present invention which is relatively safe and simple, which minimizes trauma to the eye, and which results in materially improved vision under die particular conditions and circumstances of the individual case. Such methodologies are contemplated by the present invention. Listed below is but a small sampling of possible procedures that the skilled artisan could choose from, with the understanding that an ophthalmologist would be able to weigh the advantages and disadvantages accordingly.
  • U.S. Patent No. 4,253,199 discloses various methods and apparatus for eye implants widiin the posterior chamber of the eye. In the procedure, the entire anterior portion of the lens capsule is apparently removed and, in some cases, the posterior portion of the lens capsule was also removed. An implant of the capsule is attached to the ciliary body by means of sutures.
  • U.S. Patent No. 4,42,762 discloses an implant within the lens capsule, whereby a triangular opening is formed in the anterior capsule. Similarly, in U.S. Patent No. 4,243,510, the jagged margins represent an opening made in the lens capsule for fragmentation and removal of the lens.
  • U.S. Patent No. 4.251 ,887 discloses an intracapsular implant in which a triangular capsulectomy (mat is, a removal of a portion of the capsule) is performed and d ere is inserted within die eye a lends widi two slide loops, each of which forms the shape of a kidney.
  • U.S. Patent No. 4,888,015 discloses a method having as a first step, the displacement of the cornea and conjunctiva to provide access to the iris.
  • the iris is then positioned to provide access to die anterior lens capsule.
  • a generally horizontal capsulectomy incision is performed in the anterior lens capsule.
  • the superior portion of the capsular incision is folded back to expose the apex of die lens.
  • a first portion of a removal tool is inserted between the anterior lens capsule and the anterior surface of the lens while inserting a second portion of the removal tool between the posterior lens capsule and the posterior surface of the lens.
  • the lens is then grasped with the first and second portions of the removal tool and rotated to dislodge it from the capsule and partially remove it from the capsule.
  • the lens is then removed while the capsule is kept open.
  • a replacement lens is then inserted into the capsule, the tool is withdrawn from the capsule, and the superior capsule flap, the iris the cornea and the conjunctiva are all repositioned.
  • the tool for use in accordance with the foregoing method includes a tweezer body having a normally open position and first and second ends. Gripping means are mounted to the first and second ends, which gripping means are substantially circular and have concave inner surfaces facing each other and convex outer surfaces. The concave inner surfaces are constructed and arranged to grasp a lens.
  • Pax-6 directs induction of the lens.
  • Pax-6 can induce the expression of the lens-specific marker ⁇ Bl-crystallin without inducing the general neural marker NCAM.
  • Ectopic Pax-6 expression also results in the formation of ectopic lenses in whole embryos indicating that in vertebrates, as well as Drosophila [Haider, et al, Science, 267: 1788-1792 (1995)], Pax-6 can direct the development of major components of the eye. According to NCAM staining, ectopic lenses formed in the whole embryo are sometimes associated with neural tissue.
  • Cloning of Xenopus B1 crystallin Degenerate primers for RT-PCR amplification and cloning of ⁇ -crystallins were: upstream 5'-CGGGATCCCGGGA/GTAC/TTGA/GTANCCNCKA/GTA-3' and downstream 5 ' -GGAAATCTCG AGGGNGAA/GT AC/TCCNGNTGGGA-3 ' . Specific primers used for routine ⁇ Bl-crystallin RT-PCR were upstream, 5-TGCCTGGAGTGGAACAATGC -3 and downstream, 5-TGTTGAACCATCCCATAGCC -3.
  • Embryos were labeled according to established protocols [Hemmati-Brivanlou, et al, Development, 106:611-617 (1989); Hemmati-Brivanlou, et al. Development 110:325-30 (1990)].
  • Antisense probes for in situ hybridization were prepared using rUTP-digoxigenin (Boehringer Mannheim Biochemicals) from Xenopus ⁇ Bl-crystallin or Xenopus Pax-6 templates [Hirsch, et al., 1996, supra]. Antiserum to human ⁇ -crystallins (a gift from S. Zigler) was used at 1 : 1000 dilution.
  • Neural staining was detected using the supernatant of 6F11 hybridoma cells (Gift of W. A. Harris, UCSD) at a 1 : 1 dilution in PBT + 20% goat serum.
  • the neural staining seen in sections was performed on frozen sections that had been stained for ⁇ -crystallin prior to sectioning [Hemmati- Brivanlou, et al, Cell, 77:273-81 (1994); Wilson, et al, Curr Biol 4:676-86 (1994)].
  • the RT-PCR protocol and sequence of primers were performed as described earlier [Hemmati-Brivanlou et al , Cell 77:273-281 (1994)] .
  • a critical role for the transcription factor Pax-6 in development of the eye is indicated by the mutant phenotypes of eyeless in Drosophila, small eye in mouse and ANIRIDIA in man that are all caused by mutations in the Pax-6 gene [Quiring, et al, Science, 265:785-789 (1994)]. More striking is the demonstration that ectopically expressed Drosophila Pax-6 can induce d e formation of ectopic eyes in imaginal disks [Haider, et al, 1995, supra]. Consistent with a role in the earliest stages of vertebrate eye development, in Xenopus, Pax-6 is first expressed at midgastrula stage (stage 11.5).
  • Pax-6 expression is seen in a crescent-shaped region encompassing the border of the presumptive anterior neural plate and epidermis [Hirsch, et al, J. Neurobiol , 32:45-61 (1996)].
  • expression resolves into two laterally-placed areas coincident with the presumptive lens ectoderm.
  • Pax-6 expression coincides in time and space with all the previously proposed steps [Grainger, Trends Genet, 8:349-55 (1992)] of Xenopus lens development.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • RNA encoding Pax-6 was injected into Xenopus at the 2 cell stage and the consequences were examined in regard to the expression of a variety of tissue-specific markers including mesodermal, neural and lens in the context of ectodermal explants (animal caps).
  • Expression of Pax-6 RNA resulted in the induction of ⁇ Bl-crystallin in a dose-dependent fashion ( Figure IE, lanes 1-3).
  • the induction of the lens marker occurs in the absence of neural induction as die general neural marker NCAM [Kintner, et al, Development, 99:311-325 (1987)] was not induced ( Figure IE). Muscle actin.
  • Pax-6 could induce a lens-specific marker in embryonic explants
  • a test of whether Pax-6 had the same activity in the whole embryo was performed.
  • a tagged (FLAG-tag [Pricket, et al , Biotechniques, 7:580-589 (1989)]) version of Pax-6 was generated.
  • First the activity of this tagged version in the animal cap assay was tested, which demonstrated that the addition of the tag did not alter the ability of Pax-6 to induce expression of ⁇ Bl-crystallin ( Figure IE, lane 4, Figures 1G and 1H).
  • RNA encoding Pax-6-FLAG was injected into the animal poles of one of two blastomeres. Embryos were allowed to develop to tadpoles (stage 38) and stained as whole mounts for ⁇ -crystallin. Ectopic lenses were detectable both in whole mount embryos and in sections of whole mounts. When ectopic lenses were observed in an embryo, there were frequently many ( Figure 2A-2D) and mostly restricted to the anterior region of the embryo. The proportion of embryos with one or more ectopic lenses was optimally 47% at 0.12 ng of RNA injected (Table 1). In embryos assayed for ectopic lenses at earlier stages, endogenous lenses could be detected, but development of significant numbers of ectopic lenses was delayed.
  • This table includes data from two representative experiments showing the relationship between the dose of Pax-6 RNA used and the number of ectopic lenses induced in vivo.
  • the optic cup has a role later in lens development in enhancing lens fibre cell differentiation, in one such manner through the action of FGF [Chow, et al, Develop., 121:4383-4393 (1995); Robinson, et al, Development, 121:3959-3967 (1995)].
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION /desc - "oligonucleotide primer'
  • HYPOTHETICAL NO

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Abstract

La présente invention a trait à un cristallin de vertébré produit par provocation sélective du développement du cristallin dans des cellules ectodermiques de vertébrés. Les cristallins de vertébrés de la présente invention peuvent être produits ectopiquement de façon fiable, ce qui permet d'obtenir une source abondante de cristallins de vertébrés, utiles pour des transplantations de cristallin ainsi que pour d'autres actes médicaux. L'invention se réfère à des procédés d'utilisation de cristallins produits selon la présente invention. L'invention a également trait à des méthodes de traitement d'un vertébré présentant une malformation de voie de développement du cristallin, et à des compositions pharmaceutiques associées destinées à ces traitements. En outre, des procédés permettant d'identifier un agent qui provoque de façon sélective la formation d'un cristallin sans provoquer de formation de tissu neuronal sont également décrits.
PCT/US1998/008592 1997-05-01 1998-04-29 Cristallin de vertebre obtenu a partir d'une cellule ectodermique et procedes d'utilisation de celui-ci WO1998048829A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999057294A1 (fr) * 1998-05-07 1999-11-11 The Rockefeller University Elements de regulation transcriptionnelle du cristallin et procedes d'utilisation de ces elements
US6337392B1 (en) 1998-05-07 2002-01-08 The Rockefeller University Lens transcriptional control elements and methods of use thereof
EP1200133A1 (fr) * 1999-07-12 2002-05-02 Walter Gehring Manipulation de type de tissu ou d'organe par utilisation de la voie notch
US8084258B2 (en) 1999-07-12 2011-12-27 University Of Basel Manipulation of tissue of organ type using the notch pathway

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALTMANN C R ET AL: "Lens induction by Pax -6 in Xenopus laevis.", DEVELOPMENTAL BIOLOGY, (1997 MAY 1) 185 (1) 119-23, XP002075159 *
CVEKL A. ET AL: "Lens development and crystallin gene expression: many roles for Pax-6", BIOESSAYS, vol. 18, no. 8, August 1996 (1996-08-01), pages 621 - 630, XP002075157 *
GRINDLEY J C ET AL: "The role of Pax -6 in eye and nasal development.", DEVELOPMENT, (1995 MAY) 121 (5) 1433-42, XP002075158 *
MIZUNO M ET AL: "Pax -6 gene expression in newt eye development.", DEVELOPMENT GENES AND EVOLUTION 207 (3). 1997. 167-176, XP002075160 *
WILLIAMS, SONYA C. ET AL: "A highly conserved lens transcriptional control element from the Pax -6 gene", MECH. DEV. (1998), 73(2), 225-229, XP002075161 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999057294A1 (fr) * 1998-05-07 1999-11-11 The Rockefeller University Elements de regulation transcriptionnelle du cristallin et procedes d'utilisation de ces elements
US6337392B1 (en) 1998-05-07 2002-01-08 The Rockefeller University Lens transcriptional control elements and methods of use thereof
EP1200133A1 (fr) * 1999-07-12 2002-05-02 Walter Gehring Manipulation de type de tissu ou d'organe par utilisation de la voie notch
EP1200133A4 (fr) * 1999-07-12 2003-05-28 Walter Gehring Manipulation de type de tissu ou d'organe par utilisation de la voie notch
EP1772514A2 (fr) * 1999-07-12 2007-04-11 Walter Gehring Manipulation de type de tissu ou d'organe par utilisation de la voie Notch
EP1772514A3 (fr) * 1999-07-12 2007-06-20 Walter Gehring Manipulation de type de tissu ou d'organe par utilisation de la voie Notch
US8084258B2 (en) 1999-07-12 2011-12-27 University Of Basel Manipulation of tissue of organ type using the notch pathway

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