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WO1998030677A1 - CATARACTOGENESE ET INTERRUPTION DU GENE DE CONNEXINE α3 CHEZ DES MAMMIFERES, ET PROCEDES D'UTILISATION ASSOCIES - Google Patents

CATARACTOGENESE ET INTERRUPTION DU GENE DE CONNEXINE α3 CHEZ DES MAMMIFERES, ET PROCEDES D'UTILISATION ASSOCIES Download PDF

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Publication number
WO1998030677A1
WO1998030677A1 PCT/US1998/000340 US9800340W WO9830677A1 WO 1998030677 A1 WO1998030677 A1 WO 1998030677A1 US 9800340 W US9800340 W US 9800340W WO 9830677 A1 WO9830677 A1 WO 9830677A1
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WIPO (PCT)
Prior art keywords
ice
lens
connexin
inhibitor
gene
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PCT/US1998/000340
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English (en)
Inventor
Norton B. Gilula
Xiaohua Gong
Nalin M. Kumar
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The Scripps Research Institute
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Priority to AU60184/98A priority Critical patent/AU6018498A/en
Publication of WO1998030677A1 publication Critical patent/WO1998030677A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the invention relates to cataratogenesis and the disruption of 3 connexin gene. More specifically, the invention concerns the insertion of an exogenous disrupted 3 connexin gene into the genomic DNA of a non-human mammals resulting in the lack of 3 connexin protein, the absence of which leads to age-related cataract formation.
  • the invention further relates to vector constructs containing the disrupted ⁇ 3 connexin gene. Methods of making and using the ⁇ 3 disrupted connexin gene for preparing mammals containing mutant ⁇ 3 alleles that result in the absence of functional 3 connexin protein are described. Screening methods are also described using such mammals to study cataract biology.
  • the invention also relates to in vitro and in vivo screening methods to identify compounds useful in preventing or treating cataracts. This invention further relates generally to the prevention of cataract formation or the treatment of existing cataracts with interleukin-l ⁇ converting enzyme (ICE) inhibitors.
  • ICE interleukin-l ⁇ converting enzyme
  • the present invention now provides an in vivo animal model system and compositions to produce such for studying the role of ⁇ 3 connexin protein in lens development and for identifying drugs useful in the treatment of cataracts that develop postnatally in ⁇ 3-connexin-deficient mammals.
  • the invention provides a non-human mammal, preferably a mouse, and its progeny either containing a nonfunctional ⁇ 3 connexin protein or lacking functional ⁇ 3 connexin protein altogether.
  • the resultant non-human mammal is referred to as a ⁇ 3 connexin-deficient non-human mammal.
  • the ⁇ 3 connexin deficiency is generated by the insertion of a nucleic acid sequence comprising at least a portion of the ⁇ 3 connexin gene into the genome of the mammal .
  • the inserted ⁇ 3 connexin nucleic acid sequence present in the genome is referred to as a disrupted c ⁇ connexin gene.
  • a disrupted ⁇ 3 connexin gene lacks at least one transmembrane- encoding region and more preferably two regions and most preferably four regions.
  • an ⁇ 3 connexin gene lacks one or more nucleotides in the protein coding region, the presence of which results in a nonfunctional ⁇ 3 connexin protein.
  • an 3 connexin gene contains one or more nucleotide substitutions, the presence of which also results in a nonfunctional 3 connexin protein.
  • the invention provides a nucleic acid sequence corresponding to the above-described disrupted ⁇ 3 connexin gene operably linked to a plasmid vector.
  • a preferred plasmid comprises a nucleic acid sequence that is an ⁇ 3 connexin gene lacking four transmembrane domains.
  • An 3 connexin gene- containing plasmid further comprises at least a selectable marker, preferably the gene encoding neomycin resistance.
  • an ⁇ 3 connexin gene-containing plasmid also comprises a marker gene for use in detecting the presence of an o;3 mutant allele in a mammal of this invention.
  • a preferred marker gene encodes lacZ .
  • a preferred disrupted ⁇ 3 connexin gene-containing plasmid is the plasmid, designated p ⁇ 3-GZK, on deposit with American Type Culture Collection having ATCC Accession Number 97848, the deposit of which is further described below.
  • the invention provides an embryonic stem cell, preferably a JS-1 or an ES cell, containing a disrupted ⁇ 3 connexin gene.
  • ICE interleukin-l ⁇ converting enzyme
  • One preferred approach is based on the known cleavage pattern of a crystallin lens protein, preferably ⁇ , by ICE or functional ICE analog.
  • An inhibitor of ICE or an analog is then identified by detecting an alteration in the cleavage pattern of the lens protein. In other words, an inhibitor is identified by detecting an uncleaved substrate that is normally cleaved in the absence of an inhibitor.
  • Figure ID shows the genotypes of the ⁇ 3 'knockout' mice that were determined also by PCR, using three primers termed PI, P2 and P3.
  • PI three primers termed PI, P2 and P3.
  • a 350 bp PCR fragment was generated from the wild- type 0(3 allele, the size predicted to result from the PI and P2 primers, and a 500 bp fragment from the mutant ⁇ 3 allele was generated as predicted from using PI and P3 primers.
  • Figure IE shows total RNAs that were isolated from 3 (+/+) , ⁇ 3 (+/-) and o(3 (-/-) lenses from one month old mice, and analyzed by Northern blot using a cDNA probe covering the encoding DNA sequence for the four transmembrane domains of ⁇ 3 connexin. Fifteen ⁇ g RNA was loaded in each lane. The equal intensity of the rRNAs confirms that each lane has an equivalent amount of RNA.
  • Figure 2 shows the expression pattern of the lacZ-nls gene and lens development .
  • FIG. 2B a section along the equatorial plane of an eye from an 0(3 (-/-) embryo at stage 14 dpc, after whole mount lacZ staining, contains blue stained lens, surrounded by a circle of retinal pigmented epithelium.
  • Figure 3C shows a frozen section, along the anterior- posterior axis from the eye of a 2 week old ⁇ 3 (-/-) mouse, was stained for lacZ-nls activity.
  • the nuclei of the newly formed fiber cells contained a strong blue staining, which gradually disappeared towards the interior fibers.
  • a weak blue staining was also observed in the nuclei of the lens epithelial cells located at the anterior surface of the lens (indicated by arrows) .
  • Figure 2D is a histological section of the eye from an 3 (+/+) embryo at stage 18.5 dpc.
  • Figure 2E is a histological section of the eye from an 3 (-/-) embryo at stage 18.5 dpc.
  • Figure 3 illustrates the nuclear cataract phenotype of 3 (-/-) mice.
  • Figure 3A contains the lenses (unfixed) dissected from fresh eyes of 0(3 (+/+) mice and ⁇ 3 (-/-) mice at the age of two months, and photographed from their anterior surface with a dissection microscope.
  • Figure 3B contains a side view (anterior side of the lens on the right and posterior side on the left) of similar lenses embedded in LR white resin after fixation with 2.5% glutaraldehyde .
  • the black staining on the lens surface is the retinal pigmented epithelial cells attached to the lenses.
  • Nuc represents a 2X enlargement of the nuclear region of the cataract lens .
  • a large aggregate associated with the cataract is indicated by an arrow in upper right panel.
  • the scale bar is 1 mm.
  • Figure 4 illustrates the histological and immunocytochemical analysis of adult mice.
  • Figure 4A are histological sections from the anterior bow region (left) and the nuclear region (right) of the same cataract lens embedded in LR white resin shown in Figure 3B.
  • the arrow denotes an aggregate of undefined composition in the WO 98/30677 - 9 - PCTVUS98/00340
  • the sections were stained with methylene blue.
  • the scale bar is 10 ⁇ m.
  • Figure 6C shows the elution profiles of the total water soluble crystallins from the lenses of ⁇ 3 (+/+) and ⁇ 3 (-/-) mice at the age of 4 months .
  • Figure 6D shows the elution profiles of the total water soluble crystallins from the cortical regions of the lenses of 0(3 ( +/+) and ⁇ 3 (-/-) mice at the age of 4 months.
  • Figure 7 illustrates the electrophoretic characterization of lens NaOH insoluble proteins and crystallins.
  • Figure 7A a Coomassie blue stained gel is shown of the NaOH- insoluble proteins from the lenses of ⁇ 3 (+/+) (lane 1 and 2) , ⁇ 3 (+/-) (lane 3 and 4) and ⁇ 3 (-/-) (lane 5 and 6) littermates at the age of 2 months. Both lenses from a single mouse were used to prepare the NaOH-insoluble proteins; these proteins were divided into two equal portions, which were dissolved in an equal volume of sample buffer, with and without 10 mM DTT. Equal volumes of the samples were WO 98/30677 - 11 - PCTVUS98/00340
  • Figure 7B shows the comparison of crystallin protein profiles in the lenses of ⁇ 3 (-/-) and ⁇ 3 (+/+) mice by Western blot analysis.
  • Protein extracts from ⁇ 3 (+/+) lenses (lanes 1-4) or ⁇ 3 (-/-) lenses (lanes 5-8) were subjected to SDS-PAGE and Western blotting. All samples were analysed in sample buffer containing 10 mM DTT.
  • Lanes 1 and 5 contained the total lens cortical protein; lanes 2 and 6 contained the water-soluble fraction from the lens nucleus; lane 3 and 7 contained the NaOH-soluble fraction from the lens nucleus; and lanes 4 and 8 contained the NaOH-insoluble fraction from the lens nucleus.
  • Knockout A partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell.
  • Knockout Construct A nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell.
  • the nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell .
  • Murine Includes any and all members of the family Muridae, including rats and mice.
  • Progeny or Offspring Includes any and all future generations derived and descending from a particular mammal, i.e., a mammal containing a knockout construct inserted into its genomic DNA.
  • progeny of any successive generation are included herein such that the progeny, the FI, F2 , F3 generations and so on indefinitely are included in this definition.
  • Analog Refers to a molecule substantially similar in function to interleukin-l ⁇ converting enzyme (ICE) .
  • Homolog refers to a molecules that is structurally or functionally equivalent to a molecule of the present invention.
  • Therapeutically Effective Amount refers to an amount effective in treating or ameliorating a cataract condition in a subject characterized by growth including formation, progression and regression.
  • Prophylactically Effective Amount refers to an amount effective in preventing the formation of a cataract.
  • the present invention is based in part on the ability to decrease or completely suppress the level of expression of a particular gene in a mammal by introducing into the genomic DNA of that mammal a new exogenous DNA sequence that serves to interrupt some portion of the endogenous DNA sequence to be suppressed. Another term for this type of suppression is "knockout".
  • the exogenous nucleic acid is also referred to as a "knockout construct" .
  • the knockout construct in this case the plasmid or expression vector referred to as ⁇ 3 connexin targeting vector or pcx3-GZK described below and in the Examples, is first prepared and then inserted into an embryonic stem cell in which the construct subsequently becomes integrated into that cells' genomic DNA, usually by the process of homologous recombination.
  • the embryonic stem cell containing the exogenous DNA is then subjected to selection methods as described in the Examples, for example, by selection for neomycin resistance. Selected resistant cells are then analyzed for the presence of a mutant allele.
  • An embryonic stem cell containing a mutant allele is then injected into a blastocyst that is implanted into the uterus of a pseudopregnant foster mother for integration into a developing embryo .
  • Offspring that are born to the foster mother are then screened for the presence of the mutant allele, for example, as is described more completely in the Examples.
  • the mutant allele is present in the germ line allowing for the generation of homozygous mutant animals from crossing heterozygotic animals containing the desired mutant allele.
  • the invention provides a mammal, preferably a mouse, and its progeny either containing a nonfunctional 3 connexin protein or lacking functional ⁇ 3 connexin protein altogether.
  • the resultant mammal is referred to as a c ⁇ connexin-deficient mammal.
  • the ⁇ 3 connexin deficiency is generated by the insertion of a nucleic acid sequence comprising at least a portion of the ⁇ 3 connexin gene into the genome of the mammal .
  • the inserted ⁇ 3 connexin nucleic acid sequence present in the genome is referred to as a disrupted ⁇ 3 connexin gene .
  • Exemplary disrupted ⁇ 3 connexin gene nucleic acid sequences from non-human mammals are discussed in Example 1.
  • a disrupted ⁇ 3 connexin gene lacks at least one transmembrane-encoding region and more preferably two regions and most preferably four regions.
  • an c(3 connexin gene lacks one or more nucleotides in the protein coding region, the presence of which results in a nonfunctional ⁇ 3 connexin protein.
  • an ⁇ 3 connexin gene contains one or more nucleotide substitutions, the presence of which also results in a nonfunctional ⁇ 3 connexin protein.
  • the invention provides a nucleic acid sequence corresponding to the above-described disrupted 3 connexin gene operably linked to a plasmid vector also referred to as a targeting vector.
  • a preferred plasmid comprises a nucleic acid sequence that is an 3 connexin gene lacking four transmembrane domains.
  • An ⁇ 3 connexin gene- containing plasmid further comprises at least a selectable marker, preferably the gene encoding neomycin resistance.
  • an 3 connexin gene-containing plasmid also comprises a marker gene for use in detecting the presence of an ⁇ 3 mutant allele in a mammal of this invention.
  • a preferred marker gene encodes lacZ.
  • a preferred disrupted ⁇ 3 connexin gene-containing plasmid is the plasmid, designated p ⁇ 3-GZK, on deposit with American Type Culture Collection having ATCC Accession Number 97848, the deposit of which is further described below and in Example 4.
  • the invention provides an embryonic stem cell, preferably a JS-1 or an ES cell, containing a disrupted ⁇ 3 connexin gene.
  • Exemplary expression systems comprising suitable vectors and host cells for use in the present invention are described in Example 1 and further supported by ICE-related US Patents 5,552,536, 5,578,705 and 5,654,146, the disclosures of which are hereby incorporated by reference.
  • the non-human mammals of this invention homozygous for the disrupted ⁇ 3 connexin gene developed nuclear cataracts at the age of 2 to 3 weeks.
  • Gap junctions containing ⁇ 3 connexin were found in the homozygous non-human mammals, but they could not compensate for the functional loss of 0(3 connexin in the lens nucleus.
  • the nuclear cataracts resulted from light scattering of high molecular weight aggregates formed by the lens proteins linked together via disulfide bonds.
  • the ⁇ 3 connexin gene is shown to be essential for providing a cell-cell signaling pathway or structural component for maintaining the organization of lens membrane and cytoplasmic proteins that is required for normal lens transparency. Further, the invention provides the discovery that a loss of ⁇ 3 connexin initiates a process in the lens that may involve an apoptosis pathway.
  • compositions and methods to Prevent or Treat Cataracts with Inhibitors of Interleukin-l ⁇ Converting Enzyme Activity The discovery of the ⁇ -crystallin degradation in the lenses of the ⁇ 3 connexin gene-disrupted non-human mammals of the present invention provides for therapeutic compositions and methods of use thereof for affecting growth of cataracts in a subject mediated by loss of ⁇ 3 protein, nonfunction of o;3 protein or a degradation of critical crystallin lens proteins.
  • ICE interleukin-l ⁇ converting enzyme
  • a cataract growth affecting amount of an inhibitor of this invention is any amount that is effective at inhibiting the formation of cataracts in a subject predisposed to cataract formation, such as in the 3 connexin-deficient mammal described herein.
  • the inhibitor acts prophylactically at preventing cataract formation.
  • a cataract growth affecting amount is any amount that is effective at altering the progression of cataract formation including delaying the complete formation of a cataract, inhibiting the complete formation of a cataract, and reversing a progressing or a completely formed cataract .
  • An inhibitor of the present invention is one that functions to inhibit the degradation of the lens crystallin proteins, preferably ⁇ -crystallin lens protein. Accordingly, inhibitory compounds of this invention are capable of targeting and inhibiting degradation of lens proteins, the degradation of which plays a critical role in cataract development. Such inhibitors include but are not limited to inhibitors of interleukin-l ⁇ converting enzyme (ICE) and analogs thereof.
  • ICE interleukin-l ⁇ converting enzyme
  • ICE inhibitors are well known to those of skill in the art and include but are not limited to Bcl2, Bcl-XL, CrmA (cowpox virus protein) , ZVAD-fluromethyIketone (a permeant ICE inhibitor) , lactacystin, dichloroisocourmarin, peptides Ac-YVAD-CMK and CPP32/Yama, and DEVD-CHO, and those described in US Patents 5,656,627, 5,552,536, 5,672,500 and WO 98/30677 - 19 - PCTYUS98/00340
  • the candidate inhibitor is administered orally, intravenously, topically, or the like routes.
  • the compound is preferably providied in association with an opthalmalogically acceptable carrier.
  • opthamalogical modalities are provided in US Patents 5,401,880, 5,422,376, 5,519,054, 5,578,578 and 5,628,801, the disclosures of which are hereby incorporated by reference.
  • the effect of the inhibitor is evaluated most easily by direct examination of the lens optometrically.
  • the compounds of this invention may be employed in a conventional manner for the treatment of cataractogenesis mediated by the disruption of 3 connexin gene, loss or nonfunction of ⁇ 3 connexin protein and degradation of lens proteins.
  • Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques.
  • a compound of this invention may be combined with an acceptable pharmaceutical carrier or an opthalmalogically acceptable carrier for a time period and in an amount effective to affect cataract growth in a subject.
  • the inhibitors of this invention may be used in compositions and methods for treating or protecting a subject against cataractogenesis over extended periods of time.
  • An inhibitor may be employed in such compositions either alone or together with other WO 98/30677 - 20 - PCT7TJS98/00340
  • compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically/opthalmalogically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle.
  • pharmaceutically acceptable As used herein, the terms “pharmaceutically acceptable”, “opthalmalogically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as burning, irritation, shock, nausea, dizziness, gastric upset and the like.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation.
  • Pharmaceutically/opthalmalogically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride-mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxy- methylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum album
  • compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the preferred route is topically directly to the eye for either pharmaceutical or opthalmalogical formulations.
  • Such formutions are well known in the art using suitable dispersing or wetting agents and suspending agents.
  • the formulations are preferably sterile solutions or suspensions in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol .
  • acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides .
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • a therapeutically effective amount of large molecule inhibitors of this invention is typically an amount such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.01 microgram ( ⁇ g) per milliliter (ml) to about 100 ⁇ g/ml, preferably from about 1 ⁇ g/ml to about 5 ⁇ g/ml, and usually about 5 ⁇ g/ml.
  • the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days .
  • a therapeutically effective amount of a small molecule inhibitors of this invention is typically an amount of polypeptide such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 microgram ( ⁇ g) per milliliter (ml) to about 200 ⁇ g/ml, preferably from about 1 ⁇ g/ml to about 150 ⁇ g/ml.
  • the preferred plasma concentration in molarity is from about 2 micromolar ( ⁇ M) to about 5 millimolar (mM) and preferably about 100 ⁇ M to 1 mM polypeptide antagonist.
  • the dosage per body weight can vary from about 0.1 mg/kg to about 300 mg/kg, and preferably from about 0.2 mg/kg to about 200 mg/kg, in one or more dose administrations daily, for one or several days.
  • the discoveries of the present invention also provide a variety of screening methods both in vivo and in vi tro .
  • the screening methods are useful in identifying inhibitors of ICE or ICE-like activity occurring in the lens that may be responsible for the generation of cataract growth.
  • a screening method to identify drugs that block, diminish or reverse the development or severity of cataracts relies on using the ⁇ 3 connexin- deficient mouse model of this invention.
  • This method involves the administration of a candidate drug over a range of doses to the mouse by various routes of delivery, preferably by eye drops or intravenously. Exemplary teachings are in Example 2.
  • the effect of a compound on a lens is also assessed through an in vi tro system in which the lens of the mouse described above is cultured according to methods well known in the art for explant organ culture.
  • the compound is directly applied to the medium in which the lens is cultured. Effects on opacification are assessed visually, morphologically or biochemically as previously described.
  • the present invention also describes methods to identify an inhibitor of interleukin-l ⁇ converting enzyme (ICE) or functional ICE analog.
  • Inhibitors are identified with a variety of approaches.
  • Two exemplary methods utilize the non- human mammal of the present invention and the discovery of the degradation pattern of the ⁇ -crystallin lens protein as a result of the ⁇ 3 connexin gene disruption in the non-human mammal.
  • a ⁇ -crystallin lens protein is contacted with a solution of ICE or functional ICE analog to form a mixture in the presence of a candidate inhibitor of ICE or functional ICE analog under conditions described in Example 1 for assessing cleavage patterns of ⁇ -crystallin.
  • the effect of the inhibitor is then assessed by comparing the cleavage patterns of untreated and treated samples.
  • An effective inhibitor is identified with the presence of an altered cleavage pattern compared to that of undegraded ⁇ -crystallin.
  • the lens protein is in either purified or unpurified form.
  • the ICE or functional ICE analog is either commericially obtained or is providedin a homogenate of lens isolated from the non-human mammal as described in Example 1.
  • ICE substrates include y- crystallin and precursor interleukin-l ⁇ (pIL-l ⁇ ) and are described in US Patents 5,607,831 and 5,656,627, the disclosures of which are hereby incorporated by reference.
  • the substrate is mixed with a homogenate of lens isolated from the non-human mammal prepared in Example 1.
  • the homogenate contains ICE or functional ICE analog as described in Example 1 that results in the novel cleaveage pattern of ⁇ -crystallin.
  • the above mixture is prepared in the presence of a candidate inhibitor of ICE or functional ICE analog under conditions where ICE or functional ICE analog is known to cleave the substrate generating detectable cleavage products.
  • the cleavage pattern obtained with the test compound is compared to that with a control thereby identifying the effectiveness of the test compound as an inhibitor of the novel protease in the lens.
  • Exemplary teachings of screening methods applicable to the present invention are provided in US Patents 5,656,627.
  • the present invention contemplates a kit containing packaging means that include a first container containing a ⁇ -crystallin lens protein in an amount sufficient for at least one assay, and further including a packaging material comprising a label indicating that the crystallin lens protein can be used to identify the inhibitor by detection of an altered cleavage pattern.
  • a kit containing packaging means that include a first container containing a ⁇ -crystallin lens protein in an amount sufficient for at least one assay, and further including a packaging material comprising a label indicating that the crystallin lens protein can be used to identify the inhibitor by detection of an altered cleavage pattern.
  • Exemplary teachings of the cleavage patterns are provided in the Examples.
  • the enzymatic component of the kit is further provided in the form of a second container having ICE or functional ICE analog that is either commerically available or is present in a homogenate of lens isolated from a non-human mammal of this invention. Preparation of such homogenates are detailed in the Examples
  • the invention further contemplates a kit for identifying an inhibitor of interleukin-l ⁇ converting enzyme (ICE) or functional ICE analog in which a substrate, preferably a ⁇ - crystallin lens protein, of ICE or functional homolog and a homogenate of lens isolated from a non-human mammal of this invention having disrupted alleles of 3 connexin gene are provided in separate containers .
  • the kit further contains a packaging material comprising a label indicating that the crystallin lens protein can be used to identify the inhibitor by detection of an altered cleavage pattern.
  • various regions of the ⁇ 3 connexin protein can be deleted at the nucleotide level thereby interfering with the ability of the protein to perform its intended function as a gap juntion forming protein.
  • the transmembrane domains of 3 connexin protein are thought to be critical for the creation of functional gap junctions.
  • four TMDs exist in ⁇ 3 connexin protein.
  • a disrupted ⁇ 3 connexin gene encoding a nonfunctional 3 connexin protein is generated by deletion of at least one TMD.
  • the disruption encompasses at least two TMDs and more preferably, four TMDs as described below.
  • the oligo deoxyribonucleotide 5'- CCCAGGCTCTACCTCAGGTT3 ' (SEQ ID NO 2) was used as a 5' primer for detecting both the wild type allele and the ⁇ 3 knockout allele;
  • the oligo deoxyribonucleotide (5'- CTTTGCCGATGACTGTAGAG-3 ' (SEQ ID NO 3) was used as the 3' primer for detecting the wild type gene allele;
  • the oligo deoxyribonucleotide (5 ' -CAGGGTTTTCCCAGTCACGAC-3 ' (SEQ ID NO 4) from the lacZ gene sequence was used as the 3' primer for detecting the 3 gene disrupted allele.
  • the effect of the inhibitor is evaluated most easily by direct examination of the lens optometrically.

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Abstract

Cette invention concerne la cataractogénèse liée à l'interruption d'un gène de connexine α3 endogène et à la dégradation des protéines du cristallin plus particulièrement des protéines de cristallin η. D'une manière plus spécifique cette invention concerne la production d'un mammifère non humain dans lequel l'expression de la protéine de connexine α3 est interrompue par l'insertion dans l'ADN génomique d'un mammifère non humain d'un gène de connexine α3 interrompu exogène, ceci produisant un mammifère non humain à qui il manque la protéine de connexine α3 fonctionnelle ou la protéine de connexine α3 dont l'absence conduit à la formation d'une cataracte liée à l'âge. Cette invention concerne également des compositions et des procédés de production et d'utilisation de ces dernières pour le diagnostic et la thérapie des cataractes.
PCT/US1998/000340 1997-01-10 1998-01-09 CATARACTOGENESE ET INTERRUPTION DU GENE DE CONNEXINE α3 CHEZ DES MAMMIFERES, ET PROCEDES D'UTILISATION ASSOCIES WO1998030677A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60184/98A AU6018498A (en) 1997-01-10 1998-01-09 Cataractogenesis and disruption of d3 connexin gene in mammals and methods of use

Applications Claiming Priority (4)

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US3473797P 1997-01-10 1997-01-10
US60/034,737 1997-01-10
US4651897P 1997-05-15 1997-05-15
US60/046,518 1997-05-15

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WO1998030677A1 true WO1998030677A1 (fr) 1998-07-16

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040667A1 (fr) * 2000-08-25 2002-05-23 Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences Procede pour diagnostiquer et traiter des lesions du cristallin a l'aide du gene crygs et d'un produit codant de celui-ci
US7247208B2 (en) 2001-07-09 2007-07-24 Mallinckrodt Baker, Inc. Microelectronic cleaning compositions containing ammonia-free fluoride salts
US7393819B2 (en) 2002-07-08 2008-07-01 Mallinckrodt Baker, Inc. Ammonia-free alkaline microelectronic cleaning compositions with improved substrate compatibility

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Title
BRUZZONE R., WHITE T. W., PAUL D. L.: "CONNECTIONS WITH CONNEXINS: THE MOLECULAR BASIS OF DIRECT INTERCELLULAR SIGNALING.", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 238., 1 January 1996 (1996-01-01), GB, pages 01 - 27., XP002913288, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1996.0001q.x *
CAPECCHI M. R.: "TARGETED GENE REPLACEMENT.", SCIENTIFIC AMERICAN., SCIENTIFIC AMERICAN INC., NEW YORK, NY., US, vol. 270., no. 03., 1 March 1994 (1994-03-01), US, pages 34 - 41., XP002913290, ISSN: 0036-8733 *
GONG X. H., ET AL.: "DISRUPTION OF ALPHA 3 CONNEXIN GENE LEADS TO AGE-RELATED CATARACT FORMATION IN MICE.", MOLECULAR CELL BIOLOGY, XX, XX, no. 07., 7 December 1996 (1996-12-07), XX, pages 509A., XP002913289 *
GONG X., ET AL.: "DISRUPTION OF ALPHA3 CONNEXIN GENE LEADS TO PROTEOLYSIS AND CATARACTOGENESIS IN MICE.", CELL, CELL PRESS, US, vol. 91., 12 December 1997 (1997-12-12), US, pages 833 - 843., XP002913292, ISSN: 0092-8674, DOI: 10.1016/S0092-8674(00)80471-7 *
NELLES E., ET AL.: "DEFECTIVE PROPAGATION OF SIGNALS GENERATED BY SYMPATHETIC NERVE STIMULATION IN THE LIVER OF CONNEXIN32-DEFICIENT MICE.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93., 1 September 1996 (1996-09-01), US, pages 9565 - 9570., XP002913286, ISSN: 0027-8424, DOI: 10.1073/pnas.93.18.9565 *
PAUL D. L., ET AL.: "CONNEXIN46, A NOVEL LENS GAP JUNCTION PROTEIN, INDUCES VOLTAGE- GATED CURRENTS IN NONJUNCTIONAL PLASMA MEMBRANE OF XENOPUS OOCYTES.", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 115., no. 04., 1 November 1991 (1991-11-01), US, pages 1077 - 1089., XP002913285, ISSN: 0021-9525, DOI: 10.1083/jcb.115.4.1077 *
REAUME A. G., ET AL.: "CARDIAC MALFORMATION IN NEONATAL MICE LACKING CONNEXIN43.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 267., 24 March 1995 (1995-03-24), US, pages 1831 - 1834., XP002913287, ISSN: 0036-8075, DOI: 10.1126/science.7892609 *
WESTPHAL H.: "TRANSGENIC MAMMALS AND BIOTCHNOLOGY.", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 03., 1 January 1989 (1989-01-01), US, pages 117 - 120., XP002913291, ISSN: 0892-6638 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040667A1 (fr) * 2000-08-25 2002-05-23 Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences Procede pour diagnostiquer et traiter des lesions du cristallin a l'aide du gene crygs et d'un produit codant de celui-ci
US7662552B2 (en) 2000-08-25 2010-02-16 Shanghai Institutes Of Biological Science, Chinese Academy Of Sciences Method of diagnosing and treating lesion of crystalline lens using human CRYGS gene and coding product thereof
US7247208B2 (en) 2001-07-09 2007-07-24 Mallinckrodt Baker, Inc. Microelectronic cleaning compositions containing ammonia-free fluoride salts
US7718591B2 (en) 2001-07-09 2010-05-18 Mallinckrodt Baker, Inc. Microelectronic cleaning compositions containing ammonia-free fluoride salts for selective photoresist stripping and plasma ash residue cleaning
US7393819B2 (en) 2002-07-08 2008-07-01 Mallinckrodt Baker, Inc. Ammonia-free alkaline microelectronic cleaning compositions with improved substrate compatibility

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