EP1135518A4 - Animaux transgeniques servant de bioreacteurs urinaires pour la production de proteines dans l'urine, constructions d'adn recombine pour l'expression specifique du rein, et procedes d'utilisation de ces dernieres - Google Patents

Animaux transgeniques servant de bioreacteurs urinaires pour la production de proteines dans l'urine, constructions d'adn recombine pour l'expression specifique du rein, et procedes d'utilisation de ces dernieres

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Publication number
EP1135518A4
EP1135518A4 EP99958952A EP99958952A EP1135518A4 EP 1135518 A4 EP1135518 A4 EP 1135518A4 EP 99958952 A EP99958952 A EP 99958952A EP 99958952 A EP99958952 A EP 99958952A EP 1135518 A4 EP1135518 A4 EP 1135518A4
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Prior art keywords
uromodulin
promoter
kidney
urine
gene
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EP99958952A
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German (de)
English (en)
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EP1135518A1 (fr
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Xue-Ru Wu
Tung-Tien Sun
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New York University NYU
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New York University NYU
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Publication of EP1135518A1 publication Critical patent/EP1135518A1/fr
Publication of EP1135518A4 publication Critical patent/EP1135518A4/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
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • TRANSGENIC ANIMALS AS URINARY BIOREACTORS FOR THE PRODUCTION OF PROTEIN IN THE URINE, RECOMBINANT DNA CONSTRUCT FOR KIDNEY-SPECIFIC EXPRESSION, AND METHOD OF USING SAME
  • the present invention relates to transgenic animals as urinary bioreactors for the expression and production of proteins in the urine.
  • the present invention further relates to a recombinant DNA construct for kidney-specific expression of proteins in the urine and to a method for producing such proteins in the urine.
  • Description of the Related Art Significant progress has recently been made in using transgenic animals as bioreactors to produce large quantity and high quality pharmaceuticals.
  • the overall strategy entails the use of tissue-specific promoters to drive the expression of genes encoding medically important molecules. When those molecules are expressed in the target tissue of transgenic animals and secreted into body fluids, they can be harvested, purified and used for treating human diseases.
  • the most notable example is the milk-based bioreactor system, taking advantage of mammary gland-specific gene promoters.
  • U.S. Patent No. 5,476,995 was one of the first patents directed to transgenic female sheep as milk- based bioreactors that expressed the transgene in the mammary gland so as to produce the target protein
  • a number of proteins have been produced in milk- based bioreactor systems, such as protein C (U.S. Patent No. 5,589,604), blood coagulation factors (U.S. Patent No. 5,322,775), fibrinogen (U.S. Patent No. 5,639,940), antibodies (U.S. Patent No. 5,625,126) and hemoglobin (U.S. Patent No. 5,602,306) , some of which are now being used in clinical trials.
  • a milk-based bioreactor system has several limitations. The first relates to its relatively low degree of cost-effectiveness. For instance, the lactation of transgenic livestock does not occur until an average of one and a half years old.
  • lactation only occurs in female animals and lasts for a limited period of time.
  • purification of target proteins from milk often requires the development of complicated purification schemes (Wilkins et al, 1992) .
  • leakage of biologically active proteins from the mammary gland into the blood stream commonly occurs with the possibility of leading to pathological conditions in transgenic animals.
  • Another potential bioreactor system that can circumvent some of the above-mentioned limitations is a urine- based system where urine is an easily collectable fluid from transgenic livestock animals.
  • This bioreactor system has been recently tested by Kerr and colleagues (1998) , among whom is one of the present inventors, in transgenic mice using a urothelium-specific promoter (uroplakin II promoter) to drive human growth hormone (hGH) expression and production. They found that hGH could indeed be found in the urine of these transgenic mice at a concentration of 0.1 mg/ml, indicating that the urothelium can serve as an alternate bioreactor.
  • uroplakin II promoter human growth hormone
  • the present invention provides a recombinant DNA molecule containing a kidney-specific promoter operably linked to a heterologous gene, which kidney-specific promoter is capable of expressing the heterologous gene in the kidney of a host animal to produce a recombinant biologically active protein in the urine .
  • the present invention also provides for a method for producing a recombinant biologically active protein in vivo using a urine-based bioreactor system in transgenic animals. Further provided are transgenic animals, all of whose somatic cells and preferably all of whose germ cells contain a recombinant construct or transgene from which a biologically active protein is produced in recoverable amounts in the urine.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a restriction digestion of five phage clones (lanes 1-5) on agarose gel electrophoresis .
  • M represents lanes of molecular weight markers .
  • Figure 2 shows a Southern blot corresponding to the agarose gel shown in Fig. 1 hybridized separately with each of the 5' -end, middle region, and 3 '-end probes.
  • Figure 3 shows an agarose gel electrophoresis of PCR reaction products using the sets of primers for the 5'- end, the middle region, and the 3 '-end of the uromodulin gene.
  • Figures 4A and 4B show agarose gel electrophoresis (Fig. 4A) of EcoRI restriction digests of genomic DNA from various animal species and Southern blot hybridization (Fig. 4B) of the restriction digested genomic DNA with the middle region probe .
  • Figure 5 is a schematic representation of the uromodulin gene structure in the human, bovine and rat genome.
  • the open boxes represent exons with the exon numbering provided, and the thick bars represent the introns, the lengths of which are variable.
  • Figure 6 shows Southern blot hybridization of BAC plasmid clone 1 digested with the restriction enzymes, Pstl (lane 4) , Apal (lane 6) , EcoRI (lane 7) , Sad (lane 8) , and Kpnl (lane 10) and hybridized separately with 5' -end, middle region and 3 '-end probes.
  • Figures 7A-7H show the nucleotide sequence of the mouse uromodulin promoter region (SEQ ID NO:l) which is 9,345 bp upstream of the first mouse uromodulin coding exon.
  • Figure 8 is a schematic presentation of the mouse uromodulin promoter in which the arrow denotes the transcription initiation site, the letters denote restriction sites (A, Apal; P, Pstl; B, BamHI; H, Hindlll; S, Spel), and the short bar denotes the relative size of the DNA.
  • Figure 9 shows the partial cDNA sequence of goat uromodulin gene (SEQ ID NO: 2) .
  • the location of primers AS14 , AS15 and AS17 used for isolation of goat uromodulin genomic DNA is shown in uromoduline.
  • Figure 10A and 10B show the nucleotide sequence of goat uromodulin gene intron 1 (Fig. 10A; SEQ ID NO: 3) and exon 3 (Fig. 10B, SEQ ID NO : 4 ) .
  • the present invention relates to the development of a bioreactor system in a transgenic mammal where a recombinant biologically active protein is to be produced and secreted into the urine by the kidney-specific expression of a heterologous gene under the direction of a kidney-specific promoter, such as the uromodulin promoter.
  • This urine-based mammalian bioreactor system is obtained by producing a transgenic mammal in which an isolated DNA molecule containing a recombinant construct or "transgene" for kidney- specific expression and production of the biologically active protein of interest is stably introduced.
  • any method known in the art for introducing a recombinant construct or transgene into an embryo such as microinjection, cell gun, transfection, liposome fusion, electroporation, and the like, may be used.
  • the most widely used method for producing transgenic animals is microinjection, which involves injecting a DNA molecule into the male pronucleus of fertilized eggs (Brinster et al, 1981; Costantini et al, 1981; Harbers et al, 1981; Wagner et al, 1981; Gordon et al, 1976; Stewart et al, 1982; Palmiter et al, 1983; Hogan et al, 1986; U.S.
  • the present invention for producing a biologically active protein in a urine-based mammalian bioreactor system is not limited to any one species of animal, but provides for any appropriate non-human mammal species.
  • mouse is a mammal species that is routinely used for producing transgenic animals and, thus, serves as a model system to test the transgene
  • farm animals such as pigs, sheep, goats, horses and cattle, which generate large quantities of urine, may be suitably used.
  • the success rate for producing transgenic animals by microinjection is highest in mice, where approximately 25% of fertilized mouse eggs into which the DNA has been injected, and which have been implanted in a female, will develop into transgenic mice.
  • the introduction of a DNA containing a transgene sequence at the fertilized oocyte stage ensures that the introduced transgene will be present in all of the germ cells and somatic cells of the transgenic animal.
  • the presence of the introduced transgene in the germ cells of the transgenic "founder" animal means that all of the founder animal's offspring will carry the introduced transgene in all of their germ cells and somatic cells.
  • any plasmid or viral sequences there is no need for incorporating, along with the gene being introduced, any plasmid or viral sequences (Jaenisch, 1988) , although the vector sequence may be useful in some instances. In many cases however, the presence of vector DNA has been found to be undesirable (Hammer et al, 1987; Chaka et al, 1985 and 1986; Kollias et al, 1986; Shani 1986; Townes et al, 1985) .
  • the transgene construct can be excised from the vector used to amplify the transgene in a microbial host by digestion with appropriate restriction enzymes.
  • the transgene is then recovered by conventional methods, such as electroelution followed by phenol extraction and ethanol precipitation, sucrose density gradient centrifugation, chromatography, HPLC, or combinations thereof. It has been reported in U.S. Patent No. 5,589,604 that high transformation frequencies, on the order of 20% or more, in both mice and pigs were obtained by microinjection with HPLC-purified DNA. In order for the introduced gene sequence to be capable of being specifically expressed in the kidney of the transgenic animal, the gene sequence must be operably linked to a kidney-specific promoter.
  • a DNA molecule is said to be "capable of expressing" a protein if it contains nucleotide sequences which contain cis-acting transcriptional regulatory information, and such sequences are “operably linked” to nucleotide sequences which encode the protein.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • the cis-acting regulatory regions needed for gene expression in general include a promoter region, and such regions will normally include those 5' -non- coding sequences involved with initiation of transcription.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the gene encoding a protein of interest is operably linked to a kidney-specific promoter to generate a recombinant construct or "transgene" that is then introduced into the fertilized embryo.
  • nucleotide sequences that encode the signal sequences that direct secretion of the expressed biologically active protein of interest into the urine of the transgenic animal.
  • Both endogenous and heterologous signal sequences can be used, although the endogenous signal sequence of the heterologous protein of interest is preferred.
  • other regulatory sequences in addition to the promoter such as enhancers, splice signals, ribosome binding sites and polyadenylation sites, etc., may be useful in the transgene construct.
  • Uromodulin also named Tamm-Horsfall protein
  • This -90 kDa glycoprotein has several important features that are relevant to its use in a kidney-expressed urine-based bioreactor system.
  • the protein is synthesized by the epithelial cells of the ascending limb of Henle's loop and the beginning portion of the distal convoluted tubule, delivered exclusively to apical membrane and secreted into the urine (Sikri et al, 1981; Bachmann et al, 1990) . Rindler et al
  • uromodulin is a cell surface protein anchored onto the apical plasma membrane via a glycosylphosphatidyl inositol (GPI) tail, where phospholipase C cleavage in vi tro of the GPI linkage completely releases the molecule into the culture medium.
  • GPI glycosylphosphatidyl inositol
  • Uromodulin is highly tissue-specific, being expressed only in the kidneys and not in any other epithelial and mesenchymal tissue. Moreover, uromodulin is evolutionarily conserved throughout placental animals.
  • the cDNA sequences reported for rat uromodulin (Fukuoka et al, 1992) and human uromodulin (Hession et al, 1987; Pennica et al, 1987) were found to be 91% and 77% identical with the mouse uromodulin cDNA sequence, respectively (Prasadan et al, 1995) .
  • Prasadan and colleagues (1995) also reported that an alignment of uromodulin amino acid sequences from mouse, rat and human showed 91% similarity and 86% identity between mouse and rat, and 79% similarity and 70% identity between mouse and man.
  • the present inventors have isolated and sequenced a 9,345 base pair region including about 7 Kb upstream of the coding region of the mouse uromodulin gene, which region contains the mouse uromodulin promoter.
  • This DNA promoter region, or a fragment thereof which retains the tissue specific promoter activity thereof, can be used for construction of a transgene with a biologically active protein of interest, i.e., human growth hormone (hGF) .
  • hGF human growth hormone
  • uromodulin gene promoters can be isolated using the genomic walking procedure described for the isolation of the mouse and goat uromodulin gene promoters in the Examples herein. Although there is an abundance of evidence suggesting that many important regulatory elements are located 5' to the mRNA cap site (e.g., McKnight et al, 1982; Payvar et al , 1983; Renkowitz et al, 1984; Karin et al , 1984), it also appears that important regulatory elements, particularly those mediating tissue-speci ic expression, may reside within the structural gene or even 3' to it (Charnay et al, 1984; Gillies et al, 1983; Reecy et al, 1998; James- Pederson et al, 1995; Sternberg et al, 1988; Belecky-Adams et al, 1993) .
  • alternate constructs can also be made in which the intron sequences of the uromodulin gene and, if necessary, the 3 ' -untranslated sequences are present in the event that such sequences are needed.
  • the approach to alternate constructs is outlined in the example provided herein and would be well recognized by those of skill in the art.
  • the 3 ' -untranslated sequences of the uromodulin gene can be added downstream of the coding sequence for the biologically active protein of interest.
  • Uromodulin has already been reported to be evolutionarily conserved, being detectable immunologically in all placental mammals (Kumar et al, 1990) .
  • the laboratory of the present inventors has shown by Southern blot hybridization that the uromodulin gene is present as a single copy in many mammals, including all important livestock, such as cattle, sheep, goat, horse and pig.
  • the uromodulin cDNAs from human, mouse and rat share a high level of identity (on the order of 80% or more) , but even the high mannose glycosylation of uromodulin is highly conserved among different species of mammals. This strongly suggests that the promoter sequences of uromodulin are also likely to be conserved among mammals .
  • the uromodulin promoter from one mammal species is believed to be functional in another species. Accordingly, the mouse uromodulin promoter identified herein can be used directly in transgenic livestock to drive kidney-specific expression of the biologically active protein of interest in a urine-based bioreactor system. Alternatively, the uromodulin promoter used in the transgenic livestock to drive kidney-specific expression of the biologically active protein can be its own endogenous uromodulin promoter or an interchangeable uromodulin promoter from another species of livestock.
  • the bovine and rat uromodulin promoter regions have already been identified in Yu et al (1994) , the entire contents of which are hereby incorporated herein by reference. Specifically, Fig. 5 of Yu et al (1994) shows the nucleotide sequence of the bovine and rat uromodulin promoter regions. These promoter regions, or a fragment thereof with kidney-specific promoting activity, can be used to drive the kidney-specific expression of a heterologous gene in those respective species.
  • bovine and the rat uromodulin promoters will also be active to promote the kidney-specific expression of a heterologous gene in any other mammal, such as goat, horse or sheep, which might be used as a urinary bioreactor.
  • Uromodulin promoters from other mammalian species can be isolated using the same approaches outlined in the examples provided herein, or using the same approach used in Yu et al (1994) , or by hybridization or PCR amplification of genomic libraries or genomic DNAs using probes or primers from the genomic clones of the mouse, rat or cow uromodulin gene. If the need to use a uromodulin promoter from another livestock animal species arises, then information generated from the mouse uromodulin promoter or from the bovine and rat uromodulin promoter region of Yu et al (1994) can be used to facilitate this process.
  • oligonucleotide primers based on these sequences can be designed for PCR reactions. Long-range PCR can be performed to directly isolate uromodulin promoters from a pool of genomic DNAs extracted from various livestock animal species.
  • DNA fragments containing the uromodulin promoter from livestock animal species can also be identified by hybridization of genomic libraries of corresponding species with mouse, bovine, rat or human uromodulin promoter probes under hybridization conditions similar to or the same as that used for the Southern blots (Zoo-blots of genomic DNA from various species) as disclosed in Example 1 provided herein.
  • the uromodulin promoter or any other kidney-specific promoter used in the transgene for directing kidney-specific expression of the biologically active protein of interest can include relatively minor modifications, such as point mutations, small deletions or chemical modifications that do not substantially lower the strength of the promoter or its tissue-specificity.
  • RNAs are isolated from stomach, intestine, colon, liver and brain, and Northern blot analysis of these RNAs using an actin cDNA as a probe is used to demonstrate the intactness of the actin mRNA in all of these preparations.
  • Kidney cDNAs are then used as the "tester”, and the cDNAs of all the other non- kidney tissues, referred to as the "drivers", are subtracted from the kidney cDNAs .
  • the laboratory of the present inventors had earlier probed the cDNAs of the non-subtracted and the subtracted libraries with actin cDNA or uroplakin lb cDNA, and the results indicated that the original (non-subtracted) bovine bladder cDNA preparation contained abundant actin mRNA and relatively little uroplakin lb mRNA.
  • the subtracted library contained almost no detectable actin mRNA (at least 50 fold reduction) but greatly increased uroplakin lb mRNA (>10 to 15 fold enrichment) .
  • Multiple cDNA clones have been isolated from the subtraction library and used to probe the mRNAs of various bovine tissues. For example, a uroplakin lb probe confirmed its bladder specificity.
  • kidney-specific genes can be isolated, and any gene that is involved in the structure and function of the excretory tract of the kidney, including proximal, distal tubules, Henle's loop, collecting duct system can be applied in this system to isolate its promoter for use in expressing and producing a biologically active protein in a urine-based kidney bioreactor.
  • kidney-specific genes can also be identified through other well-known methods, including biochemical methods, protein chemistry, monoclonal antibody production, two-dimensional gel electrophoresis, cDNA library screening, expression library screening, differential display, phage display, etc.
  • biologically active protein refers to a protein capable of causing some effect within an animal and preferably not within the animal having the transgene.
  • proteins include, but are not limited to, adipokinin, adrenocorticotropin, blood clotting factors, chorionic gonadotropin, corticoliberin, corticotropin, cystic fibrosis transmembrane conductance regulators, erythropoietin, folliberin, fcllitropin, glucagon gonadoliberin, gonadotropin, human growth hormone, hypophysiotropic hormone, insulin, lipotropin, luteinizing hormone-releasing hormone, luteotropin, melanotropin, parathormone , parotin, prolactin, prolactoliberin, prolactostatin, somatoliberin, somatotropin, thyrotropin, tissue-type plasminogen activator, vasopressin, antibodies, peptides, and antigens (for use in vaccines) . It will be appreciated by those of skill in the art that the above list is not exhaustive. In addition, new genes for biologically active proteins that
  • Proteins which degrade or detoxify organic material may also be produced by means of the present invention. Such proteins may be those discussed in WO 99/28463, the entire contents of which is hereby incorporated by reference.
  • the biologically active protein produced in the urine-based bioreactor system according to the present invention can be isolated from the urine of these transgenic animals. Accordingly, the present invention provides a means for isolating large amounts of biologically active proteins from the urine of transgenic animals which can be used for a variety of different purposes. Furthermore, the biologically active protein can be readily recovered and purified from the urine as would be well within the skill of those in the art. While the production of transgenic animals by the introduction of the transgene into germ line cells is most preferred, it is also contemplated that the transgenic animals, which serve as a urine-based bioreactor system, can be produced by vectors that are useful for transforming the kidney into a bioreactor capable of producing a biologically active protein in the urine for isolation. The transformed cells may be germ line or somatic cells.
  • the vector according to the present invention includes a system which is well received by the cells lining the excretory tract of the kidney, including proximal, distal tubules, Henle's loop and collecting duct system.
  • An example of a useful vector system is the Myogenic Vector System (Vector Therapeutics Inc., Houston, TX) .
  • the heterologous gene of the biologically active protein linked to a viral promoter construct capable of directing kidney-specific expression and carried in the vector is introduced into the kidney of an animal in vivo. Introduction of the vector can be carried out by a number of different methods routine to those of skill in the art.
  • Vectors of the present invention can also be incorporated into liposomes and introduced into the animal in that form.
  • the transgene is absorbed into one or more epithelial cells capable of expressing and secreting the biologically active protein into the urine collecting in the bladder. It may be preferred for some biologically active proteins to also engineer a signaling sequence into the vector to ensure that the protein is secreted from the apical surface into the lumen. Use of signaling sequences, such as the GPI linkage in anchoring proteins to a selected surface is well known in the art .
  • the biologically active protein is then voided from the lumen where it can be collected and separated from other components in the urine.
  • Another alternative embodiment for generating a transgenic animal as a kidney-based bioreactor is through the use of targeted homologous recombination, where one copy of the endogenous uromodulin gene is disrupted by insertion of a heterologous gene encoding a biologically active molecule of interest, which heterologous gene is flanked by sequences complementary to the endogenous uromodulin gene.
  • flanking complementary sequences which direct homologous recombination to an endogenous uromodulin gene are at least 25 base pairs in length, preferably at least 150 base pairs.
  • Patent 5,272,071 the entire contents of which are hereby incorporated by reference. Accordingly, if it is desired for the kidney to express and secrete a selected biologically active polypeptide into the urine, then a short sequence on either side of the start codon of the uromodulin coding sequence in a given species can be used as flanking sequences to create a construct that can be inserted at the specific location in the genome of the host animal species which is between the endogenous uromodulin gene promoter and the endogenous uromodulin gene coding sequence. In this way, the expression of the biologically active polypeptide of interest will be driven by the endogenous uromodulin promoter in the transgenic animal.
  • bovine genomic uromodulin sequence has already been reported (Yu et al . , 1994), and the mouse genomic uromodulin sequence as well as the clone containing the goat genomic uromodulin gene sequence surrounding the start codon are disclosed herein.
  • Three probes corresponding to the 5' -end, the middle region and the 3 ' -end of the full-length uromodulin cDNA were generated using the reverse transcription-polymerase chain reaction (RT-PCR) method, with three pairs of oligonucleotide primers chemically synthesized based on the published uromodulin cDNA sequence.
  • the set of primers for the 5' -end are 5 ' -TGGACCAGTCCTGTCCTGGTTCAG-3 ' (SEQ ID NO: 5; sense), and 5 ' -GGGTGTTCACACAGCTGCTGTTGG-3 ' (SEQ ID NO: 6; antisense) .
  • the set of primers for the middle region are 5 ' -AGGGCTTTACAGGGGATGGTTG-3 ' (SEQ ID NO: 7) and 5'- GATTGCACTCAGGGGGCTCTGT-3' (SEQ ID NO: 8)
  • the set of primers for the 3 '-end are 5 ' -GGAACTTCATAGATCAGACCCGTG-3 ' (SEQ ID NO: 9) and 5 ' -TGCCACATTCCTTCAGGAGACAGG-3 ' (SEQ ID NO: 10).
  • These three pairs of oligonucleotide primers were used to amplify uromodulin cDNA fragments using, as a template, a pool of cDNAs reversed transcribed from mouse kidney RNAs .
  • PCR conditions included the first cycle of 94 °C for 1 min, 55 °C for 1 min, and 72 'C for 2 min; 35 cycles of 95 °C for 2 min, 55°C for 1 min, and 72 °C for 2 min; and the last cycle of 94 °C for 2 min, 55 °C for 1 min, and 72 'C for 8 min.
  • Agarose gel electrophoresis revealed a 400 bp, a 440 bp and a second 400 bp PCR product for the three sets of primer amplifications, 5' -end, middle region, and 3 '-end, respectively.
  • These PCR products were purified by extraction and chromatography using a QIAEX II method (QIAGEN, Valencia, CA) .
  • a mixture of the above three uromodulin cDNA probes were 32 P-labeled and used to screen a BALB/c mouse kidney cDNA library (Clontech, Palo Alto, CA) .
  • a total of 2 x 10 s phage clones from the cDNA library were plated, lifted onto nylon membrane and hybridized with the mixture of probes at 42 * C for 16 hours in a solution containing 50% Formamide, 5X SSPE, 5X Denhardt's solution, 0.1% SDS and 100 mg/ml denatured salmon sperm DNA. After hybridization, the nylon filters were washed at 65 °C for 1 hour in IX SSC and 0.1% SDS, and autoradiographed.
  • phage clones Five phage clones were identified from the primary screening, and they were plaque-purified and subjected to the secondary screening using the same conditions as the primary screening. Purified phage clones were amplified by plate lysate and analyzed by EcoRI restriction digestion and agarose gel electrophoresis . On agarose gel, the five clones are of different sizes, ranging from 0.2 kb to 2.7 kb (Fig. 1) . A 2.7 kb clone hybridized with all three probes indicating that this band likely represented the full-length mouse uromodulin cDNA clone (Fig. 2) .
  • This 2.7 kb band was excised from the bacteriophage with EcoRI restriction enzyme, gel-purified, subcloned into the same site of pBluescript KS " (Stratagene, LaJolla, CA) , and sequenced. The sequence matched precisely with the published mouse (uromodulin cDNA sequence of Prasadan et al, 1995) , further establishing the authenticity of this as mouse uromodulin.
  • a commercial genomic screening service (Genomic System, St. Louis, MO) was used. Briefly, two pairs of PCR primers located in exon 3 (exon information derived from human uromodulin gene, Pennica et al, 1987) were designed and pretested by the present inventors. These primers were then used by Genomic System to mass -screen by PCR pooled genomic (BAC) plasmid clones of the MAC ES Mouse II library which harbors 129/SVJ mouse genomic DNAs.
  • BAC PCR pooled genomic
  • the first pair of primers sense 5'- AGGGCTTTACAGGGGATGGTTG-3 ' (SEQ ID NO: 11), and antisense 5'- GATTGCACTCAGGGGGCTCTGT-3 ' (SEQ ID NO:12) , was used for the initial screen which yielded two uromodulin clones, each about 60-70 kb in length. These clones were confirmed independently by using a second set of nested primers, sense 5'- GCCTCAGGGCCCGGATGGAAAG-3 ' (SEQ ID NO: 13) and antisense 5'- GCAGCAGTGGTCGCTCCAGTGT-3 ' (SEQ ID NO: 14) .
  • Figs. 4A and 4B An analysis of the conservation of the uromodulin gene sequence in other animal species is shown in Figs. 4A and 4B .
  • the genomic DNA of human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast were digested with EcoRI restriction enzyme and hybridized with the uromodulin middle region probe described above, using the same Southern blot hybridization conditions used above for screening the mouse kidney cDNA library.
  • the results of the Southern blot hybridization shown in Fig. 4B show that the uromodulin gene is conserved in mammals and is present as a single copy in human, monkey, rat, mouse, dog, cow and rabbit.
  • Pennica et al (1987) and Yu et al (1994) reported that the gene structure (exons and introns) of human, bovine and rat uromodulin are highly conserved (Fig. 5) .
  • Southern blotting was performed to identify DNA fragments containing the uromodulin promoter sequence. This approach is based on the differential reactivity of DNA restriction fragments of BAC clone 1 DNA with three different uromodulin probes located in the 5' -end, middle region, and 3 '-end of the uromodulin cDNA.
  • BAC plasmid clone 1 was digested with the restriction enzymes Notl, BamHI, Hindlll, Pstl, EcoRI, Apal, Ncol, Sad, Xhol and Kpnl .
  • DNA fragments were transferred onto nylon membrane, UV-crosslinked and hybridized with the 5'- end, middle region, and 3 '-end cDNA probes.
  • a 6.9 kb Pstl DNA fragment (Fig. 6, lane 4), an 8.3 kb Apal DNA fragment (Fig. 6, lane 6), and an 8.5 kb Sad DNA fragment (Fig. 6, lane 8) reacted with only the 5' -end probe, but not with middle region probe or the 3 '-end probe. This strongly indicates that these three DNA fragments contain portions of the 5 '-end of the uromodulin coding sequence and, more importantly, a large fragment of the 5 '-upstream region of the uromodulin gene.
  • a genomic walking method was employed to sequence the entire uromodulin promoter from both 5'- and 3'- ends by sequentially walking the sequence and synthesizing the new primers based on newly obtained sequences . Sequences were determined by the dideoxynucleotide chain termination method of Sanger et al (1977) on an automatic DNA sequencer. Listed below are sense- and anti-sense primers used for the sequencing purposes .
  • AS1 5 ' -AAGTCAGACTGTGTTAGGAT- 3 ' (SEQ ID NO : 20 )
  • AS2 5' -ATTGACTGAGCAGGAAGCAT-3' (SEQ ID NO: 21)
  • AS3 5' -ATTTTATAACCTCCCTCTAG-3' (SEQ ID NO:22)
  • AS4 5' -ATGCATTCCAGTCTCAGTGC-3' (SEQ ID NO: 23)
  • the 9,345 bp nucleotide sequence of the promoter region and the genomic coding region including exon 3 of the mouse uromodulin gene is shown in Fig. 7.
  • These results (1) establish the authenticity of the isolated uromodulin clone, (2) indicate that a 7 kb uromodulin promoter has been obtained which is more than adequate to be used in the urine- based transgenic bioreactor system.
  • This mouse promoter can be used in other mammalian species, such as farm animals, to drive the kidney-specific expression of any heterologous gene.
  • Uromodulin Promoter Having identified the uromodulin promoter region, this region can be subcloned for further amplification, and for constructing transgenes . Since the clone containing the uromodulin promoter region is at least 70 kb in size, restriction digestion of each of this clone gives rise to multiple bands. Although the relative sizes of uromodulin promoter-containing bands can be determined by Southern blotting using the 5' -end probe, this does not allow for pinpointing a specific band for subcloning, as most bands are not well-resolved.
  • the plasmid pBluescript (Stratagene, LaJolla, CA) , which was used as the cloning vector, is to be restriction- digested using Pstl, Apal and Sad, respectively, phosphatase-treated, and the linearized pBluescript cloning vectors will be mixed with the correspondingly digested inserts, ligation buffer, T4 DNA ligase, and incubated at 16 "C for 16 hours. Half of this ligation mixture will be used to transform CaCl 2 -prepared competent JM109 bacterial cells and then screened using small-scale plasmid preparations, which are carried out using mini-prep columns (Promega) and then restriction-digested to release the inserts. Through these procedures the DNA fragments containing mouse uromodulin promoter are to be subcloned. Detailed Restriction Mapping of Uromodulin Promoter
  • Restriction mapping of the 5 '-flanking sequence of uromodulin an important step for determining the restriction fragments for constructing transgenes has been performed. Although the detailed restriction map is not shown here, such a restriction map can be generated quite readily using any of the numerous publicly or commercially available DNA analysis software programs .
  • EXAMPLE 2 ISOLATION OF GOAT UROMODULIN GENE PROMOTER Isolation of Goat Uromodulin cDNA
  • the goat uromodulin cDNA was isolated using reverse transcriptase/polymerase chain reaction (RT-PCR) approach (Wu, et al . , 1993) .
  • RT-PCR reverse transcriptase/polymerase chain reaction
  • a sense and an antisense primer were synthesized based on the mouse uromodulin gene sequence that was isolated in the laboratory of the present inventors. The sequences of these two primers are:
  • RNA was isolated from goat kidneys using the guanidine isothiocyanate method, reverse-transcribed using AMV reverse transcriptase, and the second strand of cDNA was synthesized using DNA polymerase I. PCR amplification was performed using total kidney cDNAs as templates and the two mouse uromodulin as primers, in the presence of NTP, Taq polymerase, and PCR buffer.
  • the PCR reaction was performed for 35 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C and the resulting PCR products were resolved by agarose gel.
  • the products having the predicted size were subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced.
  • the PCR product was subcloned and sequenced.
  • a Blast search of Genbank of the PCR product sequence showed that the top four hits were uromodulin sequences from several species.
  • the sequence of the PCR product shared a 96% identity (287 bp/297 bp) with bovine uromodulin, 90% identity (218/241) with human uromodulin, a 78% identity (239/304) with rat uromodulin, and an 80% identity in a shorter stretch (125/156) with mouse uromodulin.
  • the high degree of sequence identity of the PCR product with known uromodulin sequences firmly established that the product is a partial goat uromodulin cDNA.
  • Genomic Walking Cloning and Sequencing
  • genomic DNA was isolated from goat kidneys and used as templates for PCR-based genomic walking (Clontech, Palo Alto, CA) .
  • the genomic DNA was digested using five restriction enzymes (Dral, Seal,
  • the PCR was performed for 1 cycle of denaturation at 99°C for 5 sec, annealing and extension at 68°C for 4 min., followed by 7 cycles of denaturation at 94 °C for 2 sec, annealing and extension at 68°C for 4 min., followed by 32 cycles of denaturation at 94 °C for 2 sec, annealing and extension at 63°C for 4 min., and followed by 1 cycle at 63°C for 4 min.
  • the products were used as templates and subjected to a second round of PCR amplification using two new, nesting sense and anti-sense primers. The specific products were subcloned into the TA cloning vector and the identity of the goat uromodulin gene was confirmed by DNA sequencing of both ends of the product.
  • the two above anti-sense primers were designed for genomic walking using goat genomic DNA to identify DNA sequences that are located in the upstream region. After the first and second rounds of PCR and nesting PCR amplifications, a 1.5 kb, single PCR product was obtained. Subcloning and sequencing of this product revealed that its 3 '-end shares 94% identity (494/522) with bovine uromodulin cDNA sequence, thus confirming that the PCR product is a portion of the goat uromodulin gene. The 5' -sequence did not share any significant homology with any of the known uromodulin cDNA sequences and therefore most likely represents intron sequences.
  • 5 '-end of the genomic clone that was isolated from the first round of genomic walking was used to design new antisense "walking primers" located in intron 1.
  • the five primers are: 5 ' -AAGATTTACCAGCCCGGGCCGTCGACC-3 ' (SEQ ID NO : 32 ; AS1) 5 ' -AATAAAGTGCCAGGGCAGGGGGGCTTA-3 ' (SEQ ID NO : 33 ; AS2 )
  • PCR and nesting PCR yielded a highly specific, 1.0 kb product in three independent primer combinations.
  • EXAMPLE 3 CONSTRUCTION OF KIDNEY-BASED BIOREACTOR SYSTEM Construction of Chimeric Genes
  • a chimeric gene containing the uromodulin promoter and a gene encoding a pharmaceutically- important protein is to be constructed.
  • the human growth hormone gene (hGH) whose expression has been recently assessed in a uroplakin II -based, bladder bioreactor system (Kerr et al, 1998) will be tested first.
  • hGH human growth hormone gene
  • Such a potential limitation may possibly be associated with the less than optimal secretory activity of the urothelium.
  • uromodulin is normally synthesized in the ascending limb of Henle's loop and the distal tubules where active secretion takes place, the present inventors expect that there will be an active secretion of synthesized hGH into the urine of mice, resulting in high protein yield.
  • the presence of this uromodulin/hGH gene in transgenic mice will allow a comparison of the efficiency between the kidney-based and the bladder-based reactor systems.
  • the uromodulin/hGH chimeric gene will be constructed using a pBluescript cloning vector in two steps.
  • the first step is to clone the restriction-mapped uromodulin 5' -flanking region (above) into pBluescript, and the second is to clone the hGH gene downstream of the uromodulin 5'- flanking region so that the transcription of hGH is under the exclusive control of the uromodulin promoter. Efforts will be made to select restriction sites that would permit efficient cohesive ligation. If necessary, however, blunt- end ligations could be performed. The proper uromodulin/hGH orientation will be verified by restriction mapping, PCR and sequencing using a sense primer at the 3 '-end of the uromodulin promoter and an antisense primer located inside the hGH coding region.
  • the uromodulin/hGH fusion gene will then be excised en bloc using two restrictions enzymes at the 5'- and 3 '-ends of the fusion gene and gel-purified for use in generating mice transgenic for this uromodulin/hGH fusion.
  • Mouse strain C57BL/6XDB2 that has been well adopted by a NYU Transgenic Mouse Facility, will be used for transgenic mouse production. All transgenic techniques, including embryo manipulation, fusion gene microinjection, implanting of embryos into pseudopregnant recipients, will be based on standard procedures, such as Hogan et al (1986), where the transgene is microinjected into the pronuclei of fertilized eggs, which are then implanted into pseudopregnant mice. The offspring are then analyzed for transgene incorporation by Southern blotting of DNA isolated from the tails of mice. Two probes are to be used, one corresponding to the mouse uromodulin promoter, and the other corresponding to hGH. The mouse uromodulin probe will reveal an endogenous uromodulin gene, which will allow calculation of the copy number of the transgene by comparing their relative intensity.
  • intron I sequences were found to be important for high-level and tissue-specific expression of an alpha-skeletal actin gene, a beta-globin gene and a peripherin gene (Reecy et al, 1998; James-Pederson et al, 1995; Belecky-Adams et al, 1993) .
  • the constructs to be made will include intron I sequences of the uromodulin gene and, when necessary, 3 ' -untranslated sequences.
  • a fragment will be isolated that spans the 5' -flanking region, the first exon and the first intron, followed by the hGH gene.
  • the translation initiation codon of the uromodulin gene could also be mutated to avoid translation of truncated uromodulin, and other regions of the uromodulin gene could also be used to ensure the tissue-specific and high-level expression of the uromodulin transgene. Expression of hGH in Mouse Kidney
  • hGH human growth factor
  • RT-PCR is to be performed to determine the expression of mRNA using primers specific for hGH.
  • Total RNAs will be extracted from transgenic mouse kidneys and from control tissues, including rat liver, skin, intestine, stomach, brain, skeletal muscle, thymus, thyroid gland, bladder, lungs, heart, pancreas, spleen, prostate, seminal vesicles, uterus and ovaries.
  • the total RNAs are to be reverse-transcribed, PCR amplified and analyzed by agarose gel electrophoresis. The results will reveal whether hGH is expressed in kidney- dependent fashion.
  • Fukuoka et al, GP-2/THP gene family encodes self-binding glycosylphosphatidylinositol-anchored proteins in apical secretory compartments of pancreas and kidney, Proc . Natl. Acad. Sci. USA 89:1189-1193 (1992)
  • tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene, Cell 33:717-728 (1983)
  • Uromodulin Tamm-Horsfall glycoprotein: a renal ligand for lymphokines, Science 237:1479-1484 (1987)
  • Uromodulin (Tamm-Horsfall glycoprotein/ uromucoid) is a phosphatidylinositol-linked membrane protein, J. Biol. Chem. 265:20784-20789 (1990)
  • Uroplakins la and lb two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins, J. Cell Biol . , 125:171-182 (1994)

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Abstract

L'invention concerne des constructions d'ADN recombiné; une méthode de production de protéines biologiquement actives recombinées in vivo dans l'urine d'un mammifère non humain au moyen d'un promoteur spécifique du rein, tel que le promoteur d'uromoduline; et des mammifères non humains transgéniques servant de bioréacteurs urinaires pour la production de protéines.
EP99958952A 1998-11-13 1999-11-12 Animaux transgeniques servant de bioreacteurs urinaires pour la production de proteines dans l'urine, constructions d'adn recombine pour l'expression specifique du rein, et procedes d'utilisation de ces dernieres Withdrawn EP1135518A4 (fr)

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JP2013526840A (ja) 2009-12-21 2013-06-27 ファーマシーネ，インコーポレイテッド 組み換えブチリルコリンエステラーゼおよびその切断型
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