WO1994005782A1 - Procede de production in vivo d'un organe transgenique par introduction du transgene dans une lumiere - Google Patents

Procede de production in vivo d'un organe transgenique par introduction du transgene dans une lumiere Download PDF

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Publication number
WO1994005782A1
WO1994005782A1 PCT/US1993/008618 US9308618W WO9405782A1 WO 1994005782 A1 WO1994005782 A1 WO 1994005782A1 US 9308618 W US9308618 W US 9308618W WO 9405782 A1 WO9405782 A1 WO 9405782A1
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Prior art keywords
gland
lumen
transgene
dna
animal
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PCT/US1993/008618
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English (en)
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Karl M. Ebert
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Trustees Of Tufts College
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a method for making an organ or gland having a transgene, and nonhu an transgenic animals made in accordance with the present invention.
  • DNA molecules have been introduced into cultured cells by calcium phosphate precipitation or electroporation, wherein the DNA enters the cell cytoplasm, with a fraction of the molecules entering the nucleus.
  • Graham and Van der Ebb Virology 52:456-467 (1973); Perucho et al, Cell 22:9-17 (1980); Chu et al Nucleic Acids Research 15:1311-1326 (1987); E.J. Robertson, "Teratocarcinomas and Embryonic Stem Cells, A Practical Approach,” (IRL Press 1987); and Bishop & Smith, Molecular Biology Medicine 6:283-298 (1989).
  • DNA molecules have also been introduced into the nucleus of cells in culture by direct microinjection.
  • Gordon et al Proceedings of the National Academy of Science, USA 77:7380-7384 (1980); Gordon and Ruttel, Methods In Enzymology 101:411-433 (1983); and Wagner and Hoppe, U.S. Patent No. 4,873,191 (1989).
  • DNA molecules have been incorporated into animal genomes by a retroviral vector infection. Jaenisch et al, Cell 24:519 (1981); Soriano et al, Science 234:1409-1413 (1986); Stewart et al, Embo. J. 6:383-388 (1987).
  • the term “gene transfer” refers to the introduction of new genetic material, either RNA or DNA into the cell.
  • the term “germ cells” refers to cells with genetic material that can be passed on to offspring.
  • “somatic cells” refer to body cells from which genetic material is not normally passed to offspring.
  • “Somatic gene transfer” refers to the introduction of genes not normally present, into somatic cells to provide gene functions that are not normally present in such cell.
  • Somatic gene therapy refers to the introduction of normal genes into somatic cells to provide gene functions that are deficient because of genetic or acquired disease.
  • Recombinant DNA refers to DNA fragments that have been joined by artificial means through genetic manipulation.
  • transformation refers to changes in the property of cells by the introduction of genetic material.
  • “Viral mediated gene transfer” refers the introduction of new genetic material into cells by infection with recombinant viruses or fragments of the viral genome containing foreign genes.
  • Transcription is a process by which DNA is copied into messenger RNA.
  • Translation is a process by which messenger RNA is processed into proteins.
  • a regulatory region is a region through which transcription of a gene is controlled or regulated. Such regulatory regions include a cis-acting DNA sequence. A function of this sequence is to be recognized by regulatory elements such as proteins.
  • a regulatory region may include promoters, enhancers, and repressors.
  • a promoter is a DNA sequence which signals the start of RNA synthesis.
  • An enhancer is a DNA sequence that alters the efficiency of transcription.
  • a repres ⁇ or is a DNA sequence that reduces the efficiency of transcription.
  • the regulatory region can direct the cell or cell type in which the adjacent promoter can function.
  • the regulatory region may be derived from a regulatory region that is normally endogenous to the cell which will receive the transgene, or from a regulatory region that is exogenous to the target cell. "Endogenous” is used to denote material which is normally found in the cell to which it refers. The term “exogenous” is used to denote material that is not normally found in the cell to which it refers.
  • coding region is a sequence of DNA that encodes for a product and that is free of a regulatory region.
  • the coding region can code for any RNA or polypeptide product.
  • the product may be a full length gene product or it may be a subfragment thereof, or it may be part of a fusion product.
  • a coding region can be sequences which include exon ⁇ and introns, as well as those which include exons and some introns, or only exons.
  • the coding region may be derived from a coding region that is normally endogenous to the target cell, or from a coding region that is exogenous to the target cell.
  • the coding region may include altered sequences to impart desired features to the encoded protein.
  • intron refers to intervening sequence within a gene for the gene product which do not constitute protein coding sequences. In eukaryotic cells, introns are removed from the primary RNA transcript to produce the mature messenger RNA.
  • a stop signal is a sequence recognized by a cell to stop transcription.
  • a well known stop signal is a polyadenylation signal.
  • transgenic product refers to the cellular products produced in response to the presence of a transgene.
  • characteristic genotype refers to the complement of genes carried by the somatic cells of the animal, including, without limitation, animals which may have one or more mutations or additional genes in the complement of genes carried by the somatic cells.
  • Gland is used in the sense of an organized aggregation of cells functioning as a secretory or excretory organ.
  • epidermal cells refers to glandular types cells capable of secretion or excretion.
  • lumen refers to the space in the interior of a tubular or cavity type structure.
  • One method involves the introduction of recombinant genes into primary cultures of bone marrow, skin, fibroblasts, or hepatic, pancreatic cells then transplanting the transformed cells into live animals.
  • small clusters of nonproliferating cells may be transplanted to provide a reservoir of cells producing the recombinant gene product or performing the deficient function normally performed by cells of its type.
  • Chronic expression of transgenic products may have profound effects on the animal carrying the gene.
  • Superphysiological levels of pharmacologically active molecules may have a detrimental effect on the animal.
  • chronic expression of homologous and heterologou ⁇ growth hormone has profound effects on the fat content of domestic species.
  • a short term expression system is desired to effectively alter the fat distribution within the animal without causing adverse physiological conditions.
  • Such a short term expression system may be considered nontoxic to the food chain.
  • a delivery system that can provide transient expression of the transgenic product from 30 to 40 days would have great utility.
  • a delivery system that can transform a selected tissue, gland or organ capable of making a transgenic product would have utility particularly where the gland or organ can secrete the transgenic product.
  • the gross phenotypic changes associated with other types of transformation systems are avoided.
  • the organ can be removed to eliminate foreign DNA from the food chain, and to terminate the production of the transgenic product.
  • Embodiments of a present invention feature as an article of manufacture, a nonhuman animal, which animal has a characteristic genotype and has at least one organ or gland which has a lumen having cells which cells are capable of introducing secretions into the lumen or, such cells are, or are capable of forming, germ cells.
  • the animal has at least one or more such cells having a transgene which transgene is absent from the characteristic genotype of nonepithelial somatic cells of the nonhuman animal.
  • the animal or its progeny is capable of expressing the transgene to make a transgenic product.
  • the organ or gland may be selected from the group of glands and organs, comprising by way of example, without limitation, the elementary epithelium, Cowper' s gland, intestinal wall, liver, mammary, nasal mucosa, pancreas, prostate, salivary, sebaceous, seminal vesicles, stomach epithelium, sweat, lacrimal, uterine endometrium, and tonsils.
  • glands and organs comprising by way of example, without limitation, the elementary epithelium, Cowper' s gland, intestinal wall, liver, mammary, nasal mucosa, pancreas, prostate, salivary, sebaceous, seminal vesicles, stomach epithelium, sweat, lacrimal, uterine endometrium, and tonsils.
  • glands which may have application in the present invention include the musk gland, oil gland, and egg producing glands.
  • the organ or gland is the mammary gland.
  • the mammary gland provides an efficient production system for pharmaceutical drugs.
  • the transfected epithelial cells of the mammary gland can be induced to secrete the transgenic product during the lactation period of the animal .
  • the transgene encodes for a gene selected from the group of genes encoding for biologically active molecules.
  • biologically active molecules refer to molecules capable of causing some effect within an animal, not necessarily within the animal having the transgene. Such molecules include, by way of example, without limitation, molecules identified in Table I below.
  • the tran ⁇ gene compri ⁇ es a coding ⁇ equence for the biologically active molecule, a promoter, and a ⁇ top signal.
  • the transgene may also encode for introns to facilitate the handling of the transgene by the molecular machinery of the transformed cell, and enhancers and repre ⁇ or ⁇ to regulate the formation of the tran ⁇ gene product.
  • the promoter may exhibit ti ⁇ ue ⁇ pecificity.
  • the beta casein promoter, the mou ⁇ e mammary tumor virus promoter, beta lactoglobulin promoter, and whey acid protein promoters are tissue specific for the mammary gland.
  • the transgene is carried in a vector which is well received by the epithelial cells lining the lumen and does not have long term effects.
  • the Myogenic Vector System (Vector Therapeutics Inc., Hou ⁇ ton, Texas) may provide transient expres ⁇ ion of the tran ⁇ gene in transformed epithelial cells.
  • a further embodiment of the present invention features a method of imparting a desired trait to an animal having a characteristic genotype.
  • the animal has at least one gland which gland has a lumen lined with epithelial cells, and the epithelial cells are capable of introducing secretions into the lumen.
  • the method comprises the step of introducing a transgene into said lumen in vivo.
  • the transgene is absorbed into one or more epithelial cells capable of expressing the transgene to make a transgenic product.
  • the transgene is expressed when desired, by imposing expressing conditions.
  • Expressing conditions will be determined by the choice of promoter, enhancer and repre ⁇ or.
  • Expressing conditions may include, by way of example, lactation and exogenous switches such as particular food additive ⁇ .
  • the tran ⁇ genic product is ⁇ ecreted by the epithelial cells into the lumen.
  • the transgenic product is preferably voided by the lumen where it is capable of being collected, and separated from the other constituents of the lumen secretions.
  • Preferred animals for the pre ⁇ ent method include rodent ⁇ , ⁇ uch as lagamorphs, mice and rat ⁇ and domestic animals, such as cow, swine, goat and other milk producing domestic animals.
  • Organ ⁇ and glands can be transformed to replace the normal functions of a disea ⁇ ed organ.
  • a pancrea ⁇ incapable of producing insulin could receive a transgene for insulin.
  • a gland other than the organ or gland which would normally make a biologically active molecule, may be transformed to supply is ⁇ ing product.
  • Table I lists examples of pharmacologically active molecules capable of being made by transformed epithelial cells.
  • Figure 1 shows the structure of a mammary specific expression vector.
  • the 17.8 kilobase icroinjected fragment contains the entire human CFTR cDNA cloned between exons 2 and 7 of the goat beta casein gene.
  • the solid line depicts the goat beta casein gene and the block identified with angled hatch marks represents the CFTR cDNA.
  • Figure 2 ⁇ how ⁇ the structure of the WAP-LAtPA expression vector and its restriction enzyme sites.
  • H represents Hind III
  • R represent ⁇ EcoRI
  • X represent ⁇ Xbal
  • B represents BamHI
  • K represents Kpml .
  • the present invention is described in detail as a method for transforming epithelial cells of a gland or organ of animals which animals are then capable of producing a transgenic product.
  • the present Example present ⁇ an overview of the process applied to selected DNA, animals and glands.
  • the DNA may be cDNA or genomic.
  • cDNA clones may be obtained from the American Type Culture Collection, universities, and corporate and private research groups.
  • the coding sequence can be obtained from natural sources in a manner known in the art. The manner of isolating such genes is not necessarily simple; however, the methodology of the present invention applies equally to such coding sequences which have been identified as relating to a particular product as well as coding sequences which are likely to be discovered in the future.
  • the DNA, whether cDNA or genomic DNA may be modified by molecular cloning techniques.
  • the coding sequences for the transgenic product is coupled with appropriate regulatory regions.
  • the choice of regulatory region and signal peptide will be influenced by the animal and gland to be transformed.
  • promoters specific for the mammary gland have been isolated which function to cause secretory epithelial cells to release transgenic products in milk.
  • promoters appear to function in species from which such promoter ⁇ were i ⁇ olated and others.
  • a DNA vector i ⁇ prepared by ⁇ olubilizing the naked DNA comprising the coding sequence and regulatory region into a solution of Tris-HCl (pH 7.4) and EDTA.
  • the DNA vector may co pri ⁇ e a pellet of precipitated DNA.
  • DNA can be precipitated from solutions with ethanol.
  • the pellet may be encapsulated to provide slow release of the DNA.
  • the DNA vector may also have features of a gel . DNA imparts viscosity to solutions in which it is dis ⁇ olved. By adju ⁇ ting the concentrations of solutions containing DNA, the DNA solution may assume gel-like features.
  • the DNA may have features of a gel due to the addition of excipients ⁇ uch a ⁇ methyl cellulo ⁇ e, ⁇ odium alginate and the like.
  • the gel-like feature ⁇ may provide a ⁇ low relea ⁇ e of the DNA into epithelial cell ⁇ of gland ⁇ .
  • the gel may al ⁇ o take the form of oleaginou ⁇ gel having a ba ⁇ e of white petrolatum, oleic acid white wax or paraffin and admixture ⁇ and variou ⁇ combination ⁇ of ⁇ uch ba ⁇ e.
  • DNA may be ab ⁇ orbed or encap ⁇ ulated in lipid ve ⁇ icles or liposome ⁇ .
  • the lipid ve ⁇ icles may also comprise binding agents to convey specificity to particular epithelial cells of the gland. For example, coupling the lipid ve ⁇ icle ⁇ to vitellogenin or very low density lipoprotein may convey specificity for chicken oocytes. Barber et al, "The Receptor for Yolk Lipoprotein Depo ⁇ ition in the Chicken Oocyte," J. Biol. Chem. , Vol 266, No. 28:18 761-18770 (1991).
  • DNA vector will be influenced by the feature ⁇ of the gland in which the epithelial cell ⁇ are to be tran ⁇ formed.
  • the oocyte of bird ⁇ may be tran ⁇ formed with a DNA vector encap ⁇ ulated in lipid ve ⁇ icles.
  • the lipid vesicles are injected into the maternal arterial supply during vitellogenesis.
  • Transformation of an embryo in utero or germ cells may be performed by direct injection into the embryonic yolk sac in the region where the primordial germ cells will migrate to the genital ridge.
  • a preferred DNA vector system is comprised of DNA in solution.
  • the mammary of gland of mammals may be transformed by injection of DNA ⁇ olution ⁇ through ⁇ terile teat infu ⁇ ion cannula ⁇ that fit Luer-lock syringes.
  • Suitable DNA vectors for the transformation of the mammary gland compri ⁇ e naked DNA in solution, naked DNA in a pelleted form, or naked DNA in a viscous gel.
  • urethra of animals may be transformed with the aid of a rubber urethral catheter or Foley catheter.
  • Suitable vehicles for gene delivery comprise DNA in solution, DNA in a slow relea ⁇ e pelleted form, and DNA in a viscous gel.
  • the vagina may be transformed with the aid of a vaginal ⁇ peculum with light, in ⁇ emination pipette ⁇ / ⁇ emen ⁇ traws, french style straw guns with sheaths, and uterine catheters.
  • Suitable vehicles for gene delivery comprise DNA in solution, DNA in a slow release pelleted form and DNA in a viscou ⁇ gel.
  • the oviduct may be transformed with the aid of standard surgical approach requiring a surgical pack, glass pipette and metal pipettor; or, in the alternative, a laparoscopic approach requiring a ⁇ traight endoscope, laparo ⁇ copy holding forcep ⁇ , ⁇ tainless steel trocar and cannula, (7 mm X 7 cm), paravertebral needle, and a siliconized gla ⁇ pipette attached to a ⁇ yringe.
  • Suitable DNA vectors for each approach comprise DNA in solution, DNA in a slow release pelleted form, and DNA in a viscou ⁇ gel.
  • Epithelial cells of the uterus may be tran ⁇ formed in at least three ways. Fir ⁇ t, epithelial cell ⁇ of the uterus can be transformed with surgical laparoscopic approaches a ⁇ set forth with respect to the oviduct. In the alternative, epithelial cells of the uterus may be transformed with uterine pipette ⁇ with drilled ends for attachment to a syringe. A third alternative to transform epithelial cells of the uterus comprise ⁇ a uterine catheter. In each of the alternatives, the vehicle for gene delivery may comprise a DNA in solution, DNA in a slow relea ⁇ e pelleted form, or DNA in a viscous gel.
  • Epithelial cells of the ovary may be transformed with standard surgical procedures involving direct injection of the DNA vector into the ovarian artery using needle and syringe.
  • Suitable gene delivery vehicles comprise DNA in ⁇ olution, DNA in ⁇ low release pelleted form, and DNA in encapsulated lipid vesicles.
  • the kidney can be transformed with the aid of standard surgical approache ⁇ with the direct injection into the ureter or renal artery; or, ureteral catheterization following cystostomy.
  • a second alternative comprises ureteral catheterization using cy ⁇ to ⁇ copy. Such technique ⁇ require an endo ⁇ cope, catheter, trocar and holding forcep ⁇ .
  • a third alternative is percutaneous nephropyelocentesi ⁇ using fluoro ⁇ copy equipment.
  • Vehicles for DNA gene delivery comprise DNA in ⁇ olution, DNA in ⁇ low release pelleted form ⁇ , and DNA in a viscous gel.
  • the colon/rectum may be transformed with the aid of a Bordex or Foley catheter of the type used to fill and hold fluid in place for barium enemas.
  • Suitable vehicles for gene delivery comprise DNA in solution, DNA in slow release pelleted form ⁇ , and DNA in vi ⁇ cou ⁇ gel.
  • the pro ⁇ tate gland may be tran ⁇ formed by direct injection by palpication through the rectum a ⁇ for biop ⁇ y examination ⁇ .
  • Suitable vehicles for gene delivery comprise DNA in solution, DNA in pelleted form, and DNA in a viscou ⁇ gel.
  • the salivary gland may be tran ⁇ formed with direct injection ⁇ into the duct using a blunt needle and syringe a ⁇ for a ⁇ ialogram.
  • Suitable vehicle ⁇ for gene delivery comprise DNA in a ⁇ olution, DNA in pelleted form ⁇ , and DNA in a viscou ⁇ gel.
  • the lacrimal gland may be transformed with a prolonged release device such as a permeable membrane-type ocular insert of the type marketed under the trademark Ocusert® (ALSA) .
  • a prolonged release device such as a permeable membrane-type ocular insert of the type marketed under the trademark Ocusert® (ALSA) .
  • An alternative comprises a DNA contained in an eye ointment/solution with a temporary clo ⁇ ure of the lacrimal duct to prevent drainage.
  • the temporary clo ⁇ ure of the lacrimal duct can be performed with nylon thread.
  • Suitable vehicle ⁇ for gene delivery compri ⁇ e DNA in ⁇ olution, DNA in a viscous gel, or DNA in a pelleted form.
  • the transformation event is performed during a period in which the organ or gland has epithelial cell ⁇ undergoing rapid proliferation.
  • epithelial cell ⁇ can take place during the fetal, new born, and infant stages of the animals' life and at other times during the development of the animal.
  • the areolar structures of the mammary gland undergo rapid proliferation prior to puberty, during induction with steroid ⁇ and during pregnancy.
  • the secretions can be collected and the transgenic product separated from the other constituent ⁇ of the secretions.
  • Example 2 outlines the production of cystic fibrosi ⁇ tran ⁇ membrane conductance regulator in the epithelial cell ⁇ of the mammary gland of goat ⁇ .
  • Other tran ⁇ genic products can be readily substituted for cystic fibrosis transmembrane conductance regulator and the construction of the tran ⁇ gene and vector ⁇ ystems can be altered as known in the art.
  • Partial CFTR cDNA clone ⁇ Til, T16-1, T16-4.5 and C-l/5 are obtainable from the American Type Culture Collection (Rockland, Maryland). Riordan et al, "Identification of the cy ⁇ tic fibrosis gene: cloning and characterization of the complementary DNA, " Science, 245:1059-1065 (1989). The alignment of the CFTR cDNA portion of these clones is pre ⁇ ented in Figure 1. The full length copy of CFTR cDNA (containing a point mutation at re ⁇ idue 936 to activate an internal cryptic bacterial promoter that otherwi ⁇ e renders the cDNA unstable) is in ⁇ erted between exons 2 and 7 of the goat beta ca ⁇ ein gene.
  • the DNA vector may take the form of high concentration solution or gel or a pellet to be placed directly into the lumen of the gland.
  • the DNA vector is directly introduced by injection via the orifice and duct leading into the lumen of the mammary gland.
  • the DNA vector is prepared by solubilizing the naked DNA into a ⁇ olution of Tri ⁇ -HCl (pH 7.4) and EDTA.
  • the DNA vector can be introduced into the lumen of the gland in a plurality of tran ⁇ formation event ⁇ .
  • the epithelial cell ⁇ take up the DNA vector and are capable of expre ⁇ ing the tran ⁇ gene. Expression is induced with lactation.
  • the transgenic product is separated from the remaining constituents of milk and can be further processed if necessary.
  • the milk i ⁇ either diluted with an equal volume of phosphate buffer saline or fractionated as described by Imam et al, "Isolation and Characterization of a Major Glycoprotein From Milk Fat Globule Membrane of Human Breast Milk," Biochem. J. , 193:47-54 (1981); and, Patton and Huston, "A Method For I ⁇ olation of Milk Fat Globules," Lipids 21:170-174 (1986).
  • Milk is solubilized and further diluted 5 fold in RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% ⁇ odium deoxycholate and 0.1% ⁇ odiu ⁇ ulphate) prior to biochemical analy ⁇ i ⁇ .
  • RIPA buffer 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% ⁇ odium deoxycholate and 0.1% ⁇ odiu ⁇ ulphate
  • wash buffer 10 mM Tris-HCl, pH 7.5, 0.25 M sucrose and 1 mM MgCl-
  • the resulting skim milk is siphoned off by inserting a needle through the layer of cream.
  • the cream i ⁇ washed twice with approximately one volume of wash buffer and dispersed in RIPA buffer.
  • the siphoned skim milk i ⁇ centrifuged at 12,000g for 10 minute ⁇ to pellet the ca ⁇ ein ⁇ .
  • the ca ⁇ ein pellet i ⁇ wa ⁇ hed once with one volume of wash buffer and re ⁇ u ⁇ pended in RIPA buffer.
  • Membrane ⁇ and ve ⁇ icle ⁇ contained in the resultant skim supernatant were collected by further centrifugation at 100,000g for 2 hours.
  • CFTR-containing immunoprecipitates is achieved by incubating with protein kinase A and [ ⁇ 32P]ATP (10 ⁇ Ci) in a final volume of 50 ⁇ l in kinase buffer (50 mM Tri ⁇ -HCl, pH 7.5, 10 mM MgCl_ and 100 ⁇ g ml bovine serum albumin) at 30°C for 60 minute ⁇ .
  • kinase buffer 50 mM Tri ⁇ -HCl, pH 7.5, 10 mM MgCl_ and 100 ⁇ g ml bovine serum albumin
  • N-glycanase, endoglycosidase H and endoglycosidase F are obtained from Genzyme Corp. (Cambridge, Mas ⁇ achu ⁇ etts) . Conditions for digestion with the re ⁇ pective glyco ⁇ idases were as specified by the manufacturer, except that incubations are performed at 37°C for 4 hours only.
  • CFTR is immunopurified u ⁇ ing the polyclonal antibody pAb Ex 13, phosphorylated in vitro with protein kina ⁇ e A and
  • [ ⁇ 32P]ATP ⁇ eparated on SDS-polyacrylamide gel ⁇ , and then extracted from the gel ⁇ by macerating the gel piece ⁇ in protein extraction buffer (50 mM ammonium bicarbonate, 0.1% SDS and 0.2% ⁇ -mercaptoethanol) .
  • Eluted protein ⁇ are recovered by trichloroacetic acid precipitation.
  • Example 3 outline ⁇ the production of cystic fibrosis transmembrane conductance regulator in the epithelial cell ⁇ of the mammary gland ⁇ of mice.
  • Other tran ⁇ genic products can be readily ⁇ ubstituted for the cystic fibrosis transmembrane conductance regulator and the construction of the transgene vector sy ⁇ tem ⁇ can be altered i ⁇ known in the art.
  • Partial CFTR cDNA clones Til, T16-1, T16-4.5 and C-l/5 are obtainable from the American Type Culture Collection (Rockland, Maryland). Riordan et al, "Identification of the cystic fibro ⁇ i ⁇ gene: cloning and characterization of the complementary DNA," Science, 245:1059-1065 (1989). The alignment of the CFTR cDNA portion of the ⁇ e clone ⁇ i ⁇ pre ⁇ ented in Figure 1.
  • the full length copy of CFTR cDNA (containing a point mutation at re ⁇ idue 936 to activate an internal cryptic bacterial promoter that otherwi ⁇ e render ⁇ the cDNA un ⁇ table) i ⁇ in ⁇ erted between exon ⁇ 2 and 7 of the goat beta casein gene.
  • the DNA vector may take the form of high concentration solution or gel or a pellet to be placed directly into the lumen of the gland.
  • the high concentration DNA solution will comprise approximately 1 ⁇ g DNA in 10 mM Tri ⁇ -HCl pH 7.6, 1 mM EDTA.
  • the molarity of the DNA can be adju ⁇ ted to create a vi ⁇ cou ⁇ gel.
  • the DNA may compri ⁇ e a pellet of DNA precipitated with ethanol.
  • the DNA vector i ⁇ directly introduced by injection via the orifice and duct leading into the lumen or the mammary gland.
  • the DNA vector can be introduced into the lumen of the gland in a plurality of transformation events.
  • the epithelial cells take up the DNA vector and are capable of expressing the transgene to produce a transgenic product.
  • mice are injected with oxytocin (250 mlU) and milked 5 minutes later with a suction device.
  • the milk is either diluted with an equal volume of phosphate buffer saline or fractionated as described by Imam et al., "Isolation and Characterization of a Major Glycoprotein From Milk Fat Globule Membrane of Human Brea ⁇ t Milk," Biochem. J. , 193:47-54 (1981); and, Patton and Hu ⁇ ton, "A Method For I ⁇ olation of Milk Fat Globule ⁇ ," Lipid ⁇ 21:170-174 (1986).
  • the casein pellet is washed once with one volume of wash buffer and resu ⁇ pended in RIPA buffer.
  • Membranes and vesicle ⁇ contained in the resultant skim supernatant were collected by further centrifugation at 100,000g for 2 hours.
  • CFTR-containing immunoprecipitates is achieved by incubating with protein kinase A and [ ⁇ 32P]ATP (10 ⁇ Ci) in a final volume of 50 ⁇ l in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl- and 100 ⁇ g ml ⁇ bovine serum albumin) at 30°C for 60 minute ⁇ .
  • kinase buffer 50 mM Tris-HCl, pH 7.5, 10 mM MgCl- and 100 ⁇ g ml ⁇ bovine serum albumin
  • N-glycanase endoglyco ⁇ ida ⁇ e H and endoglyco ⁇ ida ⁇ e F are obtained from Genzyme Corp. (Cambridge, Ma ⁇ achu ⁇ ett ⁇ ) .
  • Condition ⁇ for dige ⁇ tion with the re ⁇ pective glycosidase ⁇ were a ⁇ specified by the manufacturer, except that incubations are performed at 37°C for 4 hours only.
  • [ ⁇ 32P]ATP [ ⁇ 32P]ATP, ⁇ eparated on SDS-polyacrylamide gels, and then extracted from the gels by macerating the gel pieces in protein extraction buffer (50 mM ammonium bicarbonate, 0.1% SDS and 0.2% ⁇ -mercaptoethanol) . Eluted proteins are recovered by trichloroacetic acid precipitation.
  • Example 4 features the transformation of the mammary gland of goat ⁇ with a tran ⁇ gene encoding for ti ⁇ ue-type plasminogen activator (TPA) .
  • the expression vector WAP-TPA was generated by fusing a 2.6 kilobase EcoRI-Kpnl fragment upstream of the murine whey acid protein gene to a cDNA encoding wild-type human TPA.
  • the DNA vector i ⁇ placed as a high concentration solution, or in a pellet form into the lumen of the mammary gland for tran ⁇ fection.
  • the DNA is placed in the mammary gland of a goat prior to puberty or during rapid proliferation of the areolar structure ⁇ in the developing mammary gland during pregnancy, during the period of time when the mammary gland undergoes rapid proliferation. Proliferation of mammary gland can occur either during early pregnancy or induced with steroid hormones regimes. Alternatively, the DNA implant can be made during the first pregnancy of the female at a time where maximum proliferation occurs.
  • the vector ⁇ y ⁇ tem can be delivered ⁇ everal time ⁇ during the proliferative event to increa ⁇ e the number of tran ⁇ fected ⁇ ecretory cell ⁇ .
  • the animal produces TPA during the animal's lactation period.
  • the milk of transgenic goats is collected and frozen.
  • the transgenic goat' ⁇ milk containing LAtPA is thawed, pooled and the pH adju ⁇ ted to 4.4 using glacial acetic acid.
  • the resultant precipitate is removed by centrifugation at 8,000xg for 20 min. at 4°C.
  • the ⁇ upernate is adjusted to pH 5.5 with NaOH and filtered through a 0.22 ⁇ polypropylene filter cartridge.
  • This whey fraction i ⁇ applied at a flow rate of 100 cm/h to a Butyl-Toyopearl 650C column (2.5 x 6.5 cm) which is equilibrated in 0.02 M sodium phosphate, 0.1 M arginine-HCl, 0.01% tween 80, pH 6.0.
  • the column is ⁇ ubsequently wa ⁇ hed with equilibration buffer and the LAtPA eluted with 0.2 M ⁇ odium pho ⁇ phate, 0.1 M arginine-HCl and 70% ethylene glycol.
  • Fractions containing LAtPA, as judged by indirect amidolytic assay, are pooled and diluted 1:2 with 100 mM arginine-HCl, pH 6.0 and loaded at 15 cm/h on to an 1 x 15 cm immunoaffinity column constructed with an anti-human tPA monoclonal antibody designated 17-3.
  • the column is washed extensively and LAtPA eluted from the column with 0.1 M glycine, 0.4 M arginine-HCl, 0.5 M NaCl, 0.01% tween 80, pH 2.5. Elution of the enzyme from the column i ⁇ monitored by absorbance (280 nm) and LAtPA containing fractions are immediately pooled and the pH adju ⁇ ted to 7.0 with NaOH.
  • the ⁇ ample was then filtered through a 0.22 ⁇ Millex-GV filter and applied to a 250 x .4 mm Synchrom Syncropak pentyl HPLC column.
  • the column is equilibrated and washed with 20 mM sodium phosphate, 150 mM NaCl, 8 mM epsilon amino-n-caproic acid (EACA) , pH 6.0, developed with a 0-90% propylene glycol gradient and protein elution monitored by absorbance at 280 nm.
  • Protein containing fraction ⁇ are analyzed by SDS gel electrophore ⁇ i ⁇ , protein band ⁇ visualized by Coomassie blue staining and fractions pooled to provide a preparation of greater than 98% protein purity.
  • Purified LAtPA is ⁇ tored frozen at -80°C.
  • the blots are blocked with 5% (w/v) nonfat dry milk to reduce nonspecific binding and the LAtPA band ⁇ were vi ⁇ ualized by a combination of a rabbit anti-human tPA antibody (American Diagnostica, Chicago, IL) , a goat anti-rabbit IgG conjugated to horseradish peroxidase (Cappel Corp., We ⁇ tchester, PA) and the Enhanced Chemiluminescence System (Amersham Corp., Arlington Height ⁇ , IL) .
  • a rabbit anti-human tPA antibody American Diagnostica, Chicago, IL
  • a goat anti-rabbit IgG conjugated to horseradish peroxidase Cappel Corp., We ⁇ tchester, PA
  • the Enhanced Chemiluminescence System Amersham Corp., Arlington Height ⁇ , IL
  • Amino acid ⁇ equence analysis is performed using an Applied Bio ⁇ ystem ⁇ 477 ga ⁇ pha ⁇ e ⁇ equencer.
  • PTH amino acid analy ⁇ is i ⁇ performed with an on-line Applied Biosystems 2.1 x 220 mm PTH C-18 column.
  • Carbohydrate compositional analysi ⁇ is performed according to the method of Hardy et al, "Monosaccharide
  • sample protein ⁇ (Dionex, Sunnyvale, CA) .
  • 6 to ten nmoles of sample protein ⁇ are hydrolyzed in 2 M trifluoroacetic acid, for 2 h at 121°C.
  • Sialic acid content of the protein ⁇ i ⁇ determined by thiobarbituric acid method as modified by Powell and Hart,
  • transfection event can be facilitated with the u ⁇ e of retroviru ⁇ e ⁇ and lipo ⁇ ome ⁇ .
  • pre ⁇ ent examples feature the use of goats and mice, any domestic animal such as cow ⁇ , swine and other animals ⁇ uch as rabbits, mice, rats and guinea pigs may be used.
  • the present invention is amenable to the making of any transgenic product for which cDNA or genomic DNA can be identified.
  • the present method ⁇ for tran ⁇ forming ⁇ pecific organ ⁇ or gland ⁇ allow ⁇ the production of the tran ⁇ genic product to be controlled by the ⁇ election of regulatory region ⁇ , and by removal of none ⁇ ential glands or organs producing the tran ⁇ genic product. Thu ⁇ , the pre ⁇ ent delivery ⁇ y ⁇ tem may be used to obtain transient expression of the transgenic product from 30 to 40 days.
  • the present invention is also suitable for therapeutic applications where it is desirable to complement an animal ⁇ ' characteristic genotype with a transgene.
  • Such application ⁇ are u ⁇ eful where, by virtue of a mutation or other event, the individual lack ⁇ a particular gene; or where, by virtue of di ⁇ ea ⁇ e, the individual cannot make a gene product; for example, diabetic ⁇ cannot make in ⁇ ulin; or where, by virtue of di ⁇ ea ⁇ e, the individual make ⁇ a defective gene product.
  • any other gland or organ sy ⁇ tem having a lumen lined with epithelial cells can be transformed in a similar manner.
  • the present invention i ⁇ capable variation and modification and, therefore, the present invention ⁇ hould not be limited to the preci ⁇ e detail ⁇ ⁇ et forth, but ⁇ hould include ⁇ uch change ⁇ in alteration ⁇ a ⁇ fall within the purview of the following claim ⁇ .

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Abstract

L'invention concerne un animal non humain qui possède au moins un organe ou une glande, ledit organe ou glande présentant une lumière doublée de cellules épithéliales capables d'introduire des sécrétions dans la lumière, au moins une ou plusieurs cellules épithéliales comprenant un transgène absent du génotype caractéristique de l'animal, cette cellule étant capable d'exprimer ledit transgène afin de former un produit transgénique. L'invention porte également sur un procédé permettant de donner un trait désiré à un animal par la lumière et dans les cellules épithéliales, ces dernières étant capables d'exprimer le transgène afin de fabriquer un produit transgénique.
PCT/US1993/008618 1992-09-10 1993-09-10 Procede de production in vivo d'un organe transgenique par introduction du transgene dans une lumiere WO1994005782A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020013A1 (fr) * 1994-12-24 1996-07-04 Cambridge University Technical Services Limited Amelioration de la fonction endometriale ou s'y rapportant
WO1998015634A1 (fr) * 1996-10-07 1998-04-16 Wisconsin Alumni Research Foundation Promoteurs specifiques a la glande mammaire
US5824543A (en) * 1995-06-05 1998-10-20 New York University Method for expression and isolation of biologically active molecules in urine using a mouse uroplakin-II promoter
US6210736B1 (en) 1997-06-17 2001-04-03 Genzyme Transgenics Corporation Transgenically produced prolactin
KR100582915B1 (ko) * 1994-12-24 2006-08-30 캠브리지 유니버시티 테크니칼 서비스 리미티드 자궁내막기능또는자궁내막기능과관련된기능의개선방법
US8722384B2 (en) 1997-04-11 2014-05-13 Calgene Llc Methods and compositions for synthesis of long chain polyunsaturated fatty acids

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WO1993012756A2 (fr) * 1991-12-17 1993-07-08 The Regents Of The University Of California Transfection pulmonaire par administration transgenique aerosol
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ROSENFELD, M.A. ET AL.: "In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium", CELL., vol. 68, no. 1, 10 January 1992 (1992-01-10), CAMBRIDGE, NA US, pages 143 - 155 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020013A1 (fr) * 1994-12-24 1996-07-04 Cambridge University Technical Services Limited Amelioration de la fonction endometriale ou s'y rapportant
AU712278B2 (en) * 1994-12-24 1999-11-04 Cambridge University Technical Services Limited Improvements in or relating to endometrial function
US6472374B1 (en) * 1994-12-24 2002-10-29 Cambridge University Technical Services Limited Endometrial function
KR100582915B1 (ko) * 1994-12-24 2006-08-30 캠브리지 유니버시티 테크니칼 서비스 리미티드 자궁내막기능또는자궁내막기능과관련된기능의개선방법
US5824543A (en) * 1995-06-05 1998-10-20 New York University Method for expression and isolation of biologically active molecules in urine using a mouse uroplakin-II promoter
WO1998015634A1 (fr) * 1996-10-07 1998-04-16 Wisconsin Alumni Research Foundation Promoteurs specifiques a la glande mammaire
US8722384B2 (en) 1997-04-11 2014-05-13 Calgene Llc Methods and compositions for synthesis of long chain polyunsaturated fatty acids
US6210736B1 (en) 1997-06-17 2001-04-03 Genzyme Transgenics Corporation Transgenically produced prolactin

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