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WO1993008292A1 - Transformation induite par des particules de cellules somatiques animales - Google Patents

Transformation induite par des particules de cellules somatiques animales Download PDF

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Publication number
WO1993008292A1
WO1993008292A1 PCT/US1992/008848 US9208848W WO9308292A1 WO 1993008292 A1 WO1993008292 A1 WO 1993008292A1 US 9208848 W US9208848 W US 9208848W WO 9308292 A1 WO9308292 A1 WO 9308292A1
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WIPO (PCT)
Prior art keywords
animal
cells
protein
carrier sheet
skin
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Application number
PCT/US1992/008848
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English (en)
Inventor
William F. Swain
Ning-Sun Yang
Dennis E. Mccabe
Brian F. Martinell
Liang Cheng
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Agracetus, Inc.
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Priority to JP5507826A priority Critical patent/JPH06503479A/ja
Publication of WO1993008292A1 publication Critical patent/WO1993008292A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the present invention relates to the technologies of genetic transformation in general and relates, in particular, to strategies for the genetic transformation of the non-germ line cells of whole animals to achieve circulating levels of therapeutic proteins.
  • somatic cell transformation avoids many of the ethical and philosophical problems which would arise from human intervention with the germ lines of human beings.
  • the genetically engineered somatic cells offer the ability to make genetic corrections for inherited genetic disorders which consist of inactive or deleted enzymes or structural proteins that are necessary for normal biological functioning. It is also possible that such genetic transformations of somatic cells, and not germ line cells, may be desirable for certain therapeutic applications. For example, certain proteins offering therapeutic utility to patients must be currently injected into patients on a periodic strict time-line basis.
  • an alternative strategy might be to engineer the gene for the desired protein into somatic cells of the animal or human so that the transformed cells would produce the therapeutic protein at a consistent level while they are live.
  • somatic cells which have a pre-defined and ascertainable life expectancy, such as skin cells for example, it is possible to create such an in vivo therapeutic production system which is time limited in the administration of the protein dosage to the animal or person being treated.
  • hormones or other growth factors or proteins for animal improvement, therapeutic, or disease inhibiting purposes into somatic cell portions of the animal which are not transient but which stay with the animal for its life expectancy.
  • the transforming DNA is coated onto very small particles which are physically accelerated by actually being shot on a ballistic projectile into the tissues to be transformed. While this apparatus has been demonstrated to have utility in transforming plant cells in culture, it suffers from a deficiency in that the adjustability of the force of impact of its particles is lacking making it a difficult apparatus to use for transformation of organisms over a wide range of kinetic energies of insertion of the particles into the transformed tissue.
  • the present invention is directed toward a method of transforming the somatic cells of animals in vivo in which the exogenous DNA construct including a sequence coding for the protein desired to be expressed in the somatic animal cells, .and linked to a promoter operative in animal cells, and including a signal peptide capable of causing secretion of the protein, is coated onto small microparticles being of sufficiently small size so as to be able to enter the cells of animals without disrupting their biological function, placing an animal at a target site, and then accelerating the particles at the target animal and into the cells of the target animal to thereby genetically transform a portion of the cells so treated so as to transform in vivo in the animal a number of cells to produce and secrete the protein coded by the exogenous gene. It is a further object of the present invention to provide animals which have been treated with foreign DNA so that their somatic cells contain therein both an expressing exogenous gene construct and a very small particle of metallic material which carried the gene construct into the animal cell.
  • Fig. 1 is an exploded perspective view of apparatus used to perform the method of the present invention.
  • Fig. 2 is a top plan view of the discharge chamber of the apparatus of Fig. 1.
  • Fig. 3 is a schematic illustration of a plasmid pWRG1601 used in one of the examples below.
  • Description of the Preferred Embodiment The present invention is directed toward the transformation of the somatic cells of animals or human beings.
  • somatic cells as used herein it is meant to describe those cells of an animal or human being which when transformed do not change the genetic character or makeup of any of the germ or sex cells of the organism, so that when the animal or human reproduces through normal biological forms of reproduction, the introduced exogenous genetic material is not passed to the biological progeny of the organism.
  • genetically transforming somatic cells with a gene encoding a protein that includes a secretory signal peptide sequence By genetically transforming somatic cells with a gene encoding a protein that includes a secretory signal peptide sequence, circulating levels of therapeutic proteins can be achieved for long periods of time.
  • the animal somatic cells transformed may be of any suitable tissue type in the target animal.
  • Preferred target tissues include skin, muscle tissue and internal organ tissues, all of which may be transformed .in vivo. Somatic cells of tissues which are not normally exposed in the animal, i.e. internal organs, may be temporarily surgically exposed for the brief transformation procedure.
  • Suitable target organs for somatic cell transformations also include the liver, spleen, pancreas, heart, kidney, brain, bone marrow, breast, sex organs, thyroid, organs of the gastro-intestinal tract and circulating cells such as leukocytes.
  • the invention is directed toward the introduction of exogenous, often chimeric, genetic constructions into animal somatic cells.
  • exogenous genetic constructions consist of DNA from another organism, whether of the same or different species, which is introduced into the transformed organism through human manipulation, by the artificial introduction of genes into the cells of the transformed organism.
  • the exogenous DNA construction would normally include a coding sequence for a transcription product or a protein of interest, together with flanking regulatory sequences effective to cause the expression of the protein or the transcription product coded for by the coding sequence in the transformed cells of an organism.
  • flanking regulatory sequences are a promoter sequence sufficient to initiate transcription and a terminator sequence sufficient to terminate the gene product, coded for by the gene, whether by termination of transcription or translation.
  • Suitable transcriptional enhancers or enhancers of translational efficiency can be included in the exogenous gene construct to further assist the efficiency of the overall transformation process and expression of the protein result in the transformed cells.
  • Introns may also be included in the genetic construction to facilitate transcription and to provide for proper processing and transport of the transcribed RNA.
  • Other gene products than proteins may also be expressed by the inserted genetic construction.
  • the inserted construction could express a negative RNA strand effective either to suppress the expression of a native gene or to inhibit a disease pathology.
  • the inserted construction could itself be RNA, as an alterative to DNA, if only transient expression of the gene product was desired.
  • a signal peptide sequence is a protein-coding DNA sequence located at the 5', or upstream, end of a protein-coding DNA sequence.
  • the signal peptide itself is an amino terminal portion of the immature protein which directs sorting of the protein to various compartments in the cell, and directs co-translational and post-translational processing of the protein produced. This processing typically involves transport of the protein across cell membranes.
  • a secretory signal peptide is a signal peptide which conditions secretion of the protein from the cell, through internal cell membrane compartments and ultimately into the extracellular fluid.
  • secretory signal peptides effective in mammalian cells have been identified and the signal peptide can either be the one natively associated with the protein to be expressed or can be a heterologous signal peptide joined to a foreign protein.
  • a suitable signal peptide from human growth hormone, HuGH is set forth in SEQ ID No. : 1 below.
  • regulated secretion There are two general types of secretory pathways, termed regulated secretion and constitutive secretion. In the former, the secretory proteins are stored in an intermediate vesicle called a secretory granule and are released by fusion of the secretory granule membrane and the plasma membrane in response to a secretogogue.
  • secretory granules are not observed and although the secretory protein apparently transverses the various membranes to the outside of the cell, it is not clear how this happens.
  • the secretory hormones insulin and growth hormone are normally secreted by the regulatory pathway when produced in their normal sites, islet cells and the anterior pituitary respectively.
  • the regulatory pathway used is, however, generally dependent on cell type as much as the protein so it would be expected that human growth hormone secretion in skin cells would follow a constitutive pathway, since skin cells other than sebacious gland cells do not normally exhibit regulated secretion.
  • transformation is used to describe genetic transformation, or the process of insertion of foreign genes into living cells and the expression in the cells of proteins or other gene products encoded by the foreign genes.
  • transformation is not intended to be used to describe the onset of malignant activity by a cell or cell line, which is also sometimes referred to as a "transformation. "
  • the present invention makes particular use of an apparatus for using an adjustable electric discharge to create a gaseous shock wave to physically accelerate DNA coated onto small particles into the genetic material of somatic animal cells.
  • a suitable apparatus for use within the present invention is illustrated in Fig. 1.
  • the apparatus consists of a spark discharge chamber 12 into which are inserted two electrodes 14 which are spaced apart by a distance of approximately 1 - 2 mm.
  • the spark discharge chamber is a horizontally extended rectangle having two openings 16 and 18 out its upward end. One opening 18 is covered by an access plate 20.
  • the other opening, located opposite from the electrodes 14 is intended to be covered by a carrier sheet 22.
  • the electrodes 14 are connected to a suitable adjustable source of electric discharge voltage.
  • Such a source of electric discharge voltage would preferably include suitable electric switching connected to a capacitor of the 1 to 2 micro farad size range, with the amount of the voltage of the charge introduced into the capacitor being adjustable, such as through the use of an autotransformer, through a range of, for example, 1 to 50,000 volts.
  • Suitable switching is provided so that the capacitor can be discharged through the electrodes 14 safely and conveniently by a user.
  • the carrier sheet 22 intended to be placed upon the opening 18 on the spark discharge chamber 12 is preferably a sheet of aluminized saran coated mylar although any other light, strong, durable sheet material may also be used.
  • a retaining screen 24 Above the opening in the discharge chamber, placed approximately 5 - 10 millimeters above it, is a retaining screen 24. Placed approximately 5 - 25 millimeters above the retaining screen is a target surface 26.
  • the exogenous foreign gene construct intended to be transformed into the animal somatic cells is prepared by suitable DNA preparation techniques well known to one of ordinary skill in the art and it is coated onto small particles of a durable dense material such as gold, the particles typically being 1 to 3 microns in size.
  • the carrier particles with the DNA coated thereon is then placed upon the carrier sheet 22 which is inserted on top of the spark discharge chamber 12.
  • a target tissue such as a live and anesthetized animal, is then placed adjacent to the target surface 26.
  • a small droplet of water approximately 2 - 4 microliters in size, is placed bridging between the ends of the electrodes 14.
  • the access plate cover 20 is then placed over the top of the discharge chamber 12. At this point, the atmosphere between the carrier sheet 22 and the target is largely replaced with helium, by enclosing the apparatus and target and introducing helium in the enclosure in sufficient quantity to largely displace the atmospheric gases.
  • the initiation of the spark discharge between the electrodes may be initiated by means of the use of the appropriate electronic switching.
  • the force of the electric discharge bridges the spark discharge cap between the electrodes 14 instantly vaporizing the small droplet of water placed therebetween.
  • the force of the vaporization of that water creates a gaseous shock wave within the spark discharge chamber 12 which radiates outward in all directions.
  • the impact of the shock wave upon the carrier sheet 22 propels the carrier sheet 22 upwards with great velocity.
  • the upwardly traveling carrier sheet 22 accelerates upward in direction until contacting the retaining screen 24.
  • the presence of the helium provides less drag on the flight of the carrier sheet and on the carrier particles as well as less force for the shock wave to propagate to the target.
  • the carrier sheet 22 is retained, and the DNA-coated particles previously applied thereto fly off of the carrier sheet and travel freely on toward the target surface.
  • the particles therefor proceed into the target surface and enter the cells thereof.
  • the momentum of the particles as they impact the surface of the target organism or tissue is adjustable based on the voltage of the initial electric discharge applied to the electrodes 14.
  • the velocity by which the particles impact the target can be adjusted, and thus the depth of penetration of the particles into the tissue of a target, can be continuously adjusted over the range of adjustment of the electric discharge throughout the electrodes 14.
  • the rates of application of DNA onto the carrier particles and of application of coated carrier particles onto the carrier sheet can also be adjusted to optimize performance of the device with different cell and tissue types.
  • Fig. 1 The apparatus of Fig. 1 has been previously demonstrated to be useful for the transformation of differentiated or undifferentiated tissue in a variety of forms including cellular masses in culture and whole growing organisms. It has been found through the work discussed herein that the apparatus is equally suitable for the transformation of either animal cells in culture or for the transformation of cells of various animal somatic tissues. It is also possible to transform portions of whole animals in vivo by anesthetizing the animal, as appropriate for the species and type of animal, and then placing the anesthetized animal over a hole cut in a planar surface which will act as the target surface. The portion of the animal exposed through the hole in the target surface 26 will therefore be the treated target tissue transformed by the transformation process.
  • the exogenous gene construction includes a protein coding sequence which includes, at its 5' end, a secretory signal peptide sequence.
  • the copies of the gene construction can then be carried by the carrier particles into the tissues of the patient animal.
  • the tissue can be a surface tissue or a internal tissue or organ temporarily exposed by surgery.
  • significant levels of circulating protein in the bloodstream of a patient animal can be achieved by a particle acceleration treatment to the intact epidermis of the animal. Such treatment to the epidermis results in protein production and circulation for an extended, though perhaps not permanent, period of time.
  • the skin tissue layer may be temporarily uncovered and the transformation blast may be applied to the underside skin layer, the dermis.
  • the skin is surgically separated from underlying muscle layers, a relatively simple procedure. This separation exposes the underside of the skin tissue layer, i.e. the dermis, which may then be treated by particle acceleration. Such treatment has been found to result in at least long term gene expression. Examples a) Vectors used
  • the first examples make use of a pair of chimeric expression vectors constructed so as to express in animals the enzyme chloramphenicol acetyltransferase (CAT) , which confers resistance to the antibiotic chloramphenicol.
  • CAT chloramphenicol acetyltransferase
  • Both chimeric gene expression plasmids have been previously described and demonstrated to be effective in animal transfection studies.
  • the plasmid pSV2cat was described by Gorman et al., Mol. Cell Biol. , 2:1044-1051 (1982) and the expression vector pRSVcat was described by Walker et al., Nature, 306:557-561 (1983).
  • the plasmid pSV2cat is a chimeric cat gene construction including the Simian virus 40 (SV40) early promoter, the chloramphenicol acetyltransferase coding region from the plasmid pBR322-Tn9, the SV40 t-antigen intron, and the SV40 early polyadenylation region carried in the pBR322 vector.
  • the plasmid does not contain a complete SV40 viral genome and is not infectious.
  • the plasmid pRSVcat is also a pBR322 base plasmid that includes a chimeric Rous Sarcoma virus (RSV) long terminal repeat and promoter fragment, the cat coding region from Tn9, an intron from the mouse beta-globulin gene and the polyadenylation region from the SV40 early transcription unit. This plasmid also does not contain a complete viral genome and is not infectious.
  • a related plasmid also used is designated pRSVNPTII and includes the Rouse Sarcoma Virus promoter, the coding region for the neomycin phosphotransferase-II gene, coding for resistance to the antibiotics kanamycin and G418, and a polyadenylation region from SV40. This plasmid as well does not contain a complete viral genome and is not infectious.
  • the vector pWRG1601 includes a segment formed from pGEM3 (Promega) including oppositely oriented phage promoters, and, in an expression cassette, the cytomegalovirus immediate-early promoter (pCMVieP) followed by the transcribed and 3' flanking regions of the human growth hormone (HuGH) gene as set forth in Seldon, et al., Molec. Cell Biol.. 6:3173-3179 (1986); DeNoto, et al., Nucl. Acid Res.. 9:3719-3730 (1981); and Seeburg, DNA , 1:239-249 (1982).
  • pGEM3 Promega
  • pCMVieP cytomegalovirus immediate-early promoter
  • Human growth hormone human growth hormone
  • the HuGH protein coding sequence includes, at its 5'end, a sequence encoding a secretory signal peptide.
  • a DNA sequence of 337 nucleotides is set forth as SEQ ID NO.: 1 below, which includes two exons which together code for a 26 amino acid signal peptide and also intron A of the HuGH gene.
  • pWRG1602 was derived from pWRG1601 by deletion of the Hind III fragments that contain the EBV regions of the plasmid as follows. pWRG1601 was digested with restriction endonuclease Hind III and the ends of the fragments produced made blunt by treatment with Klenow DNA polymerase and all four deoxynucleotide triphosphates.
  • pWRG1602 The structure of pWRG1602 is shown schematically in Figure 4.
  • pWRG1602 includes the CMV-HuGH gene and pGEM3 regions from pWRG1601, but deletes the EBV regions.
  • mice were anesthetized with chloroform. On each mouse, an area of approximately 1 c ⁇ r on its side was shaved. The mouse was then placed on a petri dish having a window cut in it with the shaved patch over the window.
  • DNA of pRSVcat was then coated onto 1-3 micron gold particles at a rate of 0.1 microgram of DNA per milligram of gold.
  • the DNA was applied to the gold by precipitation with 25mM spermidine with 6% polyethylene glycol (m.w. 3,000) with the addition of CaCl2 to a final concentration of 0.6 M.
  • the DNA coated gold beads were then rinsed in 100% ethanol and applied to the carrier sheet as an ethanolic suspension at a concentration of dried gold coated beads of 0.05 mg/cm 2 of the carrier sheet.
  • the petri dish with the mouse was placed over the apparatus of Figs. 1 and 2 as the target surface. Prior to the electric spark discharge, the area between the carrier sheet and the target was flushed with helium (4 liters/min) for 15 seconds to reduce atmospheric drag on the carrier sheet and any possible shock wave damage to the animal.
  • mice After the transformation event, the animals all appeared unharmed and they seemed to recover completely. No bruising or bleeding was observed in the test animals. After 24 hours the mice were sacrificed and the skin patch was removed and assayed for CAT activity. The assay was performed by testing for acetylation activity with a radio-labeled chloramphenicol. Radioactive decay of the acetylated product could then be used as a measure of transformed enzyme activity.
  • a (Xenopus) frog was anesthetized by chilling to 4° C.
  • the chilled frog was also placed over a window cut in a petri dish lid and placed in the transformation apparatus of Figs. 1 and 2 in the same fashion as with the mice.
  • mice The conditions and procedure used for the mice were repeated for the frog except for the following.
  • the DNA used was pSV2cat.
  • the DNA coated gold beads were loaded onto the carrier sheet at a density of 0.1 mg/cm .
  • levels of CAT activity were observed greater than 50 times background.
  • delivery and expression of a foreign gene was achieved in somatic cells without any identifiable damage or injury to the animal.
  • the DNA used in the rat liver transformation procedures was pRSVcat, coated at a rate of 1 microgram per milligram onto gold particles. This was done by combining 20 micrograms DNA, 100 microliter of buffer (150 mM NaCl, 10 mM Tris 8.0), 50 microliters of CaCl2 (2.5 M) and 20 milligrams of 1 micron gold powder. The mixture was then spun down, dried, and resuspended in ethanol prior to loading onto the carrier sheet. The loading rate on the carrier sheet was 0.05 milligrams of dried coated gold per square centimeter.
  • the space of the particle travel was flooded with 2 liters per minute helium at atmospheric pressure. No vacuum containment was used.
  • the rat livers were subjected to transformation events with spark discharge voltages of 10 or 14 kilovolts.
  • the excised liver tissues were analyzed for CAT activity.
  • the gold particles were found to have penetrated up to 300 microns into the liver tissue. The following is a summary of the results of the procedure, with the level of CAT activity indicated by percentage of substrate catalyzed, and also indicated as a percentage of a defined standard unit of CAT activity.
  • mice abdominal muscle tissues were similarly treated for gene transfer as described above for liver, and the results are shown in the following.
  • This example was intended to demonstrate circulating levels of a potentially therapeutic protein in blood achieved by gene transfer to skin.
  • BALB/c mice, 7 to 8 weeks old, (approx. 20 grams) were treated.
  • the human growth hormone expression plasmid pWRGl ⁇ Ol described above was used to express HuGH in the mice.
  • mice were anesthetized using a Ketamime and Ro pun mixture (10 ml and 2ml, respectively) by intraperitoneal injection of .05 ml. The lower half of the animals were shaved. Nair hair remover was used to remove remaining hair in the treatment area.
  • the copies of DNA of plasmid pWRG1601 were loaded on amorphous gold (Engehard 1740) carrier particles at a rate of 0.5 micrograms per milligram.
  • the DNA was precipitated on the carrier particles with calcium chloride and spermidine as described above.
  • the coated carrier particles were then loaded onto carrier sheets at a rate of 0.5 mg/cm 2 .
  • the electric discharge apparatus of Figs. 1 and 2 was set for 23 kV discharge.
  • a target surface was formed by an inverted cup-shaped target support with a hole cut in its top to correspond to the target area, and which was adjusted to keep the target animals at a constant height above the retaining screen.
  • the animal was placed on the target surface with the treatment area positioned over the hole.
  • a vacuum (15 mm of mercury) was drawn on the inside of the cup-shaped support, then the particle bombardment was performed. After treatment, the animals were examined. Some redness and occasional fragments of mylar were observed, but the animals otherwise seemed healthy.
  • the animals were sacrificed at 2, 4, 5 and 7 hours after treatment. After sacrifice, the treated area of skin, was removed, weighed and placed in 1 volume (g/ml) of TES buffer and minced until fine. The solids were concentrated and frozen until assayed. Whole blood was collected from each animal by cardiac puncture resulting in 750 microliters of blood per mouse. The blood was placed at 4°C overnight to clot and spun down the next day. The serum was frozen for analysis.
  • the tissues were later analyzed using the Allegro (TM) Human growth hormone radioimmunoassay (RIA) system from Nichols Institute. To calibrate the assay, control samples spiked with known amounts of human growth hormone were prepared.
  • TM Human growth hormone radioimmunoassay
  • Control samples were prepared from non-transformed mouse skin which were treated in parallel to the experimental samples. The control samples averaged 1755.5 counts, an unusual background level. Controls of mouse serum samples averaged 219 counts.
  • Tissues and serum from seven treated mice were analyzed at various times after treatment. The results are given below in Table 2.
  • the HGH protein is first expressed in situ at measurable levels approximately 5 hours post-blast, and continues to rise thereafter.
  • Blood levels of circulating protein are detectable 7 hours post-treatment.
  • mice were bombarded with single or multiple blasts of pWRG1602.
  • the plasmid DNA was loaded onto carrier particles at 2u_g DNA/mg carrier particles.
  • the coated carrier particles were layered on the carrier sheet at 0.5 mg/cm 2 .
  • the particle bombardment was conducted at 22 kV either once or four times on the same animal at separate sites.
  • the skin and blood of the animals was tested 24 hours post blast.
  • the values of HuGH given in Table 2 below are net after subtraction of background.
  • Plasmid DNA was loaded onto carrier particles at 2.5 u_g DNA/mg gold particles, and the coated particles were loaded onto the carrier sheet at a rate of 0.1 mg/cm .
  • Blasting was done at discharge voltages between 16 and 18 kV.
  • Rats were anesthetized and the abdominal cavity was surgically opened.
  • the liver of the animals was treated with a particle acceleration apparatus aimed at the exposed liver. After treatment, the incisions were sutured, and the rats maintained under standard animal care procedures. One day later, the animals were sacrificed, and serum and liver samples were collected. In repeated experiments on over 20 rats, levels of circulating growth hormone were achieved over background levels. The results of three replicates are summarized in Table 5.
  • Rats were, anesthetized and abdominal skin hair removed by a hair clipper and Nair treatment.
  • the abdomen was opened by scalpel incision and the tissues teased open to separate skin from muscle.
  • the fascia lining was removed from the underneath side of the skin tissue.
  • the particle acceleration procedure was performed on the underside of the skin layer (dermis) .
  • phosphate buffered saline was added and the skin was sutured. Controls were bombarded with uncoated carrier particles. For each data point, 1 ml of fluid was collected from the subcutaneous tissue space, and 100 microliter of this sample was assayed for human growth hormone by RIA. The results of two replicates are set forth in Table 6.
  • ADDRESSEE Quarles & Brady
  • NAME Seay, Nicholas J.

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Abstract

L'invention concerne un procédé de transformation avantageuse de cellules somatiques d'animaux afin d'introduire des protéines thérapeutiques dans l'animal. La transformation de cellules somatiques est utile pour le traitement médical et vétérinaire de maladies génétiques ainsi qu'à d'autres fins thérapeutiques ou d'amélioration de l'animal. Des copies d'une construction génétique exogène peuvent être appliquées sur une particule porteuse et être accélérées vers l'intérieur des cellules de l'animal in situ. Si la construction génétique exogène comprend un peptide signal sécrétoire, on sait alors que des niveaux de circulation de la protéine peuvent être atteints par simple insertion transdermique des particules porteuses.
PCT/US1992/008848 1991-10-16 1992-10-15 Transformation induite par des particules de cellules somatiques animales WO1993008292A1 (fr)

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JP5507826A JPH06503479A (ja) 1991-10-16 1992-10-15 動物体細胞の粒子−媒介トランスフォーメーション

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GB2282139A (en) * 1993-09-24 1995-03-29 Univ Reading Introducing DNA into the germ line of birds
EP0741578A4 (fr) * 1994-01-27 1998-05-20 Human Genome Sciences Inc Hormone de croissance humaine
US6596540B2 (en) * 2000-09-22 2003-07-22 Osaka University Method for introduction of an exogenous genetic substance or a physiologically active compound
US7122354B2 (en) 1996-09-13 2006-10-17 Transkaryotic Therapies, Inc. Nucleic acid encoding a chimeric polypeptide
EP1538202A3 (fr) * 1996-09-13 2007-05-30 Transkaryotic Therapies, Inc. Production de l'alpha-galactosidase A humaine
US7833742B2 (en) 2002-04-25 2010-11-16 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2282139A (en) * 1993-09-24 1995-03-29 Univ Reading Introducing DNA into the germ line of birds
EP0741578A4 (fr) * 1994-01-27 1998-05-20 Human Genome Sciences Inc Hormone de croissance humaine
US5962411A (en) * 1994-01-27 1999-10-05 Human Genome Sciences, Inc. Human growth hormone variants and methods of administering same
US6566328B1 (en) 1994-01-27 2003-05-20 Human Genome Sciences, Inc. Human growth hormone
EP1538202A3 (fr) * 1996-09-13 2007-05-30 Transkaryotic Therapies, Inc. Production de l'alpha-galactosidase A humaine
US7122354B2 (en) 1996-09-13 2006-10-17 Transkaryotic Therapies, Inc. Nucleic acid encoding a chimeric polypeptide
EP2327775A3 (fr) * 1996-09-13 2012-10-24 Shire Human Genetic Therapies, Inc. Production de l'alpha-galactosidase A humaine
EP2374876A3 (fr) * 1996-09-13 2012-10-24 Shire Human Genetic Therapies, Inc. Thérapie pour l'insufficance d'alpha-galactosidase A
NO340408B1 (no) * 1996-09-13 2017-04-18 Shire Human Genetic Therapies Fremgangsmåte for å produsere humant alfa-galaktosidase A, og farmasøytisk preparat
US6596540B2 (en) * 2000-09-22 2003-07-22 Osaka University Method for introduction of an exogenous genetic substance or a physiologically active compound
US7833742B2 (en) 2002-04-25 2010-11-16 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency
US9267166B2 (en) 2002-04-25 2016-02-23 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency
US9523113B2 (en) 2002-04-25 2016-12-20 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency
US11116823B2 (en) 2002-04-25 2021-09-14 Takeda Pharmaceutical Company Limited Treatment of α-galactosidase a deficiency

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JPH06503479A (ja) 1994-04-21

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