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WO2000031267A1 - Production d'insuline par des cellules musculaires genetiquement modifiees - Google Patents

Production d'insuline par des cellules musculaires genetiquement modifiees Download PDF

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Publication number
WO2000031267A1
WO2000031267A1 PCT/EP1999/009132 EP9909132W WO0031267A1 WO 2000031267 A1 WO2000031267 A1 WO 2000031267A1 EP 9909132 W EP9909132 W EP 9909132W WO 0031267 A1 WO0031267 A1 WO 0031267A1
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cells
insulin
gene
muscle
dna
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PCT/EP1999/009132
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English (en)
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Fatima Bosch
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The Autonomous University Of Barcelona
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Priority to AU15570/00A priority Critical patent/AU1557000A/en
Publication of WO2000031267A1 publication Critical patent/WO2000031267A1/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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • 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
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • Insulin -dependent diabetes mellitus results from autoimmune destruction of the pancreatic islet ⁇ -cells, leading to long term insulin deficiency, hyperglycemia and the development of secondary microvascular and neurological complications (Rudderman, et al FASEB J , 1992, 6, 2905-2914)
  • the risk of complications increases with the degree of hyperglycemia Intensive insulin therapy can delay the onset and slow the progression of microvascular complications (The Diabetes Control and Complication Trial Research Group, N Engl J Med , 1993, 329, 977-986)
  • this kind of treatment cannot be easily implemented for all diabetic patients, especially in the very young and very old
  • myoblast cell lines like C2C12 have the advantage of a rapidly dividing in the myoblast stage and the potential for differentiating into non-dividing myotubes, a crucial feature for viability after transplantation.
  • These cells can be genetically engineered, and are amenable to production in large quantities in the myoblast state and then subjected to differentiation to allow production of the recombinant proteins with the use of a suitable promoter, and kept alive for long term delivery of the recombinant protein.
  • primary myoblast were isolated from a host, and then genetically engineered to produce, for example insulin, they can be stably returned to the host by simple injection into the muscle, where they will fuse with the other muscle fibers (Barr and Leiden, Science, 1991 , 254, 1507-1509; Dai, Proc Natl Acad Sci USA., 1992, 89, 10892-10895, Dhawan, Science, 1991 , 254, 1509-1512) or upon encapsulation (Deglon, Human gene Therapy, 1996, 7, 2135-2146; Rinsch, Human Gene Therapy, 1997, 8, 1881-1889) allow for the secreted products to enter the circulation.
  • An aspect of the invention features a process of treating diabetes in a subject comprising the step of administering to the subject a DNA segment or gene encoding for insulin, the gene being under the control of a promoter sequence in which the promoter sequence is operably linked to the to the insulin gene and is effective for the expression of a therapeutically effect amount of insulin in the diabetic subject. It is expected that the expression of insulin in diabetic subject will help to obtain a tighter control of the subjects glucose level in conjunction with conventional exogenous insulin treatment administered with meals in the case of type 1 diabetes mellitus or in conjunction with other antidiabetic therapies (e.g.
  • sulfonylureas biguanides, ⁇ -glucosidase inhibitors, glitazones, and other insulin secretagogues and insulin-enhancing agents) for type 2 diabetes mellitus.
  • therapeutically effective amounts means an amount of the expressed insulin which is sufficient to effect glucose uptake into cells, tissues and organs of a diabetic subject, and can be determined without undue experimentation.
  • insulin refers to a mature, active, polypeptide comprising substantially of the amino acid sequence of the natural hormone insulin. As an example of such an insulin is human insulin or porcine insulin or l-ispro insulin.
  • the insulin coding sequence of the DNA segment can be the same or substantially the same as the coding sequence of the endogenous insulin coding sequence as long as it encodes a functional insulin protein.
  • the DNA segment can also be the same or substantially the same as the insulin gene of a non-human species as long as it encodes a functional insulin protein.
  • the insulin gene in the DNA segment is preferably under the control of a promoter sequence different from the promoter sequence controlling the endogenous coding sequence, e.g. a promoter sequence which remains activated or induced during diabetic conditions in the patient.
  • promoter sequences include the muscle specific myosin light chain (MLC) promoters - MLC1 , MLC2, MLC3; creatine kinase, and myoD.
  • the present invention comprised of a transformed myoblast or muscle cell capable of producing mature insulin.
  • the term "transformed cell” refers to a cell that have been transfected with either homologous (same species) or heterologous (different species) gene coding for the insulin.
  • muscle cell refers to any cells human or non-human of muscle lineage or has the propensity to form a muscle cell, e.g. precursor muscle cell or myoblast.
  • muscle cells are the mouse lines C2C12, C3H/10T1/2, rat L6 and L8, human HISM.
  • the DNA segment is introduced to the diabetic patients in muscle or myoblast cells, wherein the cells are treated in vitro to incorporate therein the DNA fragment and, as a result, the cells express in vivo in the diabetic patient a therapeutically effective amout of insulin.
  • the DNA segment can be introduced into the cell by standard gene transfection methods, e.g. calcium phosphate precipitation or by a viral vector, e.g. adenoviral vector.
  • the cells may be introduced to the host by standard transplantation techniques, or in a neoorgan, or in a matrix, e.g. microencapsulated in sodium alginate, or contained within a immunoprotected cell factory.
  • the DNA segment is directly introduced into the muscle of the diabetic patient, e.g. not contained within a cell.
  • the DNA segment can be introduced in a vector.
  • suitable vectors include viral vectors (e.g. retroviral vectors, adenoviral vectors, adeno-associated viral vectors, Sindbis viral vectors, and herpes viral vector), plasmids, cosmids, and yeast artificial chromosomes.
  • the DNA segment can also be introduced as infectious particles, e.g., DNA-ligand conjugates, calcium phosphate precipitates, and liposomes.
  • FIG. 1 Expression of human insulin in C2C12 differentiated myotubes.
  • A Schematic representation of the MLC1/lnsm chimeric gene.
  • B Northern blot analysis of MLC1/lnsm chimeric gene expression in total RNA obtained from control C2C12neo and C2c12lnsm from day 0 to day 4 of differentiation along with the reference level of myogenin, and ⁇ -actin expression.
  • FIG. 3 HPLC analysis of insulin immunorecative products in cells extracts and in culture media of C1C12lnsm cells. After 3 days of differentiation. Culture media (A) and cell extracts (B) were subject to HPLC fractionation and RIA analysis. Arrows indicate the position of the mature insulin and proinsu ⁇ n peaks according to the elution time relative to the standards. Standard medium containing 5 X 10 " M porcine insulin and 5 X 10 "5 M human recombinant insulin (o); culture medium and cell extracts from C2C12neo (Q) and C2C12lnsm (»).
  • FIG. 4 Insulin production by differentiated C2C12lnsm myoblast cells.
  • A Myoblast cell were differentiated and at indicated times, aliquots of the culture medium were obtained and analyzed for immunoreactive insulin by RIA, C2C12neo (o); C2C12lnsm (•).
  • B Myoblast cell were maintained differentiated for 42 days. At the indicated days C2C12lnsm (•) cells were incubates for 2 hrs in serum free medium and immunoreactive insulin in the medium was maesured. Results are expressed mean + SEM of three different experiments, each performed in triplicates.
  • FIG. 1 Effect of insulin gene expression on the rate of glucose uptake and lactate production by C2C12lnsm differentiated myotubes.
  • Cells were differentiated for 3 days and then cultured overnight in serum-free medium. At the indicated times, aliquots of the medium fromC2C12neo (o) and C2C12lnsm (•) cells were obtained. Glucose concentrations (A) and lactate production (B) were determined. Results are the mean + SEM of three different experiments, each performed in triplicates.
  • Figure 6 Effect of insulin produced buy C2C12lnsm cells and exogenous insulin on PEPCK gene expression in FTO-2B hepatoma cells.
  • Total RNA from the FTO-2B cells were analyzed by Northern blot analysis using a PEPCK cDNA as a probe.
  • A A representative Northern blot is shown;
  • B Densitometric analysis of autoradiograms was performedand results expressed a % of the basal PEPCK gene expression. Results are the mean + SEM of three different experiments.
  • Figure 7. Effect of transplantation of C2C12lnsm cells into skeletal muscle of diabetic mice.
  • mice were transplanted with C2C12 (o) or C2C12lnsm (•) myoblast cells .
  • Insulin (A) and glucose (B) concentrations were measured during the three weeks after transplantation. Plasma insulin concentration of healthy C3H mice is indicated in (A) ( ⁇ ).
  • the therapeutic process of the invention allows for the production, at higher levels than pretreatment of insulin in a diabetic patient.
  • the production of insulin in the diabetic patient results in an increase in the target tissue level and or circulating levels of the hormone that results in an increased uptake of glucose into cells within the patient as evident in the increased disposal of glucose from the blood stream after a meal.
  • DNA segment herein is any exogenous DNA construct which includes a sequence encoding for a proinsulin polypeptide (Bondy & Rosenberg, Metabolic Control and Disease, Eigth Ed., 1980, pp. 284-287, WB Saunders) that may be processed to a functional insulin (Bondy & Rosenberg, Metabolic Control and Disease, Eigth Ed., 1980, pp. 284-287, WB Saunders), and the insulin is expressed by cells into which the DNA segment is introduced.
  • the DNA segment can be introduced into both somatic muscle cells of a patient, or may be introduced ex vivo into the muscle cells or precursor cells with the potential to differentiate into a muscle cells, myoblasts.
  • the muscles cells may or may not be derived from the patient treated, and may be derived from a non-human host species, e.g. mice, rat, pig.
  • the DNA segment therefore may or may not be an integral part of the patient's chromosome, and if the DNA segment is integrated into a chromosome, it may or may not be located at the same site as its corresponding endogenous gene sequence.
  • the DNA segment used to practice the therapeutic process includes an insulin gene or its complementary DNA (“cDNA”) that is substantially similar to the mammalian insulin gene from various species, e.g., mice, rat, pig, bovine, ovine and human, lispro insulin.
  • the insulin gene is an engineered proinsulin gene that will code for a proinsulin precursor peolypeptide that will be co ⁇ stitutively processed in muscle cells to a mature, active insulin (Vollenweider, et al., J Biol Chem., 1992, 267, 1429-14636; Yanagita, M., et al., FEBS Lett, 1992, 31 1 , 55-59; Groskreutz, D. J., et al., J Biol Chem., 1994, 269, 6241-6245).
  • the expression of the DNA segment is driven by a promoter which is expressed during diabetes conditions.
  • suitable promoters includes the strong muscle specific constitutive promoters, e.g. Myosin light chain promoters (Lee, et al, J Biol Chem., 1992, 267, 15875-15885; Greishammer, et al, Cell, 1992, 69, 79-93), Muscle creatine kinase promoter (Yi, T.M. et al. al., Nuc Acid Res, 1991 , 19: 3027-3033; Morlick, R.A. et al. al., Mol. Cell Biol, 1989, 9:2396-2413), Myogenin promoter (Cheng, T.C. et al.
  • the promoter is comprised of a cis-acting DNA sequence capable of directing the transcription of a gene in the appropriate environment, tissue, context.
  • Examples of cells targeted for the production of insulin includes primary muscle cells isolated from the patients or a different patient ( Salminer, et al., Human gene Therapy, 1991 , 2: 15-26;) are the mouse lines C2C12 (ATCC# CRL1772), C3H/10T1/2 (ATCC# CCL226), rat L6 (ATCC# CRL1458), L8 (ATCC# CRL 1769), human HISM (ATCC# CRL1692).
  • vectors both viral and non-viral, e.g. plasmid, cosmid, and yeast or bacterial artificial chromosomes and gene delivery systems available for either in vitro expression into cells utilized in ex vivo implantation or direct in vivo delivery of insulin into muscle cells or tissue of a patient.
  • vectors both viral and non-viral, e.g. plasmid, cosmid, and yeast or bacterial artificial chromosomes and gene delivery systems available for either in vitro expression into cells utilized in ex vivo implantation or direct in vivo delivery of insulin into muscle cells or tissue of a patient.
  • Viral Vectors for Delivery of an Insulin Gene can be used for delivery of an insulin gene.
  • viral vectors include recombinant retroviral vectors, recombinant adenoviral vectors, recombinant adeno-associated viral vectors, Sindbis viral vectors and recombinant herpes viral vectors.
  • retroviral vectors include recombinant retroviral vectors, recombinant adenoviral vectors, recombinant adeno-associated viral vectors, Sindbis viral vectors and recombinant herpes viral vectors.
  • the genome of a conventional recombinant retroviral vector is comprised of long terminal repeat ("LTR") sequences on both ends that serve a viral promoter/enhancer and transcription initiation site, a Psi site that serve as virion packaging signal and a selectable marker gene, for example a neomycin resistance gene.
  • LTR long terminal repeat
  • examples of such vectors include pZIP-NeoSV (Cepko, et al., Cell, 1984, 1953-1062).
  • the insulin gene can be cloned into a suitable cloning site in the retroviral genome. Expression is under the transcriptional control of the retroviral LTR.
  • the vector will drive the constitutive expression of insulin in myoblast or muscle cell. The level of expression is dictated by the promoter strength of the LTR.
  • the tissue selectivity is generally determined by the origin of the viral genome (for example, sarcoma virus/leukemia virus/mammary tumor virus).
  • the insulin gene can also be cloned into the vector linked to an internal promoter can confer tissue specificity to the control on gene expression (Lai, et al., PNAS USA, 1989, 86, 10006-10010; Scharfmann, et al., PNAS USA, 1991 , 88, 4626-4630).
  • Examples of internal promoter may be a general, strong constitutive promoter, for example the B-Actin promoter (Kawamoto, et al., MCB, 1988, 8, 267-272; Morishita, et al., BBA, 1991 , 1090, 216-222; Lai, et al., PNAS USA, 1989, 86, 10006-10010), the muscle specific myosin light chain 2 (Lee, et al., J Biol Chem., 1992, 267, 15875-15885; Shen, et al., MCB, 1991 , 1 1 , 1676-1685; Lee, et al., MCB, 1994, 14, 1220-1229),myosin light chain 1/3 (Grieshammer, et al., Cell, 1992, 69, 79-93; Donoghue, et al., Gene Dev., 1988, 2, 1779-1790), alpha-myosin heavy chain (Molkentin
  • retroviral vectors include, vLPGKSN (Valera, et al., Eur J Biochem., 1994, 222, 533-539), mLBSN (Ferrari, et al., Human Gene Therapy, 1995, 6, 733-742).
  • the glucokinase gene is cloned into the vector downstream from the internal promoter, generally as an expression cassette (Crystal, R.G., Science, 1995, 270, 404- 410).
  • the recombinant retroviruses capable of transducing the insulin gene into cells, in vivo, ex vivo, or in vitro and directing the synthesis of the insulin polypeptide in the infected cells are produced by transfecting the recombinant retroviral genome(s) into a suitable (helper-virus free) amphotropic packaging cell line.
  • a number of virus packaging cell lines are now available, PA317, Psi CRIP (Cometta, et al., Human Gene Therapy, 1991 , 2, 5-14, Miller & Buttimore, MCB, 1986, 6, 2895-2902; Cone & Mulligan, Proc Natl Acad Sci USA., 1984, 81 , 6349-6353).
  • the transfect virus packaging cell lines will package and produce recombinant retroviruses, shedding them into the tissue culture media.
  • the retroviruses are then harvested and recovered from the culture media by centrifugation as previously described (Compere, et al., MCB, 1989, 9, 6-14).
  • the viruses may be resuspended in a suitable buffer, for example, 10 mM HEPES (Sigma, St. Louis, MO) and stored at -70°C or under liquid nitrogen, (b) Recombinant Adenovirus vectors
  • Adenovirus vectors can be used for transducing an insulin expression cassette into cells (Berkner, et al., BioTechniques, 1988, 6, 616-629). Constitutive high levels of expression of the transduced gene products can be achieved. These vectors have the inherent advantage over the retroviral vectors in that they can infect replicating and not replicating cell, making them suitable vectors for somatic gene therapy (Mulligan, R.C., Science, 1993, 260,926-932).
  • Replication defective adenoviruses lacking the E1 region of the genome have been developed which will accomodate the insertion of 7.5 kilobases of foreign DNA (Crystal, R.G., Science, 1995, 270, 404-410; Logan & Shenk, PNAS USA, 1984, 81 , 3655-3659; Freidman, et al., MCB, 1986, 6, 3791-3797; Levrero, et al, gene, 1991 , 101 , 195-202; Imler, et al., Human Gene Therapy, 1995, 6, 71-721 ).
  • adenovirus particles can be propagated by transfecting the genome into cells engineered to express the E1 genes (Jones & Shenk, Cell, 1979,16, 683; Berkner, et al., BioTechniques, 1988, 6, 616-629).
  • This system allows the production of adenovirus particles at high titer (up to 10 13 /ml) which greatly enhance infection efficiency by enabling a higher multiplicity od infection (Crystal, R.G., Science, 1995, 270, 404-410).
  • Strategies for generating Adenoviral recombinants have been described (Berkner, et al., BioTechniques, 1988, 6, 616-629).
  • An expression cassette containing a regulatory and tissue specific promoter region for example the MLC-1 promoter linked to a DNA fragment encoding the human insulin with compatible 3' and 5' ends can be cloned into the Adeno-5 plasmid.
  • the entire recombinant Adenovirus genome is then generated by mixing the linearized Adeno-5-MLC-1 -human Insulin plasmid with a subgenomic fragment of Adenovirus DNA representing the 3.85-100 map units (prepared by digesting the In340 viral genome with C/al or Xba ⁇ ) (N.E. Biolabs, Beverly, MA) (Berkner, et al., BioTechniques, 1988, 6, 616-629).
  • the DNAs are then transfected into 293 cells (Graham, et al., J Gen Virol, 1977, 36, 59-72) essentially as described (Berkner & Sharp, Nuc Acid Res, 1983, 1 1 , 6003-6020).
  • Adeno-associated virus can also be used as a vector for transducing the insulin gene expression cassette (Fisher, K.L., et al., Nature Medicine, 1997, 3, 306-312).
  • AAV offers the advantage in that it has not been implicated in the etiology of any disease and its site specific integration on human chromosome 19 has not been shown to interferes host gene expression or promote gene rearrangements (Kotin, et al., PNAS USA., 1990, 87, 221 1 -2215; Samulski, et al., EMBO J., 1991 , 10, 3941 -1950).
  • AAV is capable of infecting postmitotic cells making it a suitable vector for delivery of genes to somatic cells.
  • the AAV genome contains two genes, rep and cap, and inverted terminal repeats
  • Such vectors are available in the plasmid form (Tratschin, et al., MCB., 1985, 5, 3251 -3260; Lebkowski, et al., MCB., 1988, 8, 3988-3996; McLaughlin, et al., J Virol., 1988, 62, 1963-1973).
  • the recombinant AAV genomes can be packaged into AAV particles by co- transfection of the vector plasmid and a second packaging plasmid carrying the rep and cap genes into an adenovirus-infected cell.
  • Such particles have been shown to efficiently transduce heterologous genes into mammalian cell lines, including muscle cells (Tratschin, et al., MCB., 1985, 5, 3251-3260; Lebkowski, et al., MCB., 1988, 8, 3988- 3996; McLaughlin, et al., J Virol., 1988, 62, 1963-1973; Flotte, et al., Am J Respir Cell Mol Biol., 1992, 7, 349-356; Fisher, K.L., et al., Nature Medicine, 1997, 3, 306-312).
  • Herpes virus vectors constitute a unique system for the delivery of genes into cells of neuronal lineage (Anderson, et al., Cell Mol Neurobiol., 1993, 13, 503-515). Herpes simplex virus(HSV)-derived vectors will infect postmitotic neurons, produce an established latent infection in some cell types, making it a suitable system for somatic gene therapy (Leib & Olivo., BioEssays., 1993, 15, 547-554).
  • HSV vectors and recombinant viruses suitable for example for the transduction of the insulin gene, has been described (Leib & Olivo., BioEssays., 1993, 15, 547-554).
  • the general method extensively used for mutagenizing endogenous viral genes Post & Roizman, Cell, 1981 , 25, 227-232 may be applied for the introduction of exogenous genes like insulin gene into the HSV genome.
  • the insulin expression cassette is cloned into a plasmid containing a portion of the HSV genome such that at least 300 bp flank the 5'- and 3' ends of the cassette (Breakfield & Deluca., New Biol ., 1991 , 3, 203-218; Efstathiou & Minson,., Brit Med Bull., 1995, 51 , 45-55).
  • the plasmid is transfected into permissive cells in culture along with the full length HSV DNA (Geller., et al., PNAS USA., 1990, 87, 8950-8954). Homologous recombination and DNA replication will result in the generation of recombinant HSV genomes that are packaged into novel virus particles by the cell. Through several round of plaque purification, a recombinant virus carrying the insulin expression cassette may be identified for large scale production.
  • Defective HSV vectors have benn successfully used to transfer exogenous genes into neuronal cells in vitro and in vivo (Geller & Freese., PNAS USA., 1990, 87, 1149- 1153; Geller & Breakfield., Science, 1988, 241 , 1667-1669; reviewed in Efstathiou & Minson,., Brit Med Bull., 1995, 51 , 45-55).
  • a variety of constitutive promoters have been used including the lytic cycle HSV promoters, the RSV LTR, the HCMV IE promoters and the neurofilament and PGK promoters for transient expression.
  • Sindbis virus vectors Sindbis virus-based vectors are intended as self-amplifying systems to enhance expression of exogenous genes in mammalian cells (Herweijer, et al., Human Gene Therapy, 1995, 6, 1 161 -1 167).
  • the 5'-two thirds of the Sindbis virus genome encodes the nonstructural genes needed for replication of the viral genome.
  • the 3'-third of the genome encode the structural proteins (Strauss, et al., Virology, 1984, 133, 92-110; Strauss & Strauss, Microbiol. Rev., 1994, 58, 491-562).
  • the subgenomic sequence coding for the structural proteins are replaced by the expression cassette of the transgene, for example, the insulin gene (Huang, et al., Virus Genes, 1989, 3, 85-91 ; Bredenbeek, et al., J Virol., 1993, 67, 6439-6446).
  • RNA genome of the recombinant Sindbis virus is generated by placing the entire genome under the control of the bacteriophage T7 or SP6 peomoters to enable transcription of the (+) strand RNA in vitro (Herweijer, et al., Human Gene Therapy, 1995, 6, 1161-1167). The resultant RNA genomes are then used to transfect target cells (Xiong, et al., Science, 1989, 243, 1188-1191 ). Infectious viruses are produced by infecting with a helper virus (Bredenbeek, et al., J Virol., 1993, 67, 6439- 6446). Modifications of this design using the Rous sarcoma virus LTR to direct the transcription of the non-structural genes have been described (Herweijer, et al., Human Gene Therapy, 1995, 6, 1 161-1167).
  • the luciferase gene cloned into the unique Xbal site in the vector pSin-Lux (Herweijer, et al., Human Gene Therapy, 1995, 6, 1161-1167) is replaced by the insulin cDNA or an expression cassette encoding giucokinase upon appropriate restriction endonuclease modifications (Sambrook, et al., Molecular Cloning- A Laboratory Manual, CSHL, CSH, 1989).
  • Sindbis virus vectors have been successfully used to transduce foreign genes into primary rat myoblasts (Herweijer, et al., Human Gene Therapy, 1995, 6, 1161-1167).
  • Viral vectors can be used to deliver the insulin coding sequence into the cells tissues of diabetic patients by in vivo infection.
  • the recombinant viral vector is administered to the organism in order to result in a tissue specific infection of the patient.
  • non-viral insulin gene constructs can also be targeted in vivo to specific tissues, for example the muscle in patients. Examples of such delivery systems include liposome encapsulation, and direct injection of non-viral expression vectors.
  • Recombinant DNA expression cassettes comprising of cellular promoters/enhamcers and regulatory regions operably linked to the glucokinase genes/cDNAs designed for expression in target mammalian tissues in the form of plasmids, linearized DNA fragments or viral DNA RNA vectors are prepared and purified as described in Sambrook, et al., Molecular Cloning- A Laboratory Manual, CSHL, CSH,
  • the DNAs are introduced into cells by one of the following methods, Calcium phosphate precipitation, DEAE-Dextran method, Electroporation (Ausudel, et al., Current Protocols in Molecular Biology, 1987, Wiley-lnterscience), lipofectin (Ausudel, et al., Current Protocols in Molecular Biology, 1987, Wiley-lnterscience) and protoplast fusion (Sandra-Goldin, et al., MCB., 1981 , 1 , 743-752).
  • the Calcium phosphate co-precipitation method (Ausudel, et al., Current Protocols in Molecular Biology, 1987, Wiley-lnterscience) is used.
  • the cells in culture are trypsinized and replated in selection media at a density of 1/10.
  • Clonal cell line that have inherited the selectable marker are picked by ring cloning, expanded in culture and analyzed for the inheritance of the transfected gene of interest by PCR (Innis, et al., PCR Protocols: A guide to methods and applications, Academic Press, 1990) and Southern blot analysis (Southern, J Mol Biol, 1975, 98, 503) of genomic DNA prepared from the clonal cells/ cell lines.
  • transfected insulin gene is examined by Northern blot analysis (Sambrook, et al., Molecular Cloning- A Laboratory Manual, CSHL, CSH, 1989) of total RNA, by RIA analysis (CIS Biointernational, Gif-sur-Yvette, France) and by insulin activity assay (Gros, L et al. al., 1999, Human Gene Therapy, 10: 1207-1217).
  • DNA can be introduced into cells by DNA mediated transduction using one of the following methods: calcium phosphate precipitation, DEAE- Dextran method, electroporation (Ausudel, et al., Current Protocols in Molecular Biology (Wiley-lnterscience, 1987), or lipofectin or protoplast fusion (Sandra-Goldin, et al., Mol Cell Biol., 1981 , 1 , 743-752).
  • the selectable marker is on a separate plasmid
  • the calcium phosphate co-precipitation method (Ausudel, et al., Current Protocols in Molecular Biology (Wiley-lnterscience, 1987) is used.
  • hepatocytes may be isolated from the liver (Ponder, et al., PNAS USA, 1991 , 1217-1221 ; Pages, et al., Human Gene Therapy, 1995, 6, 21-30), commited to short term culture (Pages, et al., Human Gene Therapy, 1995, 6, 21-30), and then transduced with a viral or plasmid vector carrying the expression cassette comprising of the glucokinase cDNA under the transcriptional control of a liver specific promoter.
  • the genetically modified hepatocytes are then harvested and transplanted into a recipient either via infusion of the cells into the portal vein (Wilson, et al., PNAS USA., 1990, 87, 6437-8441 ) or introduced intrasplenically (Ponder, et al., PNAS USA, 1991 , 1217-1221 ).
  • the genetically modified hepatocytes introduced intrasplenically were shown to replace up to 80% of the diseased liver (Rhim, et al., Science, 1994, 263, 1149-1152).
  • myoblasts may be isolated from muscle biopsies (Mendell, et al., N Engl J Med, 1995, 832-838) , expanded in culture and genetically modified to express high levels of insulin by transfection with DNA comprising of the insulin gene under the transcription control of a strong muscle specific promoter/enhancer or infected with a muscle specific recombinant retrovirus (Ferrari, et al., Human Gene Therapy, 1995, 6, 733-742).
  • the insulin expressing myoblasts can then be transferred into muscle my direct injection of the cells.
  • Previous experience with murine myoblast have demonstrated that the injected myoblasts will fuse into preexisting multinucleate myofibrils (Dhawan, et al., Science, 1991 , 254,1509-1512; Barr & Leiden, Science, 1991 , 254, 1507-1509) and the differentiated muscle fibers will maintain a high level of expression of the transgene (Yao & Kurachi, PNAS USA., 1992, 89, 3357-3361 ; Bohl, et al., Nature Medicine, 1997, 3, 299- 305).
  • the cells are then embedded into collagen coated lattices of expanded polytetrafluoroethylene (Gore-Tex) fibers as previously described (Thompson, et al., PNAS USA., 1989, 86, 7928-7932; Moullier, et al., Nature Genetics, 1993, 4, 154-159). Adsorption of of heparin-binding growth factors 1 to the collagen lattices, upon implantation into the peritoneal cavity will induce vasculahzation and formation of a neo- organ.
  • Gore-Tex expanded polytetrafluoroethylene
  • mice Such neo-organs were reported to produce a sustained expression of trangenes in mice (Salvetti, et al., Human Gene Therapy, 1995, 6, 1 153-1159) and dogs (Moullier, et al., Nature Med., 1995, 1 , 353-357). It is therefore anticipated that such neo organs comprising of fibroblast or other cell type overexpressing insulin could potentially serve to normalized blood sugars in the diabetic state.
  • the human cDNA encoding proinsulin containing the furin consensus cleavage site mutations was obtained by EcoRI digestion of pP2.4-lnsm (Gros, et al., Human gene Therapy, 1997, 8, 2249-2259).
  • the cDNA was inserted into the EcoRI unique site of a plasmid carrying a 1.3kb rat MLC1 promoter regulatory sequence along with 800bp of polyadenylation and splice signals of the SV40 small tumor antigen, and an 800bp genomic fragment of the MLC1 gene containing a strong muscle-specific enhancer known to enhance the muscle-specific expression (Donoghue, et al., Gene Dev., 1988, 2, 1779- 1790).
  • the plasmid was designated pMLC/lnsm ( Figure 1 ).
  • C2C12 mouse myoblast was grown in monolayers in Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL, Grand Isiand, NY). The cells were maintained at 37°C under 95% air, 5% C02 atmosphere.
  • C2C12 cells were co-transfected by the calcium phosphate co-precipitation method with both the pMLC/lnsm plasmid (10ug) and a pPGKneo plasmid(2ug) carrying the neomycin selectable marker. Cells expressing the neomycin-resistance gene were selected in media containing 1 mg/ml of geneticin (G418, GibcoBRL, Grand Island, NY).
  • RNA samples (30ug) were electrophoreses on 1% agarose gels containing 2.2M formaldehyde.
  • Northern blots were hybridized to the following 32P-labeled probes: a 0.4kb EcoRI -EcoRI fragment corresponding to the rabbit P-enolpyruvate carboxykinase (PEPCK) cDNA; a 1.3kb EcoRI -EcoRI fragment corresponding to the rabbit ⁇ -actin cDNA; and a 1.1 kb EcoRI -EcoRI fragment corresponding to the mouse myogenin cDNA.
  • the probes were labeled using [ ⁇ - 32P]dCTP, following the random oligopriming method (Boehringer Mannheim, Germany). Probed filters were placed in contact with Kodak XAR-5 films. The ⁇ -actin signal (internal control) was used to correct for RNA loading differences. Densitometric analysis of autoradiograms was performed at non-saturating exposures with a scanning densitometer (Fujix, BAS 1000).
  • the expression of the myogenin was used as a marker of differentiation. Myogenin was detected from the first day of differentiation with a maximal expression on day 3 ( Figure 1 B). A similar pattern of expression was observed with the insulin probe indicating that the clonal line C2C12lnsm expressed the chimeric insulin gene from day 1- 4 of differentiation. No insulin gene expression was detected in control cells C2C12-neo after 4 days of differentiation. The chimeric insulin gene was not expressed in C2C12lnsm in the undifferentiated state (day 0) ( Figure 1 B). Differentiated C2C12lnsm cells maintained their expression of insulin after one month in culture in the presence of 2% horse serum. These results indicate that with the MLC/lnsm construct, differentiation of the C2C12lnsm ceils resulted in an induction and maintenance of the insulin gene expression in myotubes.
  • Example 4 Detection of insulin polypeptide production in C2C12 cells transfected with pMLC/lnsm Control and insulin-expressing C2C12 cells were grown on culture chamber slides
  • C2C12-neo and C2C12lnsm cells were incubated in the presence of 2% horse serum. After 3 days of differentiation, the cells were cultured for 24 hr in serum free medium supplemented with 20mM glucose. Culture medium and cell extracts from the control C2C12neo and insulin expressing C2C12lnsm cell lines were analysed by high pressure liquid chromatography (HPLC). Each fraction obtained form the HPLC separation was subjected to radioimmunoassay for insulin. No insulin immunoreactive products were detected in cells extracts and the culture medium from the differentiated C2C12neo cells ( Figure 3A and 3B).
  • C2C12neo and C2C12lnsm cells were cultured overnight in serum free medium supplemented with 20mM gluocse, washed and then incubated for the various time period in the same medium containing 0.5% bovine serum albumin (BSA) and 0.1mg/ml aprotinin (Protease inhibitor). Aliquots were taken from the culture medium and immunoreactive insulin was measured. The release of insulin was linear during the first three hours and corresponded to about 100uU/10 6 cells ( Figure 4A). Thereafter insulin continued to accumulate in the medium and high levels were noted 48hr after culture in serum-free medium. As expected, no insulin was detected in the culture medium of the differentiated control C2C12neo cell line ( Figure 4A).
  • Example 7 Long term production of insulin in C2C12lnsm cell line
  • Insulin production by the C2C12lnsm cells was also studies after long term differentiation. To this end, cells were cultured in DMEM supplemented with 2%(v/v) hrose serum for up to 42 days. Several days after differentiation, cells were washed three times with serum-free medium and incubated for 2 hr in serum free medium supplemented with 20mM glucose containing 0.5% BSA and 0.1mg/ml aprotinin. Afterwards, insulin production was measured in the incubation medium ( Figure 4B) and used as a reference level of production. At any time period analyzed, C2C12lnsm cells were able to release levels of insulin comparable to that produced 3 days after differentiation ( Figure 4A and 4B).
  • C2C12lnsm cells were able to maintain a relatively constant level of insulin production in long-term culture.
  • C2C12neo and C2C12lnsm differentiated cells were evaluated in C2C12neo and C2C12lnsm differentiated cells. After three days of differentiation, C2C12neo and C2C12lnsm were cultured overnight in serum-free medium supplemented with 20mM glucose, washed and then incubated for variuos time period in the same medium containing 0.5%BSA and 0.1 % aprotinin.
  • differentiated C2C12lnsm cells showed a 2.5-fold increase in glucose uptake and consumption and a 5-fold increase in lactate production compared to the control C2C12neo cells ( Figure 5A and 5B). The results indicated that insulin production by the differentiated C2C12lnsm cells is functional and induced glucose uptake and utilization in the cells.
  • the biological activity of the secreted insulin was analyzed in FTO-2B rat hepatoma cells in culture.
  • FTO-2B cells were incubated for 2 hours in the presence of media from C1 C12lnsm and C2C12neo control cells. At the end of that period, total RNA was obtained from the FTO-2B cells and the expression of an endogenous insulin responsive PEPCK gene (expression suppressed by insulin) was analyzed.
  • Figure 6A and 6B is a Northern blot analysis using the PEPCK gene fragment as a probe. The results showed a marked reduction in the expression of PEPCK gene in FTO-2B cells exposed to media obtained from the differentiated C2C12lnsm for 2 hr.
  • Example 9 Transplantation of C2C12lnsm myoblast into skeletal muscle of diabetes mice.
  • C2C12lnsm myoblast cells were transplanted by direct skeletal muscle injection to syngeneic C3H mice made diabetic by Streptozotocin (STZ) treatment.
  • STZ Streptozotocin
  • Intraperitoneal injection of 40mg of STZ (Sigma, St Louis, MO) into 3-weeks old C3H mice for 5 consecutive days led to a decrease of plasma insulin (from 125 + 14 uU/ml to 60 + 5 uU/ml) levels with a resultant development of hyperglycemia (from 132 + 15 mg/dl to 340 + 40 mg/dl) one week after the last STZ injection, assessed by measuring blood glucose levels.
  • Diabetic mice were transplanted with either 2 x 10 6 control C2C12neo cells or 2 x 10 6 C2C12lnsm myoblast cells by direct injection into the muscles of the two hindlimbs of the 5-weeks old diabetic C3H mice.
  • Blood glucose and plasma insulin was measured in the fed animals 3 weeks after the injection of the cells, Blood glucose was determined by using the Glucometer Elite (Bayer, Germany) according to manufacturers instructions. Insulin was measure in the serum by radioimmunoassay ( (CIS Biointernational, Gif-Sur- Yvette, France).
  • mice treated with the control C2C12neo cells showed a very high levels of plasma glucose (>500mg/dl), i.e. severely diabetic, C2C12lnsm transplanted mice maintained a significantly lower level of plasma glucose (290 + 10 mg/dl), almost to normoglycemia (Figure 7B).
  • the results suggest that C2C12lnsm myoblast are able to survive, differentiate in vivo and produce biologically active insulin in a diabetic host after direct msucle transplantation.

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Abstract

Cette invention se rapporte à un procédé de traitement du diabète sucré chez un sujet, qui consiste soit à incorporer directement un gène codant l'insuline dans le muscle du sujet soit à transfecter une lignée de cellules musculaires avec un gène codant l'insuline et à introduire dans le corps du sujet ces cellules musculaires génétiquement modifiées.
PCT/EP1999/009132 1998-11-20 1999-11-19 Production d'insuline par des cellules musculaires genetiquement modifiees WO2000031267A1 (fr)

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Cited By (5)

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WO2002049423A1 (fr) * 2000-12-20 2002-06-27 Universitat Autonoma De Barcelona Utilisation conjointe du gene de l'insuline et du gene de la glucokinase dans la mise au point d'approches therapeutiques pour le diabete sucre
EP1572126A4 (fr) * 2001-12-21 2006-07-05 Smithkline Beecham Corp Compositions et methodes servant a modifier la production de glucose
EP1541674A4 (fr) * 2002-06-18 2007-01-10 Eisai Co Ltd Adipocytes de culture primaire utilises en therapie genique
JP2008131941A (ja) * 2002-06-18 2008-06-12 Eisai R & D Management Co Ltd 遺伝子治療用初代培養脂肪細胞
NO340167B1 (no) * 2002-06-18 2017-03-20 Eisai R&D Man Co Ltd Farmasøytisk sammensetning som omfatter en primær dyrket pre-adipocytt som stabilt opprettholder et fremmed gen som koder for insulin eller glukagonlignende peptid-1(GLP-1), den primære dyrket pre-adipocytten per se, en in vitro fremgangsmåte for fremstilling av pre-adipocytten for anvendelse i genterapi, samt en implantat-sammensetning for anvendelse i genterapi.

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002049423A1 (fr) * 2000-12-20 2002-06-27 Universitat Autonoma De Barcelona Utilisation conjointe du gene de l'insuline et du gene de la glucokinase dans la mise au point d'approches therapeutiques pour le diabete sucre
ES2170720A1 (es) * 2000-12-20 2002-08-01 Univ Barcelona Autonoma Utilizacion conjunta del gen de la insulina y del gen de la glucoquinasa en el desarrollo de aproximaciones terapeuticas para la diabetes mellitus.
EP1572126A4 (fr) * 2001-12-21 2006-07-05 Smithkline Beecham Corp Compositions et methodes servant a modifier la production de glucose
EP1541674A4 (fr) * 2002-06-18 2007-01-10 Eisai Co Ltd Adipocytes de culture primaire utilises en therapie genique
JP2008131941A (ja) * 2002-06-18 2008-06-12 Eisai R & D Management Co Ltd 遺伝子治療用初代培養脂肪細胞
EP2213730A1 (fr) * 2002-06-18 2010-08-04 Eisai Co., Ltd. Adipocytes primaires cultivés pour thérapie génique
US7820438B2 (en) 2002-06-18 2010-10-26 Eisai R&D Management Co., Ltd. Primary cultured adipocytes for gene therapy
US8071085B2 (en) 2002-06-18 2011-12-06 Eisai Co., Ltd. Primary cultured adipocytes for gene therapy
CN1671836B (zh) * 2002-06-18 2012-02-08 卫材R&D管理有限公司 用于基因治疗的原代培养脂肪细胞
NO340167B1 (no) * 2002-06-18 2017-03-20 Eisai R&D Man Co Ltd Farmasøytisk sammensetning som omfatter en primær dyrket pre-adipocytt som stabilt opprettholder et fremmed gen som koder for insulin eller glukagonlignende peptid-1(GLP-1), den primære dyrket pre-adipocytten per se, en in vitro fremgangsmåte for fremstilling av pre-adipocytten for anvendelse i genterapi, samt en implantat-sammensetning for anvendelse i genterapi.

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