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WO2001038535A1 - Gene chimerique permettant d'exprimer le gene ou le c-adn du facteur de croissance semblable a l'insuline de type i (igf-i) dans le pancreas et son utilisation dans la therapie genique du diabete mellitus - Google Patents

Gene chimerique permettant d'exprimer le gene ou le c-adn du facteur de croissance semblable a l'insuline de type i (igf-i) dans le pancreas et son utilisation dans la therapie genique du diabete mellitus Download PDF

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Publication number
WO2001038535A1
WO2001038535A1 PCT/ES2000/000452 ES0000452W WO0138535A1 WO 2001038535 A1 WO2001038535 A1 WO 2001038535A1 ES 0000452 W ES0000452 W ES 0000452W WO 0138535 A1 WO0138535 A1 WO 0138535A1
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WO
WIPO (PCT)
Prior art keywords
vector
igf
insulin
chimeric gene
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2000/000452
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English (en)
Spanish (es)
Inventor
Fátima BOSCH TUBERT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitat Autonoma de Barcelona UAB
Original Assignee
Universitat Autonoma de Barcelona UAB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitat Autonoma de Barcelona UAB filed Critical Universitat Autonoma de Barcelona UAB
Priority to AU17081/01A priority Critical patent/AU1708101A/en
Publication of WO2001038535A1 publication Critical patent/WO2001038535A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • Diabetes mellitus is the most common metabolic disease. It comprises a wide variety of syndromes with different etiologies that collectively affect 2 to 7% of the world population. From 5 to 10 ° OR of patients can be grouped into the category of insulin-dependent diabetes mellitus or type 1 diabetes, which usually manifests before age 40, often during adolescence, and is the result of the autoimmune destruction of the ⁇ cells of the islets of Langerhans in the pancreas, leading to insulin deficiency, hyperglycemia and the development of microvascular, acrovascular and neurological complications.
  • Diabetes Care 7, 188-199 In addition, delayed-acting preparations are not usually capable of producing adequate basal insulin levels, resulting in many cases. you are in the appearance of both hyperglycemia and hypoglycemia. Therefore, the levels of circulating insulin that are achieved with current therapies they are quite far from those that would be obtained if the ⁇ cells functioned correctly.
  • pancreas transplants and islet transplants have been attempted [Remuzzi et al. (1994). Lancet 343, 27-31]. These approaches are intended to eliminate daily insulin injections, but require chronic immunosuppression and the results have not been very successful. In addition, donors are very limited and, therefore, the treatment of a large number of patients does not seem very realistic. Another approach is based on the regeneration of the ⁇ cells from the precursors of the islet cells or on the induction of the proliferation of those ⁇ cells that have not yet been destroyed during the autoimmune process in diabetic type patients. one.
  • IGF-I insulin growth factor-I
  • IGF-I insulin-like growth factor type I
  • IGF-I insulin-like growth factor type I
  • IGF-I insulin-like growth factors: structure and biological function. Gxford Uni v. Press, Oxford]. Its main action is to stimulate proliferation and Cell differentiation. Circulating IGF-I is produced by the liver, although it has been observed that in the adult mouse the pancreas expresses high levels of IGF-I [Mathews et al. (1986) Regulation of i.nstiii. ⁇ ⁇ 1i.l ⁇ cro t_n f3.cto._r I ⁇ S ⁇ sssiorj. 3o r _ro t__ hormone. Proc. Nati Acad. Sci. USA, 83, 9343-9347].
  • the present invention relates to a chimeric gene that uses the insulin-like growth factor gene or cDNA of type I 'IGF-1 ⁇ directed by a promoter or fusion of promoters that allows expression of IGF-I in the pancreas during the diabetic process.
  • This chimeric gene may contain a promoter or fusion of romotores p allowing ex p ressure regulated IGF-I in endocrine or exocrine pancreas during the diabetic process.
  • the present invention also relates to an expression that allows to express the chimeric gene described above in pancreas.
  • Said vector may be a platinum, a viral vector or a non-viral vector.
  • it can be a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a Sindbis viral vector, a lentiviral vector or a vector derived from herpes virus.
  • the use of the chimeric gene for the development of approaches is an object of the present invention.
  • nc to Q i rom ⁇ 1 p use of the expression vector of the invention for use in the development of therapeutic approaches for diabetes mellitus
  • An in vivo approach to the gene therapy of diabetes was carried out by obtaining transgenic animals that overexpress IGF-I in pancreatic ⁇ cells under the control of the insulin gene promoter.In particular, said transgenic animal is a r ⁇ ón
  • the chimeric gene was obtained and it was icroinjected into fertilized oocytes to generate the mice
  • Transgenic mice that overexpress IGF-I have islet hyperplasia with age (more than 6 rnoc;. P "> ⁇ __ " 1 M "(- ⁇ __H'i ⁇ S al _a c ⁇ ⁇ H” in Hol f imn t- prni ⁇ -tr. of such transgenic mice in inducing conditions h " ⁇ ⁇ ⁇ diucem ara determine whether these animals could counteract diabetic disorders, caused by the treatment with Stz, by forming new islets or by inducing the regeneration of residual islets.
  • experimental diabetes was induced in both control mice and transgenic mice by injection for five consecutive days of the toxic streptozotocin (Stz) intraperitoneally. The administration of Stz at low doses for five consecutive days leads to the development of inflammation and autoimmune destruction of ⁇ cells
  • serum glucose and insulin concentrations were determined 30 days and 90 days in controls and transgenics and 180 days in transgenics after the last Stz injection as shown in. Table 1 below.
  • Histological and immunohistochemical analyzes of the pancreas of these transgenic animals show that although there is a process of insulitis in the pancreatic islets in the initial stages, it disappears later, insulin in the islets, which indicates that it has rs H ea ronl i rari ⁇ n S 1 pc - ' " 1 n 1 c; P In addition, there is a large increase in the number of new iqla r ⁇ r-hi -' - •] n 1 a H ____ 1 pe ⁇ / nnq decrease in apoptosis.
  • Example I Obtaining a chimeric gene that includes the insulin-like growth factor cDNA of
  • the plasmid pKCR3 containing the rabbit ⁇ -globin gene was used for the construction of the chimeric RIP / IGF-I gene.
  • the BamHI-XhoI fragment of said gene is introduced: o at the Ba HI-XhoI restriction sites of the polyliner of the Bluescript SK- plasmid vector.
  • This fragment of the ⁇ -globin gene contains the last two exons, the last intron, and the 3 ' region linked to the SV40 enhancer.
  • the SacI-BamHI fragment (-570 bp to +3 bp) of the rat-I insulin promoter (RIP-I) was introduced at SacI-BamHI sites in the polylinker of the Biuescript vector.
  • the EcoRI fragment (577 bp) containing the complete cDNA of the rat IGF-I was introduced into the EcoR.1 target of the second exon of the ⁇ -globin gene.
  • the resulting final plasmid was named
  • Example II Transgenic mouse expressing type I insulin-like growth factor cDNA (IGF-I) in pancreatic ⁇ cells.
  • IGF-I insulin-like growth factor cDNA
  • the transgenic mouse was obtained from microinjection of the 3.4 Kb Sacl-Xhol fragment containing the RIP / IGF-I chimeric gene described in the previous example.
  • the fertilized occites were collected by emptying the oviducts of the donor mice 12 hours after mating.
  • 2 to 3 pl of a DNA solution (4 ng / ⁇ l) was injected into the zigcto pronucleus.
  • Injected zygotes were implanted in the oviduct of pseudogestant females, approximately 10 zygotes per side [Hogan et al. (1986) Manipulating the Mouse E bryo. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York].
  • the presence of the transgene in the offspring was analyzed by Southern ⁇ Southern bl ot) transfer and hybridization analysis of 10 ⁇ g of DNA from the tail of each animal.
  • the DNA samples were digested with a suitable restriction enzyme, resulting in the fragments istic characteristics, the presence of which only ° s detectable in transgenic mice.
  • RNA was obtained using the isothiocyanate method and then O caaa - ⁇ m £ _-_ c_: + - vp; H ⁇ Ü Mü ⁇ a ri n rr ⁇ 1 H o __. ! or t-mfp ⁇ c "iq of agarose containing for aldehyde was transferred [Northern blot, to a membrane for hybridization with an IGF-I probe.
  • said probe was labeled with [a- 32p] clCTP following the oligcpriming method according to the manufacturer's instructions 5.
  • Transfer membranes were contacted with Kodak XAR-5 films.
  • the presence of hybridization in the RNA samples corresponding to the pancreas in the transgenic mice indicates the expression of the chimeric gene at 0 said tissue
  • pancreas from control and transgenic animals were obtained and included in paraffin.
  • the histological sections obtained from these pancreas were analyzed by immunohistochemical studies using a specific anti-IGF-I antibody 5. This allowed the presence of IGF protein to be detected. -I specifically in pancreatic ⁇ cells, indicating that the chimeric gene was adequately expressed.
  • Stz 10 40 mg Stz / kg live weight [Rossini et al. (1977) J. A. Diab. Assoc. 26, 916-920]. Stz was dissolved in a solution of sodium citrate containing 0.9 a of sodium chloride at pH 4.5 immediately before administration. Diabetes was confirmed by measurement

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un gène chimérique qui utilise le gène ou le c-ADN du facteur de croissance semblable à l'insuline de type I (IGF-I), dirigé par un promoteur ou une fusion de promoteurs permettant l'expression régulée du IGF-I dans le pancréas. L'invention concerne également un vecteur d'expression permettant d'exprimer l'IGF-I dans le pancréas. L'invention concerne, en outre, une cellule pancréatique exprimant ledit gène chimérique. L'invention concerne enfin l'utilisation dudit gène chimérique ou dudit vecteur d'expression dans le développement d'approximations thérapeutiques pour le diabète mellitus.
PCT/ES2000/000452 1999-11-26 2000-11-23 Gene chimerique permettant d'exprimer le gene ou le c-adn du facteur de croissance semblable a l'insuline de type i (igf-i) dans le pancreas et son utilisation dans la therapie genique du diabete mellitus Ceased WO2001038535A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU17081/01A AU1708101A (en) 1999-11-26 2000-11-23 Chimeric gene enabling expression of the gene or cdna of type i insulin-like growth factor (igf-i) in the pancreas and utilization thereof in genic therapy of diabetes mellitus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES9902696A ES2156769B1 (es) 1999-11-26 1999-11-26 Gen quimerico que permite expresar el gen o el cdna del factor de crecimiento similar a la insulina de tipo i (igf-i) en pancreas y su utilizacion para la terapia genica de la diabetes mellitus.
ESP9902696 1999-11-26

Publications (1)

Publication Number Publication Date
WO2001038535A1 true WO2001038535A1 (fr) 2001-05-31

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PCT/ES2000/000452 Ceased WO2001038535A1 (fr) 1999-11-26 2000-11-23 Gene chimerique permettant d'exprimer le gene ou le c-adn du facteur de croissance semblable a l'insuline de type i (igf-i) dans le pancreas et son utilisation dans la therapie genique du diabete mellitus

Country Status (3)

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AU (1) AU1708101A (fr)
ES (1) ES2156769B1 (fr)
WO (1) WO2001038535A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011004051A1 (fr) * 2009-07-10 2011-01-13 Universidad Autónoma De Barcelona Compositions de thérapie génique destinées à prévenir et/ou à traiter des maladies auto-immunes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024922A1 (fr) * 1996-12-02 1998-06-11 Valentis, Inc. Systeme d'expression du facteur de croissance insulinoide i (igf-i) et ses methodes d'utilisation
US5837875A (en) * 1995-09-25 1998-11-17 The Autonomous University Of Barcelona Transgenic mouse containing an IGF-1 transgene
WO1999010013A1 (fr) * 1997-08-25 1999-03-04 The Trustees Of The University Of Pennsylvania Utilisation du facteur de croissance de type insulinique dans un muscle
US5925564A (en) * 1991-11-06 1999-07-20 Baylor College Of Medicine Expression vector systems and method of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925564A (en) * 1991-11-06 1999-07-20 Baylor College Of Medicine Expression vector systems and method of use
US5837875A (en) * 1995-09-25 1998-11-17 The Autonomous University Of Barcelona Transgenic mouse containing an IGF-1 transgene
WO1998024922A1 (fr) * 1996-12-02 1998-06-11 Valentis, Inc. Systeme d'expression du facteur de croissance insulinoide i (igf-i) et ses methodes d'utilisation
WO1999010013A1 (fr) * 1997-08-25 1999-03-04 The Trustees Of The University Of Pennsylvania Utilisation du facteur de croissance de type insulinique dans un muscle

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KRAKOWSKI M L ET AL: "Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes", J OF ENDOCRINOLOGY, vol. 162, August 1999 (1999-08-01), pages 167 - 175, XP002901640, Retrieved from the Internet <URL:www.endocrinology.org> [retrieved on 20010301] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011004051A1 (fr) * 2009-07-10 2011-01-13 Universidad Autónoma De Barcelona Compositions de thérapie génique destinées à prévenir et/ou à traiter des maladies auto-immunes

Also Published As

Publication number Publication date
ES2156769A1 (es) 2001-07-01
ES2156769B1 (es) 2002-03-01
AU1708101A (en) 2001-06-04

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