WO1998005774A1 - Utilisation d'un gene pour le facteur vasculaire humain de croissance des cellules endotheliales en vue d'une therapie genique directe - Google Patents

Utilisation d'un gene pour le facteur vasculaire humain de croissance des cellules endotheliales en vue d'une therapie genique directe Download PDF

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Publication number
WO1998005774A1
WO1998005774A1 PCT/EP1997/003946 EP9703946W WO9805774A1 WO 1998005774 A1 WO1998005774 A1 WO 1998005774A1 EP 9703946 W EP9703946 W EP 9703946W WO 9805774 A1 WO9805774 A1 WO 9805774A1
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WO
WIPO (PCT)
Prior art keywords
gene
growth factor
endothelial cell
cell growth
vascular endothelial
Prior art date
Application number
PCT/EP1997/003946
Other languages
German (de)
English (en)
Inventor
Alfred Hahn
Susanne Hartmann
Uwe Jonas
Original Assignee
Knoll Aktiengesellschaft
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 Knoll Aktiengesellschaft filed Critical Knoll Aktiengesellschaft
Priority to AU41154/97A priority Critical patent/AU4115497A/en
Publication of WO1998005774A1 publication Critical patent/WO1998005774A1/fr

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Classifications

    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • 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

Definitions

  • the present invention relates to the use of a gene for human vascular endothelial cell growth factor for direct gene therapy.
  • the vascular endothelial cell growth factor is an endothelial cell-specific mitogen that is structurally related to the platelet derived growth factor.
  • the primary structure of the protein and the associated protein are described in WO 90/13649. Through alternative splicing, there are three different forms with 189, 165 and 121 amino acids, which correspond to VEGFig . , VEGF_ 65 and VEGF ⁇ _ in the literature (Tischer et al. J. Biol. Chem. Vol 266, 11947-11954, (1991); Weindel et al. Biochem. Biophys. Res. Comm. Vol 183, 1167- 1174, (1992)).
  • WO 90/13649 describes the genetic engineering of the VEGF protein and the therapeutic use of this protein for the treatment of trauma, in particular after incisions and cuts in blood vessels and in ulcers of the endothelial surface.
  • the task was to provide effective medicines and methods for the treatment of chronic ischemic conditions, in particular peripheral arterial occlusive diseases and angina pectoris, caused by coronary artery narrowing or occlusion.
  • Another object of the invention is the use of a gene for human vascular endothelial cell growth factor for the production of medicaments for the treatment of chronic ischemic conditions.
  • Direct gene therapy is to be understood as the direct introduction of nucleic acid constructs (DNA or RNA) into cells and tissues without the use of viral vector systems for the introduction.
  • the DNA can be introduced by injection, liposome-mediated or in the form of calcium phosphate precipitates or dendrimer complexes.
  • the nucleic acid is incorporated into a functional gene construct.
  • DNA in particular cDNA, is preferably used as the nucleic acid.
  • the DNA is provided with regulatory signals for efficient gene expression. These include transcription signals such as promoters, enhancers, polyadenylation sites and translation signals such as ribosomal binding sites.
  • Eukaryotic active promoters which can be of viral origin, such as CMV promoter, RSV promoter or cellular origin, such as ⁇ -actin promoter, are preferably used as promoters. Externally adjustable or controllable promoters are also well suited.
  • polyadenylation signals can be used as polyadenylation signals, for example those of SV 40 or of the human ⁇ -globin gene.
  • DNA constructs can also carry genetic markers such as antibiotic resistance and other elements such as an origin of replication, which are helpful in the cloning of the DNA constructs in microorganisms.
  • DNA constructions can be generated, for example, by cloning in microorganisms and PCR technology.
  • the sequence for the human VEGF gene is described, for example, in Tischer et al. (J. Biol. Chem. Vol 266, 11947-11954, (1991)) in Figure 4 and in Weindel et al. (Biochem. Biophys. Res. Comm. Vol 183, 1167-1174, (1992)) in FIG. 2, to which express reference is made.
  • the use according to the invention is not limited only to the sequence and parts thereof listed there, but also includes allelic variants and derivatives thereof which result from the exchange, insertion or deletion of one or more nucleotides, the activity of the encoded gene product essentially being as described above VEGF complies.
  • Particularly preferred is the use of a gene encoding the human VEGF_ 2 _.
  • the activity of VEGF is, for example, according to that of Rak et al. (Cancer Research 55, 4575-4580, 1995).
  • the DNA is dissolved in sterile, physiologically compatible, aqueous buffers.
  • the dosage depends on the age, condition and weight of the patient, the severity of the disease and the type of application. As a rule, the dose of active substance is between approximately 1 and 1000 ⁇ g VEGF gene per patient.
  • the preferred form of administration is intramuscular injection.
  • the drugs can also contain the usual additives and auxiliary agents, as are common for gene therapy drugs.
  • the DNA constructs are preferably used for the treatment of chronic ischemic conditions. These include peripheral arterial occlusive diseases, recalcifying wounds and angina pectoris.
  • the VEGF 12 ⁇ cDNA was enriched from human muscle tissue by PCR using primers which allow directed cloning of the cDNA (entire coding region).
  • the PCR amplificate was subcloned by means of the electrotransformation in pcDNA 3 (Invitrogen Corporation), a vector which is suitable for both transient and stable expression in eukaryotic host cells.
  • pcDNA 3 Invitrogen Corporation
  • a plasmid mini-preparation was carried out, which was based on the modified alkaline synthesis method according to Birnboim and Doly (Nucl. Acids Res.
  • the vector pcDNA 3 was ligated to the purified VEGF_ 2 _ cDNA in a ratio of 1: 3.
  • the 20 ⁇ l mixture consisted of: pcDNA 3 , VEGF 1 21 cDNA, T4 DNA ligase (1U / ⁇ l) and 1 ⁇ T4 ligase buffer.
  • the base sequence (sequence) of the obtained in the mini-preparation plasmid pLVEGF ⁇ 12 was determined using the fmol DNA sequencing cing ® system (Promega, Madison, WI). A correct reading frame could be verified autoradiographically both for the transition region plasmid / insert and for the remaining nucleotide sequence of human VEGF 1 21.
  • the construction of the plasmid pLVEGFj.21 is shown in Fig. 2.
  • pcDNA 3 / VEGF 121 for use in animal experiments, a maxi preparation was carried out with the EndoFree Maxi Kit (Qiagen, Hilden). A plasmid stock glycerol was incubated in ampicillin-containing Luria Broth Medium (200 ⁇ g / ml) overnight at 37 ° C. The maxi preparation was carried out according to the instructions for use of the kit. Ultimately, the concentration of the plas id DNA was determined photometrically at 260 nm.
  • ketamine hydrochloride 50 mg / kg body weight
  • pentobarbital 30 mg / kg body weight
  • a longitudinal incision was made on both hind legs below the inguinal ligament and the respective femoral artery was prepared.
  • Two ligatures were placed closely around the artery: the first at the proximal beginning of the femoral artery as a branch of the external iliac artery, the second proximal to the point where the superficial flexural artery leaves the femoral artery.
  • the femoral artery was severed in the middle of the ligatures, interrupting it for about one centimeter.
  • the intramuscular injections were made in four different thigh regions, with the right leg serving as a treatment group and the left as an intra-individual control.
  • 45 ⁇ l aliquots of 100 ⁇ g plasmid dissolved in 150 ⁇ l phosphate-buffered saline or phosphate-buffered saline alone were applied in each case: 1) caudal to the ligation point in the thigh; 2) caudal to the knee area; 3) close to the end of the internal iliac artery and 4) distal to the origin of the internal iliac artery.
  • the injection needle was inserted 5 mm deep into the skeletal muscle tissue. After sprinkling topical antibiotic powder, the wound was closed with single button sutures.
  • Fig. 3 shows an angiogram of a treated and a control animal.
  • Fig. 3A shows a control rat with bilateral A.
  • femoralis ligation and injection of buffer solution alone the injection sites are identified by white squares.
  • Fig. 3B shows a rat with bilateral A. femoralis ligation and injection of active substance (active treatment) or buffer solution alone ( solute control); the injection sites are marked by white squares).
  • Vascularization in the hind legs was determined by counting the visible vessels. An angiographic score can be calculated from this (a transparent 5 mm 2 grid is placed over the respective angiogram and the angiographic score is defined as follows: quotient of the number of grid crossing points that are intersected by a contrast medium-filled vessel and the number of total grid cross points in the The count was blinded by double analysis, the individual values were averaged. The results are presented as mean values ⁇ SEM. Significance calculations were carried out using the t-test for unconnected samples. P ⁇ 0.01 was considered significant.
  • Fig. 1 is the: Effect of intramuscularly applied pVEGF_ 2 i (100 ⁇ g) on the revascularization in rats with bilateral
  • Femoral artery ligature shown; the angiographic score is plotted after 3, 7, 14 and 42 days postoperatively. (Mean values ⁇ EM).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Vascular Medicine (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne l'utilisation d'un gène pour le facteur vasculaire humain de croissance des cellules endothéliales pour produire des médicaments en vue de traiter des états ischémiques chroniques, ainsi que des médicaments contenant ce gène.
PCT/EP1997/003946 1996-07-31 1997-07-22 Utilisation d'un gene pour le facteur vasculaire humain de croissance des cellules endotheliales en vue d'une therapie genique directe WO1998005774A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41154/97A AU4115497A (en) 1996-07-31 1997-07-22 Use of a human vascular endothelial cell growth factor gene for direct genetic therapy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19630896.8 1996-07-31
DE1996130896 DE19630896A1 (de) 1996-07-31 1996-07-31 Verwendung eines Gens für humanen vaskulären Endothelzell-Wachstumsfaktor zur direkten Gentherapie

Publications (1)

Publication Number Publication Date
WO1998005774A1 true WO1998005774A1 (fr) 1998-02-12

Family

ID=7801378

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/003946 WO1998005774A1 (fr) 1996-07-31 1997-07-22 Utilisation d'un gene pour le facteur vasculaire humain de croissance des cellules endotheliales en vue d'une therapie genique directe

Country Status (3)

Country Link
AU (1) AU4115497A (fr)
DE (1) DE19630896A1 (fr)
WO (1) WO1998005774A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19940012A1 (de) * 1999-08-24 2001-03-08 Karin Faerber Neues Mittel aus mindestens zwei Komponenten, das zum einen Gefäßneubildung (Neoangiogense) induziert des weiteren jedoch auch Gefäßverschlüsse (Restenosen) verhindert, das Verfahren seiner Herstellung und seine Verwendung

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ISNER ET AL: "ARTERIAL GENE TRANSFER FOR THERAPEUTIC ANGIOGENESIS IN PATIENTS WITH PERIPHERAL ARTERY DISEASE", HUMAN GENE THERAPY, vol. 7, 20 May 1996 (1996-05-20), pages 959 - 988, XP002048802 *
PILI ET AL: "ADENOVIRUS-MEDIATED GENE TRANSFER OF ANGIOGENIC GROWTH FACTORS AND BIOSAFETY: LACK OF INCREASED TUMORIGENICITY IN VIVO", CIRCULATION, vol. 90, 1994, pages I-147, XP002048803 *
TAKESHITA ET AL: "GENE TRANSFER OF NAKED DNA ENCODING FOR THREE ISOFORMS OF VASCULAR ENDOTHELIAL GROWTH FACTOR STIMULATES COLLATERAL DEVELOPMENT IN VIVO", LABORATORY INVESTIGATION, vol. 75, no. 4, October 1996 (1996-10-01), pages 487 - 501, XP002048805 *
TISCHER ET AL: "THE HUMAN GENE FOR VASCULAR ENDOTHELIAL GROWTH FACTOR. MULTIPLE PROTEIN FORMS ARE ENCODED THROUGH ALTERNATIVE EXON SPLICING", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 18, 25 June 1991 (1991-06-25), pages 11947 - 11954, XP002048804 *

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AU4115497A (en) 1998-02-25
DE19630896A1 (de) 1998-02-05

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