WO1995006745A1 - Vektor für leber-gentherapie - Google Patents
Vektor für leber-gentherapie Download PDFInfo
- Publication number
- WO1995006745A1 WO1995006745A1 PCT/DE1994/001003 DE9401003W WO9506745A1 WO 1995006745 A1 WO1995006745 A1 WO 1995006745A1 DE 9401003 W DE9401003 W DE 9401003W WO 9506745 A1 WO9506745 A1 WO 9506745A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- vector
- hepatitis
- liver
- gene
- vector according
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention relates to a vector for liver-specific gene therapy; Areas of application are medicine and genetic engineering.
- the aim of the invention is to construct a vector which finds liver cells highly specific in vivo, is effectively absorbed by the cells and can introduce introduced therapeutic genes into the cell nucleus.
- the vector is said to be usable for gene therapy in humans.
- a therapeutic gene which is coupled to a promoter is packaged in a polypeptide envelope and chemically, enzymatically or via antibodies coupled to protein domains of the HBV.
- the therapeutic gene used is the cDNA of a gene which is defective in the disease to be treated, i.e. H. is missing or changed by mutation. Examples of such genes are the LDL receptor gene, the absence of which causes the most common metabolic disease of the liver, familial hypercholesterolemia, and the alpha-1 antitrypsin gene.
- Liver-specific promoters preferably promoters / enhancers of the hepatitis B virus, such as e.g. the combinations core-promoter / enhancer II. In addition to their specificity, they are also small enough to be easily incorporated into an expression vector. Promoters of liver-specific genes such as albumin, PEPCK (phosphoenolpyruvate carboxykinase) or OTC (ornithine transcarbamylase) are also suitable for the construction of the vector according to the invention.
- promoters / enhancers of the hepatitis B virus such as e.g. the combinations core-promoter / enhancer II.
- Promoters of liver-specific genes such as albumin, PEPCK (phosphoenolpyruvate carboxykinase) or OTC (ornithine transcarbamylase) are also suitable for the construction of the vector according to the invention.
- the polypeptide envelope used for packaging preferably consists of chromosomal protein such as. B. purified HMG1.
- Other DNA-binding proteins such as e.g. B. Protamine or hepatitis core protein. This protein is particularly suitable because, in addition to its DNA binding and DNA condensation ability, it is a natural component of the hepatitis B virus and therefore favors incorporation into virus envelopes.
- the polypeptide shell can also be produced from polyamino acids of a type of basic amino acids, poly-L-lysine and poly-L-ornithine being primarily suitable.
- the particulate pre-SI / S protein of the hepatitis B virus used as a coupled component according to the invention can be isolated from virus-producing cells.
- pre-Sl / S protein is advantageously produced by genetic engineering for safety reasons.
- nucleic acid-free particles represent a complete virus envelope when viewed from the outer surface.
- the resulting vector thus has a high degree of homology to the natural hepatitis B virus and can therefore understand the infection mechanism.
- the invention can also be implemented with liposomes as transport vehicles.
- the surface of the liposomes used is modified by pre-Sl / S protein so that uptake via hepatitis B-specific mechanisms is possible.
- the vectors are produced in accordance with claim 10, subclaims 11 to 13 are preferred variants.
- An advantageous method is e.g. in that the coupling of the gene packaged in HMG1 to pre-S / S1 proteins of HBV takes place covalently by means of a transglutaminase reaction.
- the vector according to the invention enables a desired gene to be introduced into the tissue, in particular the liver of a patient, and to optimally design its path to the functional site. This is done, for example, by making up the vector and administering it to a patient via the bloodstream, preferably via the portal vein of the liver.
- the invention creates an essential prerequisite for therapy of genetic diseases of the liver.
- the envelope of the HBV virus consists of three proteins. They are translation products of an open reading frame in the HBV genome with different initiation sites. While the large coat protein (L: P39, GP42) contains the domains pre-S1, pre-S2 and S, the middle (M: P33, GP36) consists of pre-S2 and S and the small coat protein (S: P24 , GP27) only from the domain S.
- the genes of the small (S) and large (L) HBV coat protein are obtained by amplification from the genome of the hepatitis B virus (subtype ayw). Various variants are created for the L gene in order to facilitate the secretion of the protein.
- the recombinant plasmids and baculovirus DNA are cotransfected with lipofectin in Spodoptera frugiperda cells (Sf9).
- Sf9 Spodoptera frugiperda cells
- recombinant baculoviruses are generated, which express the HBV envelope proteins under control of the polyhedrin promoter in infected Sf9 cells. The synthesis of these proteins is demonstrated in the Western blot. Electron microscopy shows that the coat proteins associate to form particles.
- spinner cultures (10 9 Sf9 cells) are infected. 72 hours after infection, the cells are obtained by sedimentation and disrupted using ultrasound. After separation of membranes by sedimentation, the shell particles are cleaned either by centrifugation in the CsCl density gradient or by affinity chrootography. S-specific monoclonal antibodies are used here.
- the gene of the rat non-histone protein HMG1 is taken from the vector for bacterial expression pT7RNHMGl [Bianchi, E., Gene, 104 (1991) 271-275] and cloned into the baculovirus transfer vector PVL941. Recombinant baculoviruses are generated according to the method described for HBV genes. The expression detection is carried out by Coomassie staining of the proteins after SDS polyacrylamide gel electrophoresis.
- a cell lysate is generated by disruption using ultrasound, membranes are separated by sedimentation and the lysate is precipitated with 2% TCA. The supernatant is then subjected to acetone precipitation in an acidic medium. The resulting precipitate is dissolved and fractionated by ion exchange chromatography on a Mono Q column in a saline gradient. The fraction eluted at 1.7M NaCl contains electrophoretically pure HMG1.
- the DNA-protein complexes are formed by gradually adding a 20-fold excess of HMG1 to a DNA solution of 50 ⁇ g / ml in 150mM NaCl 10MM Tris / HCl at pH 8.0. DNA binding and condensation are detected by gel retardation and sedimentation in the sucrose gradient.
- HBV coat proteins are covalently cross-linked with HMG1 using transglutaminase.
- e-amino groups of the lysines in the HMG molecule act as acyl acceptors and 7-carboxamide groups of the glutamine residues in HBV-L and S protein as acyl donors.
- a confluent monolayer culture of human hepatocytes is infected with the transfer vector 5 days after installation for 12 hours.
- the Plas id pBAG [Proc. Natl. Acad. Be. 84 (1987) 156-160], which contains the gene of the ⁇ -galactosidase from E. coli under the control of a retroviral LTR, is packed into the vector. The gene transfer is detected 48 hours after infection by the in tu enzyme test for ⁇ -galactosidase.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94925337A EP0716711A1 (de) | 1993-09-03 | 1994-08-24 | Vektor für leber-gentherapie |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEP4329811.7 | 1993-09-03 | ||
DE4329811 | 1993-09-03 | ||
DEP4339922.3 | 1993-11-19 | ||
DE4339922A DE4339922C1 (de) | 1993-09-03 | 1993-11-19 | Vektor für Leber-Gentherapie |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995006745A1 true WO1995006745A1 (de) | 1995-03-09 |
Family
ID=25929205
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1994/001003 WO1995006745A1 (de) | 1993-09-03 | 1994-08-24 | Vektor für leber-gentherapie |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0716711A1 (de) |
CA (1) | CA2170882A1 (de) |
WO (1) | WO1995006745A1 (de) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5770442A (en) * | 1995-02-21 | 1998-06-23 | Cornell Research Foundation, Inc. | Chimeric adenoviral fiber protein and methods of using same |
US5895759A (en) * | 1993-09-03 | 1999-04-20 | Max-Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. | Vector for gene transfer in liver cells |
US6057155A (en) * | 1995-11-28 | 2000-05-02 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
US6127525A (en) * | 1995-02-21 | 2000-10-03 | Cornell Research Foundation, Inc. | Chimeric adenoviral coat protein and methods of using same |
US6913922B1 (en) | 1999-05-18 | 2005-07-05 | Crucell Holland B.V. | Serotype of adenovirus and uses thereof |
US6929946B1 (en) | 1998-11-20 | 2005-08-16 | Crucell Holland B.V. | Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells |
US6951755B2 (en) | 1994-09-08 | 2005-10-04 | Genvec, Inc. | Vectors and methods for gene transfer |
US6974695B2 (en) | 2000-11-15 | 2005-12-13 | Crucell Holland B.V. | Complementing cell lines |
US7235233B2 (en) | 2000-09-26 | 2007-06-26 | Crucell Holland B.V. | Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts |
US7468181B2 (en) | 2002-04-25 | 2008-12-23 | Crucell Holland B.V. | Means and methods for the production of adenovirus vectors |
US7749493B2 (en) | 1998-07-08 | 2010-07-06 | Crucell Holland B.V. | Chimeric adenoviruses |
-
1994
- 1994-08-24 WO PCT/DE1994/001003 patent/WO1995006745A1/de not_active Application Discontinuation
- 1994-08-24 CA CA 2170882 patent/CA2170882A1/en not_active Abandoned
- 1994-08-24 EP EP94925337A patent/EP0716711A1/de not_active Withdrawn
Non-Patent Citations (8)
Title |
---|
BÖTTGER M. ET AL.: "Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 950, 1988, pages 221 - 228 * |
BÖTTGER M. ET AL.: "Transfection by DNA-nuclear protein HMG1 complexes: raising of efficiency and role of DNA topology", ARCHIV FÜR GESCHWULSTFORSCHUNG, vol. 60, 1990, pages 265 - 270 * |
CHRISTIANO R. J. ET AL.: "Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 90, March 1993 (1993-03-01), WASHINGTON US, pages 2122 - 2126 * |
KURODA S. ET AL.: "Hepatitis B virus envelope L protein particles", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 267, no. 3, January 1992 (1992-01-01), BALTIMORE, MD US, pages 1953 - 1961 * |
NEURATH A. R. ET AL.: "Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus", CELL, vol. 46, 1986, CAMBRIDGE, NA US, pages 429 - 436 * |
PRICE P. M ET AL.: "Translational selection in the expression of the hepatitis B virus envelope proteins", DNA, vol. 7, 1988, pages 417 - 422 * |
WILSON J.M. ET AL.: "A novel mechanism for achieving transgene persistence in Vivo after somatic gene transfer into hepatocytes", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 16, 1992, BALTIMORE, MD US, pages 11483 - 11489 * |
YOUN B. W. ET AL.: "Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture", VACCINE, vol. 7, 1989, GUILDFORD GB, pages 60 - 68 * |
Cited By (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5895759A (en) * | 1993-09-03 | 1999-04-20 | Max-Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. | Vector for gene transfer in liver cells |
US6951755B2 (en) | 1994-09-08 | 2005-10-04 | Genvec, Inc. | Vectors and methods for gene transfer |
US6127525A (en) * | 1995-02-21 | 2000-10-03 | Cornell Research Foundation, Inc. | Chimeric adenoviral coat protein and methods of using same |
US6153435A (en) * | 1995-02-21 | 2000-11-28 | Cornell Research Foundation, Inc. | Nucleic acid that encodes a chimeric adenoviral coat protein |
US5770442A (en) * | 1995-02-21 | 1998-06-23 | Cornell Research Foundation, Inc. | Chimeric adenoviral fiber protein and methods of using same |
US6329190B1 (en) | 1995-11-28 | 2001-12-11 | Genvec, Inc. | Targetting adenovirus with use of constrained peptide motifs |
US6649407B2 (en) | 1995-11-28 | 2003-11-18 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
US6057155A (en) * | 1995-11-28 | 2000-05-02 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
US7749493B2 (en) | 1998-07-08 | 2010-07-06 | Crucell Holland B.V. | Chimeric adenoviruses |
US6929946B1 (en) | 1998-11-20 | 2005-08-16 | Crucell Holland B.V. | Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells |
US6913922B1 (en) | 1999-05-18 | 2005-07-05 | Crucell Holland B.V. | Serotype of adenovirus and uses thereof |
US7250293B2 (en) | 1999-05-18 | 2007-07-31 | Crucell Holland B.V. | Complementing cell lines |
US7270811B2 (en) | 1999-05-18 | 2007-09-18 | Crucell Holland B.V. | Serotype of adenovirus and uses thereof |
US7906113B2 (en) | 1999-05-18 | 2011-03-15 | Crucell Holland B.V. | Serotype of adenovirus and uses thereof |
US7235233B2 (en) | 2000-09-26 | 2007-06-26 | Crucell Holland B.V. | Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts |
US6974695B2 (en) | 2000-11-15 | 2005-12-13 | Crucell Holland B.V. | Complementing cell lines |
US7344883B2 (en) | 2000-11-15 | 2008-03-18 | Crucell Holland B.V. | Complementing cell lines |
US9228205B2 (en) | 2000-11-15 | 2016-01-05 | Crucell Holland B.V. | Complementing cell lines |
US7468181B2 (en) | 2002-04-25 | 2008-12-23 | Crucell Holland B.V. | Means and methods for the production of adenovirus vectors |
US7820440B2 (en) | 2002-04-25 | 2010-10-26 | Crucell Holland B.V. | Means and methods for producing adenovirus vectors |
Also Published As
Publication number | Publication date |
---|---|
EP0716711A1 (de) | 1996-06-19 |
CA2170882A1 (en) | 1995-03-09 |
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