EP1007107A2 - Procede pour amener une substance moleculaire dans des zones predeterminees de cellules eucaryotes - Google Patents

Procede pour amener une substance moleculaire dans des zones predeterminees de cellules eucaryotes

Info

Publication number
EP1007107A2
EP1007107A2 EP99939916A EP99939916A EP1007107A2 EP 1007107 A2 EP1007107 A2 EP 1007107A2 EP 99939916 A EP99939916 A EP 99939916A EP 99939916 A EP99939916 A EP 99939916A EP 1007107 A2 EP1007107 A2 EP 1007107A2
Authority
EP
European Patent Office
Prior art keywords
molecular substance
vehicle according
protein
pentamer
vehicle
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.)
Withdrawn
Application number
EP99939916A
Other languages
German (de)
English (en)
Inventor
Wolf Bertling
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.)
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Original Assignee
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
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 November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin filed Critical November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Publication of EP1007107A2 publication Critical patent/EP1007107A2/fr
Withdrawn legal-status Critical Current

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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to a method for producing a vehicle and a vehicle for transporting molecular substance, such as DNA, RNA, protein, PNA, drugs of lipophilic and lipophobic character, into the cell nucleus of a eukaryotic cell.
  • molecular substance such as DNA, RNA, protein, PNA, drugs of lipophilic and lipophobic character
  • the object of the invention is to eliminate the disadvantages of the prior art.
  • a method for producing a vehicle and a vehicle for transporting molecular substance are to be specified, with which a targeted transport of the molecular substance into a predetermined region, for example the cell nucleus, of a eukaryotic cell can be carried out.
  • a method for producing a vehicle for the transport of molecular substance into the cell nucleus of a eukaryotic cell wherein at least one viral protein is modified so that it is specific for transport into the cell nucleus, and wherein the modified viral Protein is coupled or associated with the molecular substance.
  • Molecular substance is understood here to mean, for example, DNA, RNA, protein, PNA, medicinal substances lipophilic and lipophobic character.
  • the method according to the invention has the advantage that it allows the molecular substance to be transported in a specific area within a eukaryotic cell. This enables a gentle and particularly effective influencing of cell functions. With low doses of molecular substance, a highly efficient, e.g. molecular therapeutic effect can be achieved.
  • An amino-terminal section of the viral protein is advantageously modified, it being possible for at least one, preferably six, histidine residues to be coupled thereto.
  • Polylysine and / or polyasparagine and / or polyarginine can also be coupled to the amino terminal section.
  • a cell-type or differentiation-specific enzyme preferably telomerase
  • telomerase can be used as the molecular substance. Active telomerase has been demonstrated in carcinoma and leukemia. It appears that the teleomerase is normally repressed and is only activated in the case of malignant transformations. The life of the cell can be influenced in a targeted manner by transferring telomerase into the cell nucleus using the method according to the invention.
  • An enzyme preferably for substitution in the case of lysosomal diseases, preferably in the case of mucopolysaccharidosis, preferably Sanfilippo disease, and / or a protein, preferably an antibody, can also be used.
  • a peptide can be used for immunization, in particular for T cell-specific immunization.
  • Bone Morphogenic Protein-6 (BMP-6) or a mutant or a modification thereof can also be considered as a molecular substance. Conveniently, as a molecular substance
  • Transcription factor preferably NFKB, can be used.
  • NFKB is an inducible transcription factor that is activated in many different cells in response to a number of stressful situations. It is taken up into the cell nucleus via a translocation signal. NFKB leads to the rapid induction of genes that are responsible for an early defense or inflammatory response. NFKB also plays a role in the regulation and differentiation of proteins.
  • PNA phosphorothionate-derivatized oligonucleotide
  • a chimeric from PNA and DNA or a DNA-peptide complex is also conceivable.
  • the vehicle is expediently designed such that it can be introduced into a specific cell type, preferably into chondrocytes.
  • the viral protein can advantageously be synthesized, purified and isolated.
  • the viral protein can then be complexed with the molecular substance.
  • the molecular substance can also be a viral or a non-viral, preferably having a liposome, transfer system.
  • the viral protein is Virus Protein 1 (VP1) of a papovavirus, preferably of polyavirus.
  • the VP1 can be a component of a VPl pentamer, the molecular substance expediently being coupled to or associated with one side of the VPl pentamer and / or being enclosed in a capsid or a capsid-like structure using at least one further capsomer, so that one side corresponds to the inside of the capsid or the capsid-like structure.
  • the inclusion of the molecular substance in a capsid brings about an improved absorption of molecular substance into the cell.
  • the capsid-like structure can comprise at least one virus protein 2 (VP2) and / or virus protein 3 (VP3) and / or proteins derived and / or modified from the VP2 or VP3.
  • VP2 virus protein 2
  • VP3 virus protein 3
  • proteins derived and / or modified from the VP2 or VP3 it is also possible for the molecular substance to be coupled via the N-terminal end of VP2 or VP3 or a fragment thereof and for the C-terminal end to bind to the inside of the VP1 pentamer.
  • the aforementioned measures increase the stability of a construct formed from the molecular substance and the modified VP1.
  • a structure of the VPl pentamer which interacts with the molecular substance for coupling or association expediently comprises groups which increase affinity, such as 4-iodoacetamido-salicylic acid and / or p-arsonic acid-phenyldiazonium fluoroborate and / or derivatives thereof.
  • the structure which interacts with the molecular substance can be formed in particular by epitopes of the VP1 pentamer.
  • a vehicle for the transport of molecular substance into the cell nucleus of a eukaryotic cell, at least one viral protein being modified so that it is specific for the transport into the cell nucleus, and the modified viral protein with the molecular substance is coupled or associated.
  • the vehicle has the advantage that it allows molecular substance to enter predetermined areas of eukaryotic cells, especially in the cell nucleus can be transported. Because of the targeted transport, smaller amounts of molecular substance can be used.
  • the use of the vehicle according to the invention enables highly effective molecular therapy.
  • FIG. 1 shows a light micrograph of cells which have been treated with a modified VPI oligonucleotide construct
  • FIG. 3 shows a light micrograph of cells which have been treated with an unmodified VP1 oligonucleotide construct
  • FIG. 1 shows an optical micrograph of cells which have been treated with a construct composed of modified VP1 and fluorose isothiocyanate-labeled oligonucleotide.
  • ZK denotes the cell nucleus
  • ZM the cell membrane
  • ZY the cytoplasm a of a cell Z.
  • the fluorescence-labeled oligonucleotide is clearly enriched in the cell nucleus ZK.
  • VPl is called "His Day” was used.
  • the "His tag” is an artificial sequence consisting of six histidine residues, which is coupled to the amino-terminal end of VP1.
  • FIG. 2 shows an optical micrograph of cells which have been treated with fluorescence-labeled oligonucleotide. No specific enrichment of the oligonucleotide is discernible.
  • FIG. 3 shows an optical micrograph of cells which have been treated with a construct consisting of unmodified VP1 and fluorescence-labeled oligonucleotide. It can be seen that when non-modified VP1 is used, the oligonucleotide is distributed significantly more unspecifically in cell Z. The oligonucleotide is enriched both in the cytoplasm ZY and in the nucleoli N located in the cell nucleus ZK.
  • FIG. 4 shows a light micrograph of cells which have been treated with fluorescence-labeled oligonucleotide. As in FIG. 2, it can also be seen here that no specific enrichment of the oligonucleotides can be determined.
  • the cell uptake of the capsids loaded with oligonucleotides with His tag or without His tag was tested in embryonic mouse fibroblasts, namely NIH 3T3 cells. For this, the loading mixture was incubated for 1 hour at 37 ° C., then replaced with 500 ⁇ l of new cell culture medium, for 3
  • the NIH 3T3 cells used for the loading batch were fixed with methanol. The later examination was carried out in a confocal laser scanning microscope. For this purpose, the membranes of the NIH 3T3 cells were stained with concanavalin A labeled with rhodamine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de produire un véhicule servant à amener une substance moléculaire dans le noyau (ZK) de cellules eucaryotes (Z), selon lequel au moins une protéine virale est modifiée de façon à assurer spécifiquement le transport jusqu'audit noyau (ZK), et la protéine virale modifiée est couplée ou associée à ladite substance moléculaire.
EP99939916A 1998-06-29 1999-06-19 Procede pour amener une substance moleculaire dans des zones predeterminees de cellules eucaryotes Withdrawn EP1007107A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19829005 1998-06-29
DE19829005A DE19829005C2 (de) 1998-06-29 1998-06-29 Verfahren zum Transport molekularer Substanz in vorgegebene Bereiche eukaryontischer Zellen
PCT/DE1999/001805 WO2000000224A2 (fr) 1998-06-29 1999-06-19 Procede pour amener une substance moleculaire dans des zones predeterminees de cellules eucaryotes

Publications (1)

Publication Number Publication Date
EP1007107A2 true EP1007107A2 (fr) 2000-06-14

Family

ID=7872407

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99939916A Withdrawn EP1007107A2 (fr) 1998-06-29 1999-06-19 Procede pour amener une substance moleculaire dans des zones predeterminees de cellules eucaryotes

Country Status (4)

Country Link
EP (1) EP1007107A2 (fr)
CA (1) CA2311119A1 (fr)
DE (1) DE19829005C2 (fr)
WO (1) WO2000000224A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19952957B4 (de) * 1999-11-03 2006-08-31 Acgt Progenomics Ag Modulare Transportsysteme für molekulare Substanzen und deren Herstellung und Verwendung

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1319101C (fr) * 1986-09-03 1993-06-15 Marta Iris Sabara Proteine de capside nucleique de rotavirus avec ou sans peptides liants comme porteurs de macromolecules immunologiques
US4950599A (en) * 1987-01-29 1990-08-21 Wolf Bertling Method for exchanging homologous DNA sequences in a cell using polyoma encapsulated DNA fragments
WO1992013568A1 (fr) * 1991-02-04 1992-08-20 University Of Saskatchewan Systeme d'administration d'un medicament encapsule par du vp6
DE4335025A1 (de) * 1993-10-14 1995-04-20 Boehringer Ingelheim Int Endosomolytisch wirksame Partikel
WO1996040958A1 (fr) * 1995-06-07 1996-12-19 Baylor College Of Medicine Transporteurs d'acide nucleique servant a introduire des acides nucleiques dans une cellule
GB9607899D0 (en) * 1996-04-17 1996-06-19 Scottish Crop Research Inst Virus-like particle
DE19618797C2 (de) * 1996-05-10 2000-03-23 Bertling Wolf Vehikel zum Transport molekularer Substanz
DE19750354A1 (de) * 1997-11-13 1999-05-27 November Ag Molekulare Medizin Verfahren zur Applikation von Wirkstoffen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0000224A2 *

Also Published As

Publication number Publication date
DE19829005A1 (de) 1999-12-30
WO2000000224A2 (fr) 2000-01-06
CA2311119A1 (fr) 2000-01-06
DE19829005C2 (de) 2000-08-31
WO2000000224A3 (fr) 2000-04-13

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