CA2311119A1 - Method for transporting a molecular substance in predetermined areas of eucaryotic cells - Google Patents
Method for transporting a molecular substance in predetermined areas of eucaryotic cells Download PDFInfo
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- CA2311119A1 CA2311119A1 CA002311119A CA2311119A CA2311119A1 CA 2311119 A1 CA2311119 A1 CA 2311119A1 CA 002311119 A CA002311119 A CA 002311119A CA 2311119 A CA2311119 A CA 2311119A CA 2311119 A1 CA2311119 A1 CA 2311119A1
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- 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
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- 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
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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
- 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
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- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
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- Organic Chemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (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
The invention relates to a method for the production of a vehicle for transporting a molecular substance in the nucleus (ZK) of an eucaryotic cell, whereby at least one viral protein is modified so that it specifically carries out said transport to the nucleus (NK) and whereby the modified viral protein is coupled to or associated with the molecular substance.
Description
~
Method for transporting molecular substance into predetermined regions of eukaryotic cells The invention relates to a method for preparing a vehicle and to a vehicle for transporting molecular substance, such as DNA, RNA, protein, PNA, medicinal substances with lipophilic and lipophobic characteristics, into the cell nucleus of a eukaryotic cell.
WO 97/43431 discloses coupling molecular substance to a capsomere, which is derived or originates from a virus, for transport into eukaryotic cells. It is possible with a construct of this type to transport the molecular substance into eukaryotic cells with a high rate of uptake. However, it is a disadvantage with a construct of this type that it is impossible to transport the molecular substance specifically into the cell nucleus.
Braun, H. et al., Biotechnol. Appl. Biochem. (1999) 29, 31-43 describes the possibility of packaging oligo-nulcleotides [sic] and plasmid DNA in polyoma virus-like particles. The described particles likewise permit only nonspecific transport.
It is an object of the invention to eliminate the disadvantages of the state of the art. It is particularly intended to indicate a method for preparing a vehicle, and a vehicle for transporting molecular substance, with which targeted transport of the molecular substance into a predetermined region, for example the cell nucleus, of a eukaryotic cell can be carried out.
This object is achieved by the features of Claims 1 and 24. Advantageous embodiments of the invention are evident from the features of Claims 2 to 23 and 25 to 46.
Method for transporting molecular substance into predetermined regions of eukaryotic cells The invention relates to a method for preparing a vehicle and to a vehicle for transporting molecular substance, such as DNA, RNA, protein, PNA, medicinal substances with lipophilic and lipophobic characteristics, into the cell nucleus of a eukaryotic cell.
WO 97/43431 discloses coupling molecular substance to a capsomere, which is derived or originates from a virus, for transport into eukaryotic cells. It is possible with a construct of this type to transport the molecular substance into eukaryotic cells with a high rate of uptake. However, it is a disadvantage with a construct of this type that it is impossible to transport the molecular substance specifically into the cell nucleus.
Braun, H. et al., Biotechnol. Appl. Biochem. (1999) 29, 31-43 describes the possibility of packaging oligo-nulcleotides [sic] and plasmid DNA in polyoma virus-like particles. The described particles likewise permit only nonspecific transport.
It is an object of the invention to eliminate the disadvantages of the state of the art. It is particularly intended to indicate a method for preparing a vehicle, and a vehicle for transporting molecular substance, with which targeted transport of the molecular substance into a predetermined region, for example the cell nucleus, of a eukaryotic cell can be carried out.
This object is achieved by the features of Claims 1 and 24. Advantageous embodiments of the invention are evident from the features of Claims 2 to 23 and 25 to 46.
The invention provides a method for preparing a vehicle for transporting 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 cell nucleus, and wherein the modified viral protein is coupled or associated with the molecular substance.
Molecular substance means in this connection, for example, DNA, RNA, protein, PNA, medicinal substances with lipophilic and lipophobic characteristics.
The method according to the invention has the advantage that it is possible therewith for the molecular substance to be transported specifically into a predetermined region within a eukaryotic cell. This makes it possible to influence cell functions in a nonstressful and particularly effective way. It is possible with low doses of molecular substance to achieve a highly efficient, for example molecular therapeutic effect.
It is advantageous to modify an amino-terminal section of the viral protein, in which case at least one, preferably six, histidine residues) can be coupled thereto. It is also possible to couple polylysine and/or polyasparagine and/or polyarginine to the amino-terminal section. Coupling of histidine residues to the amino-terminal section, in particular the amino-terminal end of VPl, achieves particularly efficient transport of the molecular substance into the cell nucleus of eukaryotic cells.
The molecular substance which can be used is a cell type- or differentiation-specific enzyme, preferably telomerase. An active telomerase has been detected in carcinomas and in leukaemias. It appears that teleomerase [sic] is normally repressed and is activated only in the event of malignant transformation. It is possible by transferring telomerase into the cell nucleus using the method according to the invention to influence specifically the lifespan of the cell.
It is also possible to use an enzyme, preferably for replacement in lysosomal disorders, preferably in mucopolysacharidosis [sic], preferably Sanfilippo disease, and/or a protein, preferably an antibody. It is furthermore possible to use a peptide for immunization, in particular for T-cell-specific immunization. Furthermore suitable as molecular substance is bone morphogenic protein-6 (BMP-6) or a mutant or a modification thereof. It is possible and expedient to use as molecular substance a transcription factor, preferably NFxB.
NFxB is an inducible transcription factor which is activated in many different cells as response to a number of stress situations. It is taken up into the cell nucleus via a translocation signal. NFKB leads to rapid induction of genes which are responsible for an early defence or inflammatory reaction. In addition, NFKB plays a part in the regulation and differentiation of proteins.
It is also conceivable to use a phosphorothionate-derivatized [sic] oligonucleotide, PNA, a chimera of PNA and DNA or a DNA-peptide complex. Reference is made in this connection to Braun, H. et al., Biotechnol.
Appl. Biochem. (1999) 29, 31-43, in which the transfer of oligonucleotides into the cell is described. The content of the abovementioned publication is hereby incorporated.
The vehicle is expediently designed so that it can be transferred into a specific cell type, preferably into chondrocytes.
Molecular substance means in this connection, for example, DNA, RNA, protein, PNA, medicinal substances with lipophilic and lipophobic characteristics.
The method according to the invention has the advantage that it is possible therewith for the molecular substance to be transported specifically into a predetermined region within a eukaryotic cell. This makes it possible to influence cell functions in a nonstressful and particularly effective way. It is possible with low doses of molecular substance to achieve a highly efficient, for example molecular therapeutic effect.
It is advantageous to modify an amino-terminal section of the viral protein, in which case at least one, preferably six, histidine residues) can be coupled thereto. It is also possible to couple polylysine and/or polyasparagine and/or polyarginine to the amino-terminal section. Coupling of histidine residues to the amino-terminal section, in particular the amino-terminal end of VPl, achieves particularly efficient transport of the molecular substance into the cell nucleus of eukaryotic cells.
The molecular substance which can be used is a cell type- or differentiation-specific enzyme, preferably telomerase. An active telomerase has been detected in carcinomas and in leukaemias. It appears that teleomerase [sic] is normally repressed and is activated only in the event of malignant transformation. It is possible by transferring telomerase into the cell nucleus using the method according to the invention to influence specifically the lifespan of the cell.
It is also possible to use an enzyme, preferably for replacement in lysosomal disorders, preferably in mucopolysacharidosis [sic], preferably Sanfilippo disease, and/or a protein, preferably an antibody. It is furthermore possible to use a peptide for immunization, in particular for T-cell-specific immunization. Furthermore suitable as molecular substance is bone morphogenic protein-6 (BMP-6) or a mutant or a modification thereof. It is possible and expedient to use as molecular substance a transcription factor, preferably NFxB.
NFxB is an inducible transcription factor which is activated in many different cells as response to a number of stress situations. It is taken up into the cell nucleus via a translocation signal. NFKB leads to rapid induction of genes which are responsible for an early defence or inflammatory reaction. In addition, NFKB plays a part in the regulation and differentiation of proteins.
It is also conceivable to use a phosphorothionate-derivatized [sic] oligonucleotide, PNA, a chimera of PNA and DNA or a DNA-peptide complex. Reference is made in this connection to Braun, H. et al., Biotechnol.
Appl. Biochem. (1999) 29, 31-43, in which the transfer of oligonucleotides into the cell is described. The content of the abovementioned publication is hereby incorporated.
The vehicle is expediently designed so that it can be transferred 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 may also comprise a viral or nonviral transfer system, which preferably comprises liposomes.
It is regarded as particularly advantageous that the viral protein is virus protein 1 (VP1) of a papovavirus, preferably of polyomavirus. According to one embodiment feature, the VP1 can be a constituent of a VP1 pentamer, in which case the molecular substance is expediently coupled on one side of the VP1 pentamer or is associated therewith and/or is enclosed using at least one other capsomere in a capsid or a capsid-like structure so that the one side corresponds to the inside of the capsid or of the capsid-like structure.
Enclosure of the molecular substance in a capsid in particular brings about improved uptake of molecular substance in the cell.
It has proved to be advantageous for the molecular substance to link via bifunctional groups to cysteine residues located on the one side of the VP1 pentamer.
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 VP2 or VP3. However, it is also possible for the coupling of the molecular substance to take place 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.
Covalent bonding between the viral modified protein and the oligonucleotide can be promoted by adding bifunctional coupling reagents. A structure, which interacts for coupling or association with the molecular substance, of the VP1 pentamer expediently comprises affinity-increasing groups such as 4-iodo-acetamidosalicylic acid and/or p-arsonic acid-phenyl-diazonium [sic] fluoroborate and/or derivatives thereof. The structure interacting with the molecular substance can be formed in particular by epitopes of the VP1 pentamer.
The invention further provides a vehicle for transporting 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 the modified viral protein is coupled or associated with the molecular substance.
The vehicle has the advantage that molecular substance can be transported into predetermined regions of eukaryotic cells, in particular into the cell nucleus.
The targeted transport makes it possible to employ smaller amounts of molecular substance. The use of the vehicle according to the invention makes highly effective molecular therapy possible.
The embodiment features described in relation to the achievement of the method can be applied equally to the vehicle.
The invention is described in detail by means of the following examples and depictions. These show:
Fig. 1 a light micrograph of cells which have been treated with a modified VP1-oligonucleotide construct, Fig. 2 a light micrograph of cells which have been treated with oligonucleotides, Fig. 3 a light micrograph of cells which have been treated with an unmodified VP1-oligonucleotide construct, and Fig. 4 a light micrograph of cells which have been treated with oligonucleotides.
Fig. 1 shows a light micrograph of cells which have been treated with a construct of modified VP1 and fluorescein isothiozyanath-labelled [sic] oligo-nucleotide. CN refers to the cell nucleus, CM the cell membrane and CY the cytoplasm of a cell C. It is clearly evident that the fluorescence-labelled oligonucleotide has accumulated in the cell nucleus CN.
In this case, VP1 with a so-called "His tag" was used.
The "His tag" is an artificial sequence which consists of six histidine residues and which is coupled to the amino-terminal end of VP1.
Fig. 2 shows a light micrograph of cells which have been treated with fluorescence-labelled oligo-nucleotide. No specific accumulation of the oligonucleotide is evident.
Fig. 3 shows a light micrograph of cells which have been treated with a construct consisting of unmodified VP1 and fluorescence-labelled oligonucleotide. It is evident that on use of unmodified VP1 the oligonucleotide is distributed distinctly less specifically in the cell C. The oligonucleotide has accumulated both in the cytoplasm CY and in the nucleoli N present in the cell nucleus CN.
Fig. 4 shows a light micrograph of cells which have been treated with fluorescence-labelled oligo-nucleotide. As in Fig. 2, no specific accumulation of the oligonucleotides is detectable in this case either.
_ 7 _ Example:
Recombinantly expressed (E. coli), in vitro assembled, empty polyoma VP1 capsids were used for the results shown in Figs . 1 to 4 . These VP1 capsids were prepared using both VP1 proteins with His tag and VP1 proteins without His tag. The His tag used is depicted in the sequence listing shown hereinafter.
For the loading, the VP1 capsids were present in a buffer of the following composition: 10 mM sodium acetate, 150 mM sodium chloride, 5% glyerol [sic] (pH =
5). Fluorescein isothiocyanate-labelled oligo-nucleotides were employed. The in vitro introduction of the oligonucleotides into the VP1 capsids with His tag or without His tag was carried out by an osmotic method (Barr S.M., Keck K., Aposhian H.V., 1979, Cell-free assembly of a polyoma-like particle from empty capside and DNA, Virology 96(2); 656-659). The molar ratio of VP1 capsid to oligonucleotide was 1 to 439.3.
5 ~.1 of the fluorescence-labelled oligonucleotide solution were added to 95 ul of the VP1-containing solution. This solution was incubated at 37°C for 20 minutes. The osmotic shock took place by adding 350 ~,1 of distilled water and renewed incubation at 37°C for 40 minutes. For cell uptake, the water content of the loading mixture was made isotonic with mannitol.
Cell uptake of the oligonucleotide-loaded capsids with His tag or without His tag was tested in embryonic mouse fibroblasts, namely NIH 3T3 cells. For this purpose, the loading mixture was incubated at 37°C for 1 hour, then exchanged for 500 ~,1 of new cell culture medium, incubated at 37°C for 3 hours and again replaced by 500 ~1 of new cell culture medium. After a total incubation time of 24 hours at 37°C, the NIH 3T3 cells used for the loading mixture were fixed with methanol. Subsequent inspection took place in a _ g _ confocal laser scanning microscope. For this purpose, the membranes of the NIH 3T3 cells were counterstained with rhodamine-labelled concanavalin A.
_ g _ List of reference symbols cell CN cell nucleus CM cell membrane CY cytoplasm N nucleoli SEQUENCE LISTING
<110> november AG
<120> Method for transporting molecular substance into predetermined regions of eukaryotic cells <130> 380446 <140>
<141>
<160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 16 <212> PRT
<213> Artificial sequence <220>
<223> Description of the artificial sequence: His tag <400> 1 Met Arg Gly Ser His His His His His His Gly Ser Ile Glu Gly Arg
It is regarded as particularly advantageous that the viral protein is virus protein 1 (VP1) of a papovavirus, preferably of polyomavirus. According to one embodiment feature, the VP1 can be a constituent of a VP1 pentamer, in which case the molecular substance is expediently coupled on one side of the VP1 pentamer or is associated therewith and/or is enclosed using at least one other capsomere in a capsid or a capsid-like structure so that the one side corresponds to the inside of the capsid or of the capsid-like structure.
Enclosure of the molecular substance in a capsid in particular brings about improved uptake of molecular substance in the cell.
It has proved to be advantageous for the molecular substance to link via bifunctional groups to cysteine residues located on the one side of the VP1 pentamer.
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 VP2 or VP3. However, it is also possible for the coupling of the molecular substance to take place 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.
Covalent bonding between the viral modified protein and the oligonucleotide can be promoted by adding bifunctional coupling reagents. A structure, which interacts for coupling or association with the molecular substance, of the VP1 pentamer expediently comprises affinity-increasing groups such as 4-iodo-acetamidosalicylic acid and/or p-arsonic acid-phenyl-diazonium [sic] fluoroborate and/or derivatives thereof. The structure interacting with the molecular substance can be formed in particular by epitopes of the VP1 pentamer.
The invention further provides a vehicle for transporting 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 the modified viral protein is coupled or associated with the molecular substance.
The vehicle has the advantage that molecular substance can be transported into predetermined regions of eukaryotic cells, in particular into the cell nucleus.
The targeted transport makes it possible to employ smaller amounts of molecular substance. The use of the vehicle according to the invention makes highly effective molecular therapy possible.
The embodiment features described in relation to the achievement of the method can be applied equally to the vehicle.
The invention is described in detail by means of the following examples and depictions. These show:
Fig. 1 a light micrograph of cells which have been treated with a modified VP1-oligonucleotide construct, Fig. 2 a light micrograph of cells which have been treated with oligonucleotides, Fig. 3 a light micrograph of cells which have been treated with an unmodified VP1-oligonucleotide construct, and Fig. 4 a light micrograph of cells which have been treated with oligonucleotides.
Fig. 1 shows a light micrograph of cells which have been treated with a construct of modified VP1 and fluorescein isothiozyanath-labelled [sic] oligo-nucleotide. CN refers to the cell nucleus, CM the cell membrane and CY the cytoplasm of a cell C. It is clearly evident that the fluorescence-labelled oligonucleotide has accumulated in the cell nucleus CN.
In this case, VP1 with a so-called "His tag" was used.
The "His tag" is an artificial sequence which consists of six histidine residues and which is coupled to the amino-terminal end of VP1.
Fig. 2 shows a light micrograph of cells which have been treated with fluorescence-labelled oligo-nucleotide. No specific accumulation of the oligonucleotide is evident.
Fig. 3 shows a light micrograph of cells which have been treated with a construct consisting of unmodified VP1 and fluorescence-labelled oligonucleotide. It is evident that on use of unmodified VP1 the oligonucleotide is distributed distinctly less specifically in the cell C. The oligonucleotide has accumulated both in the cytoplasm CY and in the nucleoli N present in the cell nucleus CN.
Fig. 4 shows a light micrograph of cells which have been treated with fluorescence-labelled oligo-nucleotide. As in Fig. 2, no specific accumulation of the oligonucleotides is detectable in this case either.
_ 7 _ Example:
Recombinantly expressed (E. coli), in vitro assembled, empty polyoma VP1 capsids were used for the results shown in Figs . 1 to 4 . These VP1 capsids were prepared using both VP1 proteins with His tag and VP1 proteins without His tag. The His tag used is depicted in the sequence listing shown hereinafter.
For the loading, the VP1 capsids were present in a buffer of the following composition: 10 mM sodium acetate, 150 mM sodium chloride, 5% glyerol [sic] (pH =
5). Fluorescein isothiocyanate-labelled oligo-nucleotides were employed. The in vitro introduction of the oligonucleotides into the VP1 capsids with His tag or without His tag was carried out by an osmotic method (Barr S.M., Keck K., Aposhian H.V., 1979, Cell-free assembly of a polyoma-like particle from empty capside and DNA, Virology 96(2); 656-659). The molar ratio of VP1 capsid to oligonucleotide was 1 to 439.3.
5 ~.1 of the fluorescence-labelled oligonucleotide solution were added to 95 ul of the VP1-containing solution. This solution was incubated at 37°C for 20 minutes. The osmotic shock took place by adding 350 ~,1 of distilled water and renewed incubation at 37°C for 40 minutes. For cell uptake, the water content of the loading mixture was made isotonic with mannitol.
Cell uptake of the oligonucleotide-loaded capsids with His tag or without His tag was tested in embryonic mouse fibroblasts, namely NIH 3T3 cells. For this purpose, the loading mixture was incubated at 37°C for 1 hour, then exchanged for 500 ~,1 of new cell culture medium, incubated at 37°C for 3 hours and again replaced by 500 ~1 of new cell culture medium. After a total incubation time of 24 hours at 37°C, the NIH 3T3 cells used for the loading mixture were fixed with methanol. Subsequent inspection took place in a _ g _ confocal laser scanning microscope. For this purpose, the membranes of the NIH 3T3 cells were counterstained with rhodamine-labelled concanavalin A.
_ g _ List of reference symbols cell CN cell nucleus CM cell membrane CY cytoplasm N nucleoli SEQUENCE LISTING
<110> november AG
<120> Method for transporting molecular substance into predetermined regions of eukaryotic cells <130> 380446 <140>
<141>
<160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 16 <212> PRT
<213> Artificial sequence <220>
<223> Description of the artificial sequence: His tag <400> 1 Met Arg Gly Ser His His His His His His Gly Ser Ile Glu Gly Arg
Claims (46)
1. Method for preparing a vehicle for transporting molecular substance into the cell nucleus (CN) of a eukaryotic cell (C), wherein at least one viral protein is modified so that it is specific for transport into the cell nucleus (CN), and wherein the modified viral protein is coupled or associated with the molecular substance.
2. Method according to Claim 1, wherein an amino-terminal section of the viral protein is modified.
3. Method according to Claim 2, wherein at least one, preferably six, histidine residue(s) is/are coupled to the amino-terminal section.
4. Method according to any of the preceding claims, wherein polylysine and/or polyasparagine and/or polyarginine is/are coupled to the amino-terminal section.
5. Method according to any of the preceding claims, wherein a cell type- or differentiation-specific enzyme, preferably telomerase, is used as molecular substance.
6. Method according to any of the preceding claims, wherein an enzyme, preferably for replacement in lysosomal disorders, preferably mucopoly-sacharidosis [sic], preferably Sanfilippo disease, is used as molecular substance.
7. Method according to any of the preceding claims, wherein a protein, preferably an antibody, is used as molecular substance.
8. Method according to any of the preceding claims, wherein a peptide is used as molecular substance for immunization, in particular for T-cell-specific immunization.
9. Method according to any of the preceding claims, wherein bone morphogenic protein-6 (BMP-6) or a mutant or a modification thereof is used as molecular substance.
10. Method according to any of the preceding claims, wherein a transcription factor, preferably NFkB, is used as molecular substance.
11. Method according to any of the preceding claims, wherein a phosphorothionate-derivatized [sic]
oligonucleotide, PNA, a chimera of PNA and DNA or DNA-peptide complex is used as molecular substance.
oligonucleotide, PNA, a chimera of PNA and DNA or DNA-peptide complex is used as molecular substance.
12. Method according to any of the preceding claims, wherein the vehicle is designed so that it can be transferred into a specific cell type, preferably chondrocytes.
13. Method according to any of the preceding claims, wherein the viral protein is synthesized, purified and isolated.
14. Method according to any of the preceding claims, wherein the viral protein is complexed with the molecular substance.
15. Method according to any of the preceding claims, wherein the viral protein is virus protein 1 (VP1) of a papovavirus, preferably of polyomavirus.
16. Method according to any of the preceding claims, wherein the VP1 is constituent of a VP1 pentamer.
17. Method according to any of the preceding claims, wherein the molecular substance is coupled on one side of the VP1 pentamer or is associated therewith and/or is enclosed using at least one other capsomere in a capsid or a capsid-like structure so that the one side corresponds to the inside of the capsid or of the capsid-like structure.
18. Method according to Claim 17, wherein the molecular substance is linked via bifunctional groups to cysteine residues located on the one side of the VP1 pentamer.
19. Method according to any of the preceding claims, wherein the capsid-like structure comprises at least one virus protein 2 (VP2) and/or virus protein 3 (VP3) and/or proteins derived and/or modified from VP2 or VP3.
20. Method according to any of the preceding claims, wherein the coupling of the molecular substance takes place via the N-terminal end of VP2 or VP3 or a fragment thereof, and the C-terminal end binds to the inside of the VP1 pentamer.
21. Method according to any of the preceding claims, wherein covalent bonding between the modified viral protein and the oligonucleotides is achieved by adding bifunctional coupling reagents.
22. Method according to any of the preceding claims, wherein a structure, which interacts for coupling or association with the molecular substance, of the VP1 pentamer comprises affinity-increasing groups such as 4-iodoacetamidosalicylic acid and/or p-arsonic acid-phenyldiazonium [sic]
fluoroborate and/or derivatives thereof.
fluoroborate and/or derivatives thereof.
23. Method according to any of the preceding claims, wherein the structure interacting with the molecular substance is formed by epitopes of the VP1 pentamer.
24. Vehicle for transporting molecular substance into the cell nucleus (CN) of a eukaryotic cell (C), wherein at least one viral protein is modified so that it is specific for transport into the cell nucleus (CN), and wherein the modified viral protein is coupled or associated with the molecular substance.
25. Vehicle according to Claim 24, wherein an amino-terminal section of the viral protein is modified.
26. Vehicle according to Claim 25, wherein at least one, preferably six, histidine residue(s) is/are coupled to the amino-terminal section.
27. Vehicle according to any of Claims 24 to 26, wherein polylysine and/or polyasparagine and/or polyarginine is/are coupled to the amino-terminal section.
28. Vehicle according to any of Claims 24 to 27, wherein the molecular substance is a cell type- or differentiation-specific enzyme, preferably telomerase.
29. Vehicle according to any of Claims 24 to 28, wherein the molecular substance is an enzyme, preferably for replacement in lysosomal disorders, preferably mucopolysacharidosis [sic], preferably Sanfilippo disease.
30. Vehicle according to any of Claims 24 to 29, wherein the molecular substance is a protein, preferably an antibody.
31. Vehicle according to any of Claims 24 to 30, wherein the molecular substance is a peptide for immunization, in particular for T-cell-specific immunization.
32. Vehicle according to any of Claims 24 to 31, wherein the molecular substance is bone morphogenic protein-6 (BMP-6) or a mutant or a modification thereof.
33. Vehicle according to any of Claims 24 to 32, wherein the molecular substance is a transcription factor, preferably NF~B.
34. Vehicle according to any of Claims 24 to 33, wherein the molecular substance is a phosphorothionate-derivatized [sic]
oligo-nucleotide, PNA, a chimera of PNA and DNA or a DNA-peptide complex.
oligo-nucleotide, PNA, a chimera of PNA and DNA or a DNA-peptide complex.
35. Vehicle according to any of Claims 24 to 34, wherein the vehicle is designed so that it can be transferred into a specific cell type, preferably chondrocytes.
36. Vehicle according to any of Claims 24 to 35, wherein the viral protein is prepared by synthesis, purified and isolated.
37. Vehicle according to any of Claims 24 to 36, wherein the viral protein is complexed with the molecular substance.
38. Vehicle according to any of Claims 24 to 37, wherein the viral protein is virus protein 1 (VP1) of a papovavirus, preferably of polyomavirus.
39. Vehicle according to any of Claims 24 to 38, wherein the VP1 is constituent of a VP1 pentamer.
40. Vehicle according to any of Claims 24 to 39, wherein the molecular substance is coupled to one side of the VP1 pentamer, or is associated therewith, and/or is enclosed using at least one other capsomere in a capsid or a capsid-like structure so that the one side corresponds to the inside of the capsid or of the capsid-like structure.
41. Vehicle according to Claim 40, wherein the molecular substance is linked via bifunctional groups to cysteine residues located on the one side of the VP1 pentamer.
42. Vehicle according to any of Claims 24 to 41, wherein the capsid-like structure comprises at least one virus protein 2 (VP2) and/or virus protein 3 (VP3) and/or proteins derived and/or modified from VP2 or VP3.
43. Vehicle according to any of Claims 24 to 42, wherein the coupling of the molecular substance takes place via the N-terminal end of VP2 or VP3 or a fragment thereof, and the C-terminal end binds to the inside of the VP1 pentamer.
44. Vehicle according to any of Claims 24 to 43, wherein a covalent bond between the modified viral protein and the oligonucleotides is achieved by adding bifunctional coupling reagents.
45. Vehicle according to any of Claims 24 to 44, wherein a structure, which interacts for coupling or association with the molecular substance, of the VP1 pentamer comprises affinity-increasing groups such as 4-iodoacetamidosalicylic acid and/or p-arsonic acid-phenyldiazonium [sic]
fluoroborate and/or derivatives thereof.
fluoroborate and/or derivatives thereof.
46. Vehicle according to any of Claims 24 to 45, wherein the structure interacting with the molecular substance is formed by epitopes of the VP1 pentamer.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19829005.5 | 1998-06-29 | ||
| DE19829005A DE19829005C2 (en) | 1998-06-29 | 1998-06-29 | Process for the transport of molecular substance into predetermined areas of eukaryotic cells |
| PCT/DE1999/001805 WO2000000224A2 (en) | 1998-06-29 | 1999-06-19 | Method for transporting a molecular substance in predetermined areas of eucaryotic cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2311119A1 true CA2311119A1 (en) | 2000-01-06 |
Family
ID=7872407
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002311119A Abandoned CA2311119A1 (en) | 1998-06-29 | 1999-06-19 | Method for transporting a molecular substance in predetermined areas of eucaryotic cells |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1007107A2 (en) |
| CA (1) | CA2311119A1 (en) |
| DE (1) | DE19829005C2 (en) |
| WO (1) | WO2000000224A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19952957B4 (en) * | 1999-11-03 | 2006-08-31 | Acgt Progenomics Ag | Modular transport systems for molecular substances and their preparation and use |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1319101C (en) * | 1986-09-03 | 1993-06-15 | Marta Iris Sabara | Rotavirus nucleocapsid protein with or without binding peptides as immunologic carriers for macromolecules |
| 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 (en) * | 1991-02-04 | 1992-08-20 | University Of Saskatchewan | Vp6 encapsulated drug delivery |
| DE4335025A1 (en) * | 1993-10-14 | 1995-04-20 | Boehringer Ingelheim Int | Endosomolytically active particles |
| WO1996040958A1 (en) * | 1995-06-07 | 1996-12-19 | Baylor College Of Medicine | Nucleic acid transporters for delivery of nucleic acids into a cell |
| GB9607899D0 (en) * | 1996-04-17 | 1996-06-19 | Scottish Crop Research Inst | Virus-like particle |
| DE19618797C2 (en) * | 1996-05-10 | 2000-03-23 | Bertling Wolf | Vehicle for the transport of molecular substances |
| DE19750354A1 (en) * | 1997-11-13 | 1999-05-27 | November Ag Molekulare Medizin | Covering an inductor molecule with a viral capsid, for more efficient transfection of cells |
-
1998
- 1998-06-29 DE DE19829005A patent/DE19829005C2/en not_active Expired - Fee Related
-
1999
- 1999-06-19 WO PCT/DE1999/001805 patent/WO2000000224A2/en not_active Application Discontinuation
- 1999-06-19 CA CA002311119A patent/CA2311119A1/en not_active Abandoned
- 1999-06-19 EP EP99939916A patent/EP1007107A2/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000000224A2 (en) | 2000-01-06 |
| WO2000000224A3 (en) | 2000-04-13 |
| EP1007107A2 (en) | 2000-06-14 |
| DE19829005C2 (en) | 2000-08-31 |
| DE19829005A1 (en) | 1999-12-30 |
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