WO2002009507A1 - Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen - Google Patents
Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen Download PDFInfo
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- WO2002009507A1 WO2002009507A1 PCT/EP2000/007239 EP0007239W WO0209507A1 WO 2002009507 A1 WO2002009507 A1 WO 2002009507A1 EP 0007239 W EP0007239 W EP 0007239W WO 0209507 A1 WO0209507 A1 WO 0209507A1
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Classifications
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- 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
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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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
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A01K2227/101—Bovine
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
Definitions
- Somatic cloning gene transfer for the production of recombinant proteins, cells and organs
- the present invention relates to a method for the recombinant production of substances, in which cells with a nucleotide sequence coding for the substance are transformed, the transformed cells are subjected to a cloning process, and the cells thus obtained are introduced into a recipient organism.
- the present invention relates in particular to the use of the method in the production of recombinant proteins, cells and tissues.
- the invention relates to a method in which cells of an individual are isolated, these cells are introduced into an immunocompetent animal for further growth and the cells, tissues and / or organs grown in the animal are isolated again and introduced into an individual ,
- the cells used in the first method step can be any cells isolated from an individual that can be subjected to a cloning process. Examples of these are, in particular, fetal or adult fibroblasts, primordial germ cells, granulosa cells, thymocytes, spleen cells, liver cells, macrophages, testes or ovary cells etc.
- the cells can be used as such, as isolated, or kept in culture, even before transformation may have been subjected to a cloning process.
- the cells used for the transformation are isolated from an existing cell clone, a cell culture or an organism.
- cell clones are to be understood as meaning in vitro cultured, cloned cells, fetuses from cloning or cloned animals.
- the cells are then transformed with a nucleotide sequence that encodes the substance of interest.
- All substances that can be synthesized in the body are understood as substances, such as proteins, polysaccharides, lipids etc., but also the cells, tissues and organs that have the nucleotide sequence introduced.
- the nucleotide sequence coding for the substance is therefore not only a sequence which codes the substance directly, but also a sequence which codes the various polypeptides / enzymes which form the substance of interest in or outside the cells.
- recombinant refers to genes which are either exogenous to the animal or also endogenous, but which have been modified in their expression pattern by means of recombinant genetic engineering, such as, for example, in their differentiation-specific expression pattern or the amount expressed.
- All currently known techniques can be used for the transformation, such as physical, chemical or viral techniques.
- the use of homologous recombination is preferred with regard to the directional integration of the construct at specific locations in the genome. If large gene clusters are to be introduced into the recipient cell, it is also possible to use artificial chromosomes.
- Cell hybrids, such as those used for mapping can also be created and selected so that they also contain specific chromosomal fragments from the target genome in addition to the farm animal genome. This is especially so advantageous if an entire system of the target genome, such as the immunoglobulin system, is to be transferred.
- the cells are then subjected to a cloning process.
- Cloning methods are now known and are described, for example, in PCT / EP98 / 00230 (WO 99/36510) and PCT / EP / 00229 (WO), which is incorporated by reference here.
- the cloned cells are then grown to a certain stage of development in vivo and / or in vitro, for example to the fetus, and then introduced into the recipient organism (step c) of claim 1).
- the cloned cells can be grown to such an extent that a certain differentiation is present, with certain, differentiated cells already being introduced into the recipient organism.
- the cloned cells are then introduced into a recipient organism, which can be an animal, an animal fetus, an animal embryo or an animal cell aggregate, and also cloned animals, cloned animal fetuses, cloned animal embryos and cell aggregates.
- a recipient organism which can be an animal, an animal fetus, an animal embryo or an animal cell aggregate, and also cloned animals, cloned animal fetuses, cloned animal embryos and cell aggregates.
- the parent cells that are transformed and subjected to a cloning process are of the same genotype as the recipient organism.
- An advantage of such a procedure can be seen in the fact that an already existing, ie prepared recipient organism can be provided in a very short time with cells, preferably of the same genotype, which contain genes to be newly expressed, and produce recombinant substances.
- the time saved compared to conventional germline gene transfer can be up to 5 years until animals are ready for production.
- the present method is also more efficient since the producing organisms can be generated more economically.
- the polypeptide of interest can be obtained by means of transgenic animals, at least 3 to 4.
- the method according to the invention enables a considerable reduction in the time until an existing recipient organism is transformed with a new construct using cells and expression clones are created can be opened (only takes a few months).
- the method according to the invention therefore makes it possible to arrive at the first protein amounts for the analysis in a time period comparable to the in vitro cell culture, whereby according to the invention a production system is already available with which the desired substance can be produced.
- the recipient organisms or animals can therefore be used quickly to produce the desired substances.
- the recipient organism which is preferably produced or to be produced in stock, specifically for the subsequent somatic cell gene transfer. This is particularly necessary if the applied transgenic or differently prepared cells are to be given an advantage over the cells or structures present in the recipient organism in order to achieve better expression or expression.
- certain cells, tissues or organs can be removed in the recipient organism, which can be achieved by physical, chemical, immunological, molecular genetic or surgical methods.
- animals or fetuses that are homozygous negative at crucial gene locations can be bred or kept in stock and used as required.
- One way of depleting certain cells, tissues or organs in the recipient organism is to use transgenic organisms which contain a construct which is under the control of a stage of development-specific or an inducible promoter.
- a protein is formed automatically and tissue-specifically, which leads to the death / apoptosis of the corresponding cells, for example the diphtheria toxin.
- a "suicide" gene construct which has been preceded by a suitable tissue-specific promoter, is activated by application of a specific agent, such as, for example, tetracycline or ecdysone, which leads to the freely definable, specific depletion of the cells, tissues or organs in the Recipient organism leads.
- Another example is the total or partial removal of the blood stem cells or the stem cells of the immunological system from the recipient organism by, for example, wise radiation or other methods and their replacement by transformed stem cells, which were transformed according to the invention and subjected to a cloning process and which, for example, enable the expression of humanized antibodies (AK) or AK fragments (for example bispecific AK) or other animal or human blood factors.
- AK humanized antibodies
- AK fragments for example bispecific AK
- these recipient organisms form polyclonal human-identical antibodies which are of great value for the therapy of human diseases.
- a particularly efficient and unproblematic production system can be created for factors which are present in the animals but are present in too low a concentration for economically interesting extraction.
- An example of this is the expression of several genes of heparin synthesis under very strong promoters. Since the heparin is naturally present in the organism, no immunological reactions would be elicited either against the cells, which preferably belong to the same genotype, or against the product.
- the recloning of cells can be repeated at least three times. It has been found on this side that cells can complete up to 150 divisions, i.e. the cells from the cloning process survive much longer than normal cells and can therefore survive and express genes at least as long after several cloning processes in the animal and express the life span of the animal.
- cells can be obtained from an existing animal, and embryos, fetuses and cell lines cultivated in vitro can be created. These cell lines are genetically modified using known methods. The cells are then differentiated directly in the in vitro culture or isolated after repeated cloning from fetal organs and introduced into the adult parent organism as "transgenic" but otherwise genotypically originating cells. Since these, with the exception of the transgene, do not differ immunologically from this, the cells are able to Establish animal and will produce, for example, recombinant substances according to the genetic transformation.
- the invention also relates to a method for producing cells, tissues or organs in animal organisms, in which cells of an individual are isolated in a first step, the cells are introduced into an immune-incompetent animal organism, the animal organism is grown the cells of the individual grown in the organism are isolated, and the isolated cells are introduced into an individual.
- This method is particularly suitable for culturing cells, tissues and organs, preferably of human origin, in animal organisms, since the cells in the recipient animal organism are replicated or also grow into tissues and organs.
- the cells of the starting individual, or the tissues or organs grown therefrom are returned to the same individual from which they were removed, whereby tissues or organs can be made available which are used in a return (transplantation) the individual cannot be repelled.
- Any animal or fetus or animal embryo / cell aggregate that does not reject the cells derived from the starting individual is understood as an immune-incompetent animal organism.
- organisms that are at such an early stage of development are suitable for this, during which the developing immune system has not yet learned to distinguish between its own and a foreign one. If cells of the starting individual are therefore introduced into such a recipient organism, then these cells are not rejected but essentially recognized as being their own and further developed accordingly.
- Animal organisms obtained by cloning are therefore preferably suitable as the recipient animal organism. So it is possible to have multiple fetuses or embryos
- the fetuses / embryos / cell aggregates are then up to one stage grown in which they have produced the desired effect, for example until the development of the mature organ or tissue or also until the development of a sufficient amount of cells of the starting individual.
- This tissue / organ or these cells are then obtained, for example by removing the organ from the animal, and transferring it to the starting individual. Depending on the distance of a particular organ, this will of course involve killing the animal.
- the cells of the starting individual can be introduced into a conventional recipient organism.
- the animal is expected to chimerize the cells / tissue / organ with its own cells / tissue / organ.
- the corresponding tissue / organ / the cells as a whole can be isolated and the animal cells separated, for example by means of labeling using antibodies directed against the animal cells and separation by means of FACS (Fluorescence Activated Cell Sorting) can be achieved.
- FACS Fluorescence Activated Cell Sorting
- the cells / tissues / organs in question are removed from the recipient organism beforehand, which can be done as described above.
- the corresponding organ-specific stem cells of the starting individual such as adult human stem cells which are obtained directly from the affected organ, are introduced into the fetus and will then replace the deputated cells in the fetus and form the appropriate organ.
- the organ in question represents a xenoorgan in the animal organism, which, however, is not rejected due to the lack of immune competence of the fetus.
- the use of the present method thus allows the generation of cells, tissues or organs within a short period of time, so that even certain patients themselves can be quickly supplied with organs with an essentially or absolutely identical MHC type.
- the method according to the invention therefore solves the currently existing problems of the scarcity of available organs / tissues for the transplantation.
- the following examples illustrate the invention without restricting it.
- the examples describe the production of bispecific antibodies from the serum of non-transgenic clone calves with transgenic blood stem cells.
- the following steps are carried out for the generation of calves with transgenic bone marrow cells, reference being made to PCT / EP98 / 00230 or PCT / EP98 / 0029 for the details, which are incorporated here by reference.
- BOFFs primary bovine fetal fibroblasts
- BOFFs were plated semi-confluently and the sensitivity to neomycin (G418), hygromycin and puromycin was examined in terms of concentration and effect / time.
- different Preparations from BOFFs showed clear differences (up to 2-100x) in relation to the necessary antibiotic concentrations in order to achieve the LD ⁇ 00 .
- the time / effect windows between and within the preparations were sometimes greatly shifted.
- the best results were achieved with puromycin at a concentration of 1.5 ⁇ g / ml (3-5 times the concentration compared to the selection of stable human or murine fibroblast cell lines).
- DOSPER Lipofectin, polycationic lipids
- LIPOFECTAMINETM (GBCOBRL Life Technologies, Lipofection, polycationic lipids);
- CLONfectinTM (Clontech, Lipofection, cationic and amophilic lipids);
- TransFastTM Promega, Lipofection, cationic and amphophilic lipids
- Ca 3 (PO 4 ) 2 DNA precipitation
- BOFFs cannot be plated below a certain critical density.
- BOFFs either die or go through one or more crises, which lead to changes in morphology and / or prophylactic behavior.
- the plating density of the BOFFs was therefore chosen in such a way that optimal growth was ensured.
- the antibiotic-resistant cells represent a population and are not of clonal origin.
- Subsequent isolation and subcloning generate transgenic clonal cell lines b) Transfection with ScFv and pJW ⁇ puro in BOFFs
- MAR the MAR sequences (chicken lysozyme gene matrix attachment regions; Castilla et al., Nat. Biotechnol. 16 (1998), 349) were cloned with ScFv in BlueScript (Stratagene). p77 (Brem et al., Theriogenology 43 (1995), 175) in ⁇ UC18 (Norrander et al., Gene 26 (1983), 101).
- pJW ⁇ puro (Morgenstern & Land, Nucleic Acids Res. 18, 1068) p77 and MAR :: ScFv were linearized to guarantee a functional integration of the constructs. Due to the restriction enzyme interfaces available, the vector sequences could not be separated from the gene construct sequences. pJW ⁇ puro was transfected circularly / supercoiled.
- BOFF # 32330201 pl cells were used, from which embryos had already been created more than a year before the transefection by cloning and after transfer also clone calves (10 calves born). After the trypsin treatment, the cell suspension was transferred to 10 mm culture dishes and with Dulbecco's modified Eagle's Medium (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum (Biochrom, Berlin), 2mM L-glutamine, 10-4mM 2-mercaptoethanol , 2 mM non-essential amino acids (Sigma, St. Louis, MO), 100 IU / ml penicillin and 100 ⁇ g / ml streptomycin. The cells are cultured at 37 ° C in 5% CO 2 in air until the cell lawn is subconfluent (2 to 3 days) after which part of this "passage 0" is frozen (10% dimethyl sulfoxide, Sigma) and stored in liquid nitrogen ,
- the selection medium was added 24 hours after the transfection. After 2 days of selection in 6-well culture dishes, the cells of a 0 35 mm well were divided 1: 150 into a 24-well dish (concentration 2-8 x 10 2 cells / dish). The selection was continued until the shell was subconfluently covered with puro®-BOFFs (4-9 days). The pools were expanded, cryopreserved and tested for successful transfection with p77 and MAR :: ScFv using PCR.
- Example 2 The animals were cloned according to the procedure described in PCT / EP98 / 00229 and the transgenic embryos were transferred to the recipient animals.
- Transgenic cattle fetuses are obtained in the second trimester of pregnancy. During this period, the hepato-linear period of fetal development, the liver is the main site of blood formation and the
- the hemocytoblasts After immigration into the bone marrow cavity, the B lymphocytes also develop from these hemocytoblasts.
- the fetal liver is obtained aseptically and taken up in RPMI medium with 4 ⁇ g gentamycin sulfate and 200 IU / ml heparin as an anticoagulant.
- Separation into individual cells is carried out under strictly sterile conditions.
- the transplantation of the fetal blood cells takes place in a suspension in physiological saline or medium by intravenous infusion using a catheter (14 gauge, 1.7mm x 64mm, Teruno Co. Ltd).
- Blood is obtained by puncturing the jugular vein at regular intervals after transfusion of the transgenic blood cells.
- the serum is separated from the blood cells by centrifugation of the blood (100 g, 30 min.).
- the serum is mixed with sodium azide (final concentration 0.02%) and filtered through cellulose acetate filters (0.22 ⁇ m).
- the supernatant is adjusted to pH 7.2 with 1 M NaOH.
- the Culture supernatant was added to a protein L-agarose column (# 20520 Pierce, Rockford II, USA) equilibrated with 0.1M sodium phosphate buffer (protein L binds human IgG in particular also single chain variable elements (ScFv) but not bovine IgGl and IgG2).
- the column bed is washed with 0.1 M phosphate buffer.
- the bound proteins are eluted step by step through a 0.1 M glycine buffer at pH values of 3.0 and 2.0.
- the eluate collected is dialyzed overnight against PBS, filtered 0.22 ⁇ m sterile and stored at 4 ° C.
- the biological activity of the bispecific antibodies is tested in the cytotoxicity test with 5000 SK-Mel63 or M21 tumor cells. These two melanoma cell lines are very positive for the target antigen HMWG (high molecular weight glycoprotein), which is recognized by the monoclonal antibody (only scFv portion) 9.2.27.
- HMWG high molecular weight glycoprotein
- Peripheral blood lymphocytes PBLs freshly obtained from healthy human donor and isolated by Ficoll gradient
- PBLs freshly obtained from healthy human donor and isolated by Ficoll gradient
- the non-adherent blood lymphocytes are removed by washing them several times with PBS, so that only the remaining adherent tumor cells remain in the cell culture dishes (visual control), the cell culture dishes become 100 ⁇ l fresh medium and 10 ⁇ l of the Cell Proliferation Reagent dye WST (Boehringer Mannheim, Cat. No. 1644 807) was added. This is followed by another incubation (37 ° C / 5% CO 2 ) in the incubator for 1 to 4 hours and then an evaluation using an ELISA reader (480 nm). The visual evaluation and the low optical density show a tumor cell killing of 100%. This shows that recombinant bispecific antibodies that are produced in transgenic blood cells are highly efficient.
- WST Cell Proliferation Reagent dye
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JP2002515074A JP2004518405A (ja) | 2000-07-27 | 2000-07-27 | 組換えタンパク質、細胞、および生物を産生するための体細胞クローニング遺伝子移入 |
AU2000265672A AU2000265672A1 (en) | 2000-07-27 | 2000-07-27 | Somatic cloning gene transfer for the production of recombinant proteins, cells and organs |
CA2416877A CA2416877C (en) | 2000-07-27 | 2000-07-27 | Somatic cloning gene transfer for producing recombinant proteins, cells and organs |
PCT/EP2000/007239 WO2002009507A1 (de) | 2000-07-27 | 2000-07-27 | Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen |
EP00953104A EP1303183A1 (de) | 2000-07-27 | 2000-07-27 | Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen |
US10/333,670 US8030537B1 (en) | 2000-07-27 | 2000-07-27 | Somatic cloning gene transfer for the production of recombinant proteins, cells and organs |
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PCT/EP2000/007239 WO2002009507A1 (de) | 2000-07-27 | 2000-07-27 | Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen |
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EP (1) | EP1303183A1 (de) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7129062B2 (en) | 2001-01-26 | 2006-10-31 | Selexis Sa | Matrix attachment regions and methods for use thereof |
US8012933B2 (en) | 2003-01-29 | 2011-09-06 | Lipopeptide Ab | Use of the cathelicidin LL-37 and derivatives therof for wound healing |
US8252917B2 (en) | 2003-10-24 | 2012-08-28 | Selexis S.A. | High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure of MAR sequences |
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- 2000-07-27 EP EP00953104A patent/EP1303183A1/de not_active Withdrawn
- 2000-07-27 JP JP2002515074A patent/JP2004518405A/ja active Pending
- 2000-07-27 WO PCT/EP2000/007239 patent/WO2002009507A1/de active Application Filing
- 2000-07-27 CA CA2416877A patent/CA2416877C/en not_active Expired - Fee Related
- 2000-07-27 US US10/333,670 patent/US8030537B1/en not_active Expired - Fee Related
- 2000-07-27 AU AU2000265672A patent/AU2000265672A1/en not_active Abandoned
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7129062B2 (en) | 2001-01-26 | 2006-10-31 | Selexis Sa | Matrix attachment regions and methods for use thereof |
US8012933B2 (en) | 2003-01-29 | 2011-09-06 | Lipopeptide Ab | Use of the cathelicidin LL-37 and derivatives therof for wound healing |
US8506994B2 (en) | 2003-01-29 | 2013-08-13 | Lipopeptide Ab | Use of the cathelicidin LL-37 and derivatives thereof for wound healing |
US8936807B2 (en) | 2003-01-29 | 2015-01-20 | Lipopeptide Ab | Use of the cathelicidin LL-37 and derivatives thereof for wound healing |
US9125875B2 (en) | 2003-01-29 | 2015-09-08 | Lipopeptide Ab | Use of the cathelicidin LL-37 and derivatives thereof for wound healing |
US8252917B2 (en) | 2003-10-24 | 2012-08-28 | Selexis S.A. | High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure of MAR sequences |
US9879297B2 (en) | 2003-10-24 | 2018-01-30 | Selexis Sa | High efficiency gene transfer and expression in mammalian cells by amultiple transfection procedure of MAR sequences |
US10669562B2 (en) | 2003-10-24 | 2020-06-02 | Selexis S.A. | High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure of MAR sequences |
Also Published As
Publication number | Publication date |
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US8030537B1 (en) | 2011-10-04 |
CA2416877C (en) | 2013-03-12 |
CA2416877A1 (en) | 2003-01-22 |
AU2000265672A1 (en) | 2002-02-13 |
EP1303183A1 (de) | 2003-04-23 |
JP2004518405A (ja) | 2004-06-24 |
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